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recent years have seen the development of a new class of implicit - solvent models the variational implicit - solvent model ( vism ) . coupled with the robust level - set numerical method , such models allow an efficient and quantitative description of molecular solvation . central in the vism is a mean - field free - energy functional of all possible solute solvent interfaces , or dielectric boundaries , that separate the continuum solvent from all solute atoms . in a simple setting , such a free - energy functional consists of surface energy , solute solvent van der waals interaction energy , and continuum electrostatic free energy , all depending solely on a given solute the minimization of the functional determines the solvation free energy and stable equilibrium solute solvent interfaces . mathematically , such minimization leads to highly nonlinear geometrical partial differential equations that are hard to solve in general . in our previous work , we developed a level - set method to numerically relax the free - energy functional in the three - dimensional space . our extensive numerical results with comparison with molecular dynamics ( md ) simulations demonstrated the success of this new approach to the solvation of nonpolar molecular systems in capturing the hydrophobic interaction , multiple equilibrium states of hydration , and fluctuation between such states . most of the existing , surface type , implicit - solvent models are based on various kinds of predefined solute solvent interfaces , such as the van der waals surface ( vdws ) , solvent - excluded surface ( ses ) , or solvent - accessible surface ( sas ) . such a surface is used to compute the solvation free energy as the sum of the surface energy and electrostatic energy , with the two contributions being decoupled . in contrast , those contributions are coupled in the free - energy functional in the vism , making free - energy estimation more consistent with physical processes , such as capillary evaporation , many - body hydrophobic effects , and hydrophobic hydrophilic coupling effects . consequently , stable equilibrium solute solvent interfaces determined by our level - set vism can be quite different from vdws , ses , or sas , particularly when it comes to the description of hydrophobic interactions . perhaps the most significant feature of vism is that its free - energy functional exhibits a complex energy landscape with multiple local minima corresponding to different equilibrium states . an underlying solvation system can fluctuate among these states and exhibit hysteresis ( i.e. , a relaxation - pathway - dependent ensemble of equilibrium states ) . all of these are difficult to capture with a fixed - surface type implicit - solvent model . we notice that several related issues , such as coupling the solvent boundary to the optimization of overall energy , the curvature effect to surface energy , and dewetting transition , have been discussed in the literature . other related models and methods have also been proposed . up to now , the development of level - set vism has focused only on nonpolar systems . in this work , we propose and implement an efficient approach to treat the electrostatics in the framework of vism . this approach is based on the coulomb - field approximation ( cfa ) of the electric displacement field , without solving the poisson or poisson boltzmann equation as often done in an implicit - solvent model . with such an approximation , we can express the electrostatic part of the solvation free energy as a volume integral over the solvent region that can be arbitrarily shaped . but , we do not compute generalized born radii and hence introduce no additional parameters . moreover , the cfa of electrostatics allows us to derive a simple analytical formula of the effective boundary force , defined as the negative functional derivative of the total free energy with respect to the location change of the dielectric boundary . this force is used as the normal velocity in our level - set numerical method . we emphasize that all of our level - set calculations are done in the three - dimensional space for arbitrarily shaped solute solvent interfaces . this simple radially symmetric system is used to test the convergence and accuracy of our numerical method . such a system is also used to probe the effect of charge on the equilibrium solute in addition , we consider the hydration of some single ions and compare our vism calculations with experiments . the second system consists of two methane - like particles that are treated as atoms . for this system , we test the effect of charging on the potential of mean force along the center - to - center distance between the two atoms . the third system consists of two initially hydrophobic plates , where the plate charge is a free parameter and gradually increased . we study the influence of charging on the hydrophobic interactions , the system balance between dry and wet equilibrium states , and the resulting hysteresis . in contrast to simple model systems , the complexity of such a protein in terms of size , geometry , and charge distribution , rigorously examines the applicability of our level - set vism on realistic biological systems . we focus on the dehydration for this particular system and discuss our results in light of md simulations . we review the vism functional , present the cfa of electrostatic free energy , and provide formulas of potentials of mean force . in section , we describe briefly our level - set numerical method . in section , we apply our level - set vism to various model systems and the protein bphc . we consider the solvation of molecules ( solutes ) in a solvent ( typically water ) . we divide geometrically the underlying system into three parts : the solute region m , the solvent region w , and the corresponding solute solvent interface , which is the dielectric boundary , cf . we assume that there are n atoms in the solute molecules that are located at x1 , ... , xn inside m and carry point charges q1 , ... , qn , respectively . the solute region , solvent region , and solute solvent interface are denoted by m , w , and , respectively . in vism , one minimizes the solvation free - energy functional , proposed in refs ( 1 ) and ( 2 ) , of all possible solute solvent interfaces :ii.1here , the term p vol(m ) , proportional to the volume of solute region m , is the energy of creating the solute cavity , with p being the pressure difference between the solvent liquid and solute vapor . this term can often be neglected for systems on a nanometer scale , since the pressure difference p under normal conditions is very small . the surface integral term is the surface energy , where is the surface tension . it is known for systems of nanometer scale that the surface tension is no longer a constant . , we apply the correction : where 0 is the constant macroscopic surface tension for a planar solvent liquid vapor interface , is the correction coefficient historically called the tolman length , and h is the mean curvature defined as the average of the two principal curvatures . we denote by ggeom[ ] the sum of the first two terms in eq ii.1 and call it the geometrical part of the free energy . for each i ( 1 i n ) , the volume integral in eq ii.1 over the solvent region w is the van der waals ( vdw ) type interaction energy between the solute atom at xi and the solvent molecules or ions at x that are coarse grained . the parameter w is the constant solvent density , and each ui is a pairwise interaction potential . as typically used in md simulations , we define ui to be the lennard - jones ( lj ) potentialii.2the parameters i of energy and i of length can vary with different solute atoms as in conventional force fields . we shall denote by gvdw[ ] the third term ( i.e. , the summation term ) in eq ii.1 and call it the van der waals ( vdw ) part of the free energy . the last term gelec[ ] in eq ii.1 is the electrostatic part of the solvation free energy . the electrostatic part of the solvation free energy gelec[ ] is defined by the born cycle as the difference of the energies of two states . a natural reference state is that our charged solute molecules are placed in a vacuum . in this case , the electric potential 1 in si units is given bywhere 0 is the vacuum permittivity and m is the relative permittivity of the molecule . the corresponding electric field e1 = e1(x ) and electric displacement field d1 = d1(x ) are given by e1(x ) = 1(x ) and d1(x ) = m0e1(x ) , respectively . the electrostatic energy in this state is given byii.3where the integral is taken over the entire space excluding small balls centered at singularities xi with cutoff radii . in the second state , solvent interface or dielectric boundary . the electric potential 2 = 2(x ) is the unique solution of the boundary - value problem of the poisson equationii.4ii.5where the relative permittivity or dielectric coefficient = (x ) is defined through the dielectric boundary byand w is the relative permittivity of the solvent , cf . since the dielectric boundary can be arbitrarily shaped , there is in general no analytical formula for the potential 2(x ) . the corresponding electric field e2 = e2(x ) and the electric displacement field d2 = d2(x ) are defined by e2 = 2 and d2 = 0e2 , respectively . the electrostatic energy of this state is given byii.6where again the integral is taken over the entire space excluding small balls centered at singularities xi with cutoff radii . note that the dependence of g2 on is through the dielectric coefficient = (x ) . now the electrostatic part of solvation free energy is gelec[ ] = g2[ ] g1 . invoking the coulomb - field approximation ( cfa ) d2 d1 ( cf . e.g. , ref ( 38 ) ) , we obtain from eqs ii.3 and ii.6 thathere , we assume that the respective cutoff small balls in the integrals in g1 and g2[ ] are the same . as a result , the cutoff radii do not appear in the expression of gelec[ ] . notice that for a single - atom , spherical solute , the solute region is the ball of some radius r > 0 centered at x1 . . then the boundary - value problem of eqs ii.4 and ii.5 has a unique solution : direct calculations verify that in this case d2(x ) = d1(x ) = qx/(4|x| ) for all x. hence , the cfa is exact in this case . in summary , our vism free - energy functional with the cfa of electrostatics is given byii.7where each interaction potential ui is given by the lj potential ii.2 . the solvation free - energy functional g[ ] defined on a solute solvent interface or dielectric boundary gives rise to an effective force acting on the boundary . we define this force to be g[ ] , the negative functional derivative of the free - energy functional g[ ] with respect to the location change of the boundary . it is only the normal component of this force that can affect the motion of such a boundary . we denote by n = n(x ) the unit normal vector at a point x on the boundary , pointing from the solute region m to the solvent region w , cf . we can obtain the normal component of this boundary force as a function defined on the boundary . this function isii.8where k = k(x ) is the gaussian curvature , defined as the product of the two principal curvaures , at a point x on . this force will be used as the normal velocity in our level - set numerical calculations . one is often interested in the effective interaction between solutes that stems from direct solute solute interactions and that is mediated by the solvent . the potential of mean force ( pmf ) is a general term for such effective interactions . it is usually defined with respect to a reaction coordinate as the difference between the free energy of solvated state at a given coordinate d and that at a fixed , reference coordinate dref . for our applications , we consider a solvation system in which solute atoms are divided into two groups : x1 , ... , xm and xm+1 , ... , xn . we chose the reaction coordinate d to be the distance between the geometrical centers ( i=1mxi)/m and ( i = m+1nxi)/(n m ) of the two groups of solute atoms . for instance , d is the center - to - center distance between the two atoms in a two - atom system , and it is the plate - to - plate separation distance for a system of two parallel plates . for a two - domain protein , this is the domain domain separation distance . the reference state is conveniently chosen with dref = ; i.e. , the atoms in the first group are at infinite separation from those in the second group . fix now a finite coordinate d. denote by d a corresponding vism optimal surface , i.e. , a stable equilibrium solute solvent interface minimizing locally the vism solvation free - energy functional . we define the ( total ) pmf as the sum of its separate contributionseach of them given byhere a quantity at is understand as the limit of that quantity at a coordinate d as d . the term gvdw(d ) is the sum of van der waals interaction energies between all the solute atoms . the above definition of gvdwpmf ( d ) and some simple calculations lead toii.9where ui , j is the lj interaction potential between xi and xj . the electrostatic part gelecpmf of the pmf is defined via the electrostatic free energies of the solvated states at d and . denote by g1(d ) the vacuum electrostatic energy as defined in eq ii.3 . since gelec[d ] = g2[d ] g1(d ) , we obtain by the above definition of gelecpmf(d ) that as d becomes large , the vism optimal solute solvent interface d becomes the union of two separate vism optimal solute solvent interfaces i and ii , both independent of d. they are obtained by minimizing the vism free - energy functional for the corresponding groups of fixed , solute atoms . if we denote by g[i ] and g[ii ] the corresponding minimum vism free energies for these individual groups of atoms , thensimilarly , each component of the vism free energy is the sum of that for the two groups of solute atoms , i.e. , g in the above equation can be replaced by ggeom , or gvdw , or gelec for small d , the solute solute vdw interaction , defined by the double summation term in eq ii.9 , can be very large and can therefore dominate over all other parts in the total pmf . in order to better understand the solvent influence in the pmf for small d , it is reasonable to look only at the pmf that excludes the solute solute vdw interaction . we remark that for a given reaction coordinate d there can be multiple stable equilibrium interfaces d that are local minimizers of the vism free - energy functional . different local minimizers d for the same coordinate d define multiple values g[d ] of vism local minimum free energies . therefore , the pmf can have multiple branches along the reaction coordinate d and hence can lead to hysteresis . our current level - set vism has not yet included fluctuations that in principle should allow an underlying system to get out of such a local minimizer . strictly speaking , therefore , our pmfs are different from those defined using a boltzmann average over all possible minimizers . rather , our pmfs reflect possible branches of the vism free energy along the reaction coordinate d. to numerically minimize the free - energy functional ii.7 , we begin with an initial surface that encloses all of the solute atoms located at x1 , ... , xn . the initial interface may have a very large value of the free energy . we then move the surface in the direction of steepest descent of the free energy by the level - set method until a steady state is reached . the starting point of the level - set method is the representation of a surface using the ( zero ) level set of a function = (x ) : = { x:(x ) = 0}. with this representation of the surface , the unit normal n = n(x ) , the mean curvature h = h(x ) , and the gaussian curvature k = k(x ) of a point x at the surface are then given by n = /|| , h = ( 1/2)n , and k = nadj()n , respectively , where is the 3 3 hessian matrix of the function whose entries are all the second order partial derivatives ij of the level - set function , and adj( ) is the adjoint matrix of the hessian . the motion of a moving surface = (t ) with t denoting the time is then tracked by the evolution of the level - set function = (x , t ) whose zero level - set is (t ) at each t. such evolution is determined by the level - set equationiii.1where vn = vn(x , t ) is the normal velocity of a point x on the surface at time t. to solve the level - set eq iii.1 numerically , one needs to extend the normal velocity vn to the entire computational box or a band surrounding the surface (t ) . to apply the level - set method to minimize our free - energy functional , we choose the normal velocity vn to move our surface in the steepest descent of the free energy this means that the normal velocity vn is the normal component of the effective dielectric boundary force , vn = fn , and is given by eq ii.8 . with such a choice of the normal velocity , our level - set method is in fact an optimization method of the steepest descent type . as the vism free - energy functional is quite nonconvex due to the capillary evaporation or dewetting energy barriers existing in an underlying molecular system , different initial surfaces can then lead to different local minimizers that are of practical interest . in order to capture multiple local minimizers the first one is a tight wrap : a surface that is close to the van der waals surface of the atoms . the second one is a loose wrap : a surface that loosely encloses all the solute atoms . we now briefly describe how we solve numerically the level - set eq iii.1 with our normal velocity vn = fn given in eq ii.8 . more details can be found in our previous publications . to discretize the spatial variable in the level - set eq iii.1 with the normal velocity fn given in ii.8 , we rewrite this equation aswith a = a(x ) ( dropping the time dependence in ) and b = b(x ) given bywe discretize a = a( ) by the central differencing with parameter correction . this means that we linearize a = a( ) at the that is computed in the previous time step and change the parameter so that the linearized part is elliptic ; that is , the time - dependent equation is parabolic . we discretize b|| using an upwinding scheme . in our implementation , we use a fifth - order weno ( weighted essential - no - oscillation ) scheme . we use the forward euler method to discretize the time derivative in the level - set eq iii.1:iii.2where (x ) and vn(k)(x ) are the approximations of vn(x , tk ) and (x , tk ) , respectively , at time tk = kt ( k = 1 , 2 , ... ) and t is the time step . we chooseiii.3so that the cfl condition is satisfied , where c = c( ) is the matrix defined by a( ) = 0c(): and input all the parameters p , 0 , , w , i , i , xi , and qi for all i = 1 , ... , discretize uniformly a computational box containing x1 , ... , xn with the grid size h a third or half of 1 . compute b(x ) at all the grid points x that are not any points x1 , ... , xn . generate an initial surface by defining a corresponding level - set function . choose a narrow band that is centered at the surface and that has a width of about 12 to 16 grid points . at each grid point in the band , compute the gradient , the hessian , and the curvatures h and k using central differencing schemes . we employ for efficiency the local level - set method developed in ref ( 47 ) in which the zero boundary condition is used for the level - set function at the boundary of the band . the extension of the normal velocity is necessary , since the lj potential changes rapidly near the surface , causing possibly numerical instabilities . in practice , we need only to extend the b part of the normal velocity . calculate t using iii.3 and update the level - set function using the euler scheme iii.2 . reinitialize the level - set function . to do so , we solve the equationwith the initial value = 0 at t = 0 to obtain a steady - state solution . here , 0 is the level - set function before reinitialization , and the time t is different from that in the original level - set equation . the quantity sign(0 ) is the sign of 0 and is approximated as 0/(0 + h ) with h being the spatial step size . check if a steady state is reached . if not , locate the interface by the level - set function obtained in the previous step , set k : = k + 1 , and go back to step 2 . we first consider a solute consisting of a single atom located at the origin carrying a point charge q > 0 . a solute solvent interface in this case is a sphere centered at the origin with a radius denoted r. for this system , we have an exact analytical formula of the free energy ii.7:this simple one - dimensional function can be minimized numerically with very high accuracy . we use the following parameters valid for methane - like particles in water at normal conditions : p = 0 , 0 = 0.175 kbt / with t = 300 k , = 1 , w = 0.0333 , = 1 = 0.3 kbt , = 1 = 3.5 , m = 1 , and w = 80 . we also solve the level - set eq ii.8 with the normal velocity fn given in eq ii.8 , starting from a large sphere centered at the origin . in table 1 , we display the results of our level - set vism calculations on a three - dimensional grid ( marked level - set ) and those of one - dimensional numerical optimization based on the above analytical formula g[r ] ( marked analytical ) that have basically the vanishing error . we compare the nonpolar and polar parts of the free energy ( in units kbt ) and the optimal ( equilibrium ) radius r ( in units ngstrom ) for different values of the point charge q ( in units e , the elementary charge ) . in fact , the relative error for both the minimum free energy and the optimal radius is within 0.5% . note that the total free energy for q = 1e is consistent with the typical magnitude of solvation free energies of monovalent ions , cf . , note also that the higher the charge , the smaller the resulting optimal radius r. this is because the dielectric medium ( multipolar water ) gains more and more electrostatic energy by penetrating deeper into the solute region . consequently , the hydrophobic ( geometrical ) part of the energy decreases because the radius r decreases . the vdw part of the free energy on the other hand increases significantly as the water pushes deep into the repulsive lj part . this clearly indicates that both the polar and nonpolar contributions are inherently coupled in the solvation free energy . the full 3d level - set minimization ( level - set ) is compared to the 1d numerical optimization of the free energy with the analytical formula g[r ] ( analytical ) . we now apply our vism one - particle framework to single ions k , na , cl , and f. for all these systems , we use the surface tension 0 = 0.175kbt / with the temperature t = 298 k , the water density w = 0.0333 , the dielectric coefficients m = 1 and w = 80 , and the tolman length = 1 . in our vism - cfa calculation of the hydration free energy of anions , cl and f , we use the dielectric boundary that is obtained by shrinking the solute solvent boundary by the water oh bond length of 1 to include the asymmetry effect of water as pointed out in dzubiella et al . in table 2 , we display our vism solvation free - energy values and the experimental free energies for the hydration of these ions . the second and third columns are the lj parameters , taken from horinek et al . we now consider a molecular system of two methane - like particles in water and treat these particles as atoms . we use the parameters p = 0 , 0 = 0.175 kbt / with t = 300 k , = 1 , w = 0.0333 , 1 = 2 = 0.3603 kbt , 1 = 2 = 3.4418 , m = 1 , and w = 80 . the point charges q1 and q2 are chosen to have the same magnitude , i.e. , |q1| = |q2| . we denote by d the center - to - center distance between the two atoms . to test the accuracy of the cfa , we compare the electrostatic solvation energy values computed by our cfa and that using the adaptive poisson boltzmann solver ( apbs ) . we examine a set of d values and use the same vdw surfaces for both of the cfa and apbs calculations . we also calculate the cfa values of electrostatic solvation energies using our optimal level - set vism surfaces . we see from the table that the cfa agrees with apbs well , particularly for large d where the vdw surface breaks into two disjoint parts and vdw and vism surfaces are very similar . for small d , we also notice that the energy values of cfa with vism optimal surfaces are about 10% different from those obtained using cfa with vdw surfaces . we now study the solvent - mediated pmf of the system with the center - to - center distance d between the two atoms as the reaction coordinate . for each of a few selected coordinates d and charge combinations q1 = q2 = 0.1e and q1 = q2 = 0.1 , 0.2 , and 0.5e , we relax an initial surface to a final , stable equilibrium solute solvent surface and calculate different parts of the minimum solvation free energy . in figure 2 , we plot the different components of the solvent mediated pmf . the full vdw component of the pmf , including the solute solute vdw interaction , is plotted in the inset of figure 2b . the total pmfs excluding and including the solute solute vdw interaction are plotted in the main frame and inset , respectively , of figure 2d . for comparison , the coulomb law of charge different contributions to the pmf of the two - atom system vs the center - to - center distance d for different values of charges . solute vdw interaction is excluded in the curves in the main frame but included in those in the inset . solute vdw interaction is excluded in the curves in the main frame but included in those in the inset . remarkably the nonpolar parts ( figure 2a and b ) display a desolvation barrier at d = 56 as observed in explicit water simulations . in our implicit - solvent model , it appears due to increasingly concave surface parts upon merging of the isolated solute surfaces . somehow , the magnitude of distortion of the overlapping first solvation shells is reflected in increasing concavity . the water - induced vdw interaction ( figure 2b ) displays a maximum at around d 5 because the dispersion interaction of the solutes with the water is minimal here . the electrostatic part of the pmf follows roughly the anticipated coulomb interaction as shown in figure 2c with corrections stemming from the ( image charge ) repulsion between the low - dielectric atomic cavities in the electric field . for small d 4 , the electrostatic interaction is strongly enhanced as the solvent dielectric screening is diminished for the overlapping cavities . charging influences the nonpolar ( geometric and vdw ) parts for d 7 where the solvation shells merge and are perturbed . at this fragile point , the system seems most sensitive to a change in electrostatics , even in an implicit - solvent model . as we see in figure 2d , charging thus mostly affects the total pmf below d 7 , where the interface starts overlapping . this is partially due to the subtle influence of electrostatics on surface geometry and not directly due to the electrostatic solute solute interaction . note that this phenomenon would not be describable by a simple fixed - surface ( e.g. , vdws , ses , or sas ) model , where the solvent surface is predefined . for small d 4 note again that the shown total pmf in the main frame is only the solvent - mediated part of the pmf , i.e. , it excludes the solute this interaction is included in the pmf plotted in the inset . here , we consider the strongly hydrophobic system of two parallel paraffin plates taken from the work of koishi et al . each plate consists of 6 6 fixed ch2 atoms and has a square length of about 3 nm . the two plates are placed at a center - to - center distance d. for these hydrophobic plates , capillary evaporation takes place at distances d 15 . in the following , we investigate how ( a ) the capillary evaporation , ( b ) the hydrophobic attraction , and ( c ) a possible hysteresis in the free energy are affected by charging up the plates . to this end , we assign central charges q1 and q2 to the first and second plates , respectively , with |q1| = |q2| . we study like - charged and oppositely charged plates by choosing the values of ( q1,q2 ) to be ( 0e,0e ) , ( + 0.1e,0.1e ) , ( + 0.1e,+0.1e ) , ( + 0.2e,0.2e ) , and ( + 0.2e,+0.2e ) . in figure 3 , we show 2d surface cuts through the 3d equilibrium surface for d = 10 and different charges . for q1 = q2 = 0e , charging the plates at d = 10 has different consequences on the final state depending on the sign of the charge . if the plates are charged oppositely , capillary evaporation is surpressed , and the final state is wet . this is because that the strong electrostatic potential between the plates drags the polar water into the void . if the plates are equally like - charged , then a stable capillary bubble remains with only a slightly tighter surface when compared to the case q1 this is because the oppositely directed electrostatic field cancels out in the void , and the water distribution is hardly affected . a few 3d snapshots of the vism surfaces are shown in figure 4 for d = 10 and different charges with loose initial configurations , where the mean curvature is color - coded . we observe clearly that charging displays tighter surfaces , diminished capillary evaporation ( totally surpressed for opposite charges ( q1,q2 ) = ( + 0.2e,0.2e ) ) , and more concave parts of the surface . these examples highlight the sensitive coupling between electrostatics and hydrophobicity in aqueous solvation , especially when the system is prone to capillary evaporation . the surpression of the latter between plates by introducing hydrophilic patches has been observed in recent md simulations . 2d cuts through the center of the 3d stable equilibrium solute solvent interfaces around the two plates at d = 10 with tight or loose initial surfaces for different charge combinations . solvent surfaces of the two - plate system obtained by the level - set vism calculations with loose initials at d = 10 . from left to right : atomic charges ( q1,q2 ) = ( 0e,0e ) , ( + 0.2e,+0.2e ) , and ( + 0.2e,0.2e ) . the color code represents the mean curvature being convex ( red ) , flat ( green ) , and concave ( blue ) . a more detailed , quantitative assessment is provided in figures 5 and 6 where we plot the different components of the full pmf ( including the solute solute vdw interaction ) with loose and tight initial surfaces , respectively . for the loose initials ( figure 5 ) , the geometric part displays a strong attraction below a critical distance dc at which capillary evaporation begins . the crossover distance decreases from dc 14 down to 9 for ( q1,q2 ) = ( + 0.2e,0.2e ) . note that the opposite charging has a much stronger effect than like - charging due to the electrostatic field distribution discussed above . solvent vdw part of the interaction is strongly affected by electrostatics due to the very different surface geometries induced by charging . both curves ggeompmf and gvdwpmf demonstrate the strong sensitivity of nonpolar hydration to local electrostatics when capillary evaporation occurs and very soft surfaces are present . the total pmf is not just the independent sum of nonpolar and electrostatic interactions . for the surfaces resulting from the tight initials , cf . figure 6 , the situation is a bit less sensitive to electrostatics , as the final surface is closer to the vdw surface for dc 6 . still , the nonpolar parts , in particular the vdw contributions , are affected by charging the plates as the surface geometry for distances d close to the desolvation barrier can be strongly perturbed by the electrostatics , see figures 3 and 4 . different components of the full pmf vs separation distance d between the two plates for different charge combinations ( q1,q2 ) ( see legend ) obtained by the level - set vism with loose initial surfaces . different components of the full pmf vs separation distance d between the two plates for different charge combinations ( q1,q2 ) ( see legend ) obtained by the level - set vism with tight initial surfaces . finally , figure 7 graphically displays the bimodal behavior and hysteresis by plotting the two different pmf branches stemming from the equilibria of wet and dry states . for the neutral plates ( cf . figure 7a ) , a strong hysteresis is present for 6 d 14 . adding charges influences the free - energy branches and hysteresis as shown in figure 7b and c. however , only in the case of oppositely charged plates are the changes significant , as a strong electrostatic field develops in between the hydrophobic plates . here , the water occupancy is most sensitive to local potentials . comparison of the two branches of pmf corresponding to the wet and dry states for the two plates carrying different charges ( q1,q2 ) ( see legend ) . our last example is biphenyl-2,3-diol-1,2-dioxygenase ( bphc ) , a key enzyme of biphenyl biodegradation pathway in pseudomonos sp . the functional unit of this protein is a homo - octamer , and each subunit consists of two domains . we set up a series of configurations where the two domains are increasingly separated from d = 0 to d = 20 apart , perpendicular to their interface . note that the zero domain separation corresponds to the native configuration in the crystal structure ( pdb code : 1dhy ) . to ensure the vism with cfa of electrostatics is suitable for this system , we compared electrostatic solvation energy computed by the cfa and that by apbs using the same hard sphere dielectric boundary . across the entire range of the system configurations ( i.e. , from 0 to 20 domain separations ) , md simulations reported in zhou et al . suggested that water density in the inter domain region is lower than in the bulk when two domains are within 6 . when electrostatic interactions between the water and protein are turned off , the strong dewetting transition occurs at as far as 9 domain separation . in comparison , we display in figure 8 six surfaces of our level - set vism at three different domain separations with and without atomic partial charges . at d = 8 , the level - set vism identifies the interdomain region as partially solvent excluded when atomic charges are included , and as completely solvent excluded without any atomic partial charges . the interface wraps around the protein more tightly with charges than that without them due to the attractive nature of the polar interactions between the solute and solvent . the dark blue regions on the surfaces correspond to the deep trenches created by a strong polar interaction . at d = 14 , the uncharged and charged bphc molecules pose topologically distinct solute solvent interfaces . with polar interactions , both domains are completely solvated . in contrast , the center of the domain interface still retains low water occupancy without electrostatic interactions . this is consistent with the results from atomistic simulations , where dewetting extends to a much greater region without polar interactions . compared with traditional surfaces , such as vdws , ses , or sas , the vism surfaces are topologically similar at small and large interdomain separations . however , a traditional surface would break into two independent surfaces at d > 4 regardless of charge distribution . here the stable equilibrium solute solvent interfaces of bphc at three different domain separations , obtained by the level - set vism with loose initial surfaces . the top row is with atomic partial charges , and the bottom row is without partial charges . figure 9 shows different parts of the pmf of bphc with respect to the separation of two domains , with and without partial charges , obtained by our level - set calculations using tight and loose initial surfaces . we observe bimodal hydration in this system as well . with polar interactions , the ( de)wetting process happens at smaller domain separations . this is illustrated by the general shifting of charged curves in a and b by 2 toward smaller separation . in addition , the bimodal hydration is damped by partial charges since they assist the nucleation process in the interdomain region . the sharp transitions seen in a and b for tight initials indicate a strong dewetting occurring around 68 for uncharged case , capturing the hydrophobic collapse that has been also reported in md simulations . it is noticeable that at both small and large separations the loose and tight initial conditions yield slightly different energetics . this is because the small differences remain in the final optimized surfaces when drastically different initial surfaces are used for complex systems . the optimized surface is only one of multiple local minimizers of the vism free - energy functional . the coexistence of such multiple local minima represents the fluctuating nature of the solvent solute interface . different parts of the pmf of bphc with respect to the domain separations , with or without partial charges , and with loose and tight initial surfaces . in this work , we introduce the coulomb - field approximation ( cfa ) of electrostatic free energy into the variational implicit - solvent model ( vism ) and implement the level - set numerical method for relaxing the resulting vism free - energy functional . we apply our theory and method to molecular systems of various complexities , including the protein bphc . these applications demonstrate that our level - set vism with the cfa is an efficient approach to qualitatively capturing the stable equilibrium solute solvent interfaces with the correct corresponding free energies . we have found from our extensive numerical data and detailed analysis using potentials of mean force ( pmf ) the following:(a)the coupling of the geometrical , van der waals , and electrostatic contributions in the vism is essential in qualitatively estimating solvation free energy , and capturing multiple equilibrium states of wet and dry . but they are hard to describe using implicit - solvent models of fixed - surface type.(b)charges impact strongly on the hydration / dehydration process from a simple molecular dimer to a complex protein . the detail of local electrostatic field is critical for wet and dry transitions , as shown in the example of two paraffin plates . in the more complex bphc case , polar interactions enhance wetting , which dampens the strong hydrophobic collapse propensity posed by hydrophobic residues at the center of the domain interface . while these have been observed and explained in molecular dynamics ( md ) simulations , our theory and method seem to be the first with an implicit solvent to capture such properties.(c)compared with md simulations , our level - set vism with the cfa is computationally much more efficient , and also qualitatively , often even quantitatively , accurate . it is therefore promising to apply our theory and method to study efficiently the hydration of large and realistic systems . the coupling of the geometrical , van der waals , and electrostatic contributions in the vism is essential in qualitatively estimating solvation free energy , and capturing multiple equilibrium states of wet and dry . but they are hard to describe using implicit - solvent models of fixed - surface type . charges impact strongly on the hydration / dehydration process from a simple molecular dimer to a complex protein . the detail of local electrostatic field is critical for wet and dry transitions , as shown in the example of two paraffin plates . in the more complex bphc case , polar interactions enhance wetting , which dampens the strong hydrophobic collapse propensity posed by hydrophobic residues at the center of the domain interface . while these have been observed and explained in molecular dynamics ( md ) simulations , our theory and method seem to be the first with an implicit solvent to capture such properties . compared with md simulations , our level - set vism with the cfa is computationally much more efficient , and also qualitatively , often even quantitatively , accurate . it is therefore promising to apply our theory and method to study efficiently the hydration of large and realistic systems . we notice that the cfa is known to be an efficient but not necessarily accurate approximation of the electrostatic free energy . we emphasize that the purpose of our work is mainly to couple the cfa with our level - set vism to demonstrate that the coupling of electrostatics and nonpolar contributions in vism can capture qualitatively some important features , such as the capillary evaporation in hydrophobic confinement , that are otherwise hard to capture by other fixed - surface type implicit - solvent models . more accurate continuum descriptions of electrostatics , such as the yukawa - field approximation and the poisson boltzmann theory , need to and can be included in our level - set vism . finally , we remark that finding an energy - minimizing solute solvent interface using our level - set vism approach is usually computationally more expensive and less efficient than generating a van der waals surface , solvent - excluded surface , or solvent - accessible surface . to completely relax a system with our method , it can take minutes to hours depending very much on the choice of initial surface and the numerical resolution . we are currently working on speeding up our calculations so that our approach can be combined with md simulations . we note , however , that the application of our theory and methods is not limited to the calculation of electrostatics for md simulations . as we have shown in our previous and current work , we can efficiently capture various kinds of equilibrium conformations of biomolecules and estimate the solvation free energies . we are also developing related theory and methods to include solvent fluctuations for the real dynamics of biomolecular solvation .

lichen planus ( lp ) is a chronic dermatologic disease that is relatively common among our population . it is most common among middle - aged adults with women predominance ( 3:2 ) ratio over men . this article focuses on the prevalence of oral , skin , and oral and skin lesions of lp . a study was conducted in the department of oral medicine and radiodiagnosis , pushpagiri college of dental sciences , tiruvalla , kerala . this is an ongoing study , and results of 2 years are being reported ( june 2008 to june 2010 ) . a total of 18,306 subjects who visited the department of oral medicine and radiology for diagnosis of various oral complaints over a period of 2 years were interviewed and clinically examined for oral , skin , and oral and skin lesions of lp . lp was diagnosed on the basis of clinical presentation , and confirmation was done through histopathological analysis of mucosal and skin biopsy done for all patients suspected of having lp . a pilot study of 100 randomly selected individuals was carried out to determine the feasibility of the study and the amount of time required for examination of each subject . data obtained were recorded in a proforma which was later transferred for statistical analysis done using spss ( statistical package for social sciences ) software version 14 . patients in the age group ( 1070 years ) were included in the study.both men and women participated in this study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study.lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study . lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . patients in the age group ( 1070 years ) were included in the study.both men and women participated in this study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study.lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study . lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . patients in the age group ( 1070 years ) were included in the study.both men and women participated in this study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study.lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . lesions mimicking oral lp , e.g. , lichenoid reaction secondary to dental amalgam restorations were excluded from the study . lesions mimicking oral and skin lp but not confirmed as oral and skin lp through biopsy were also excluded from the study . out of 18,306 patients screened 8,040 ( 43.9% ) were males and 10,266 ( 56% ) females , with ages ranged between 10 and 70 years . from 18,306 patients screened , lp prevalence was ( 0.64% ) with oral lichen planus ( olp ) prevalence was ( 0.4% ) , skin lichen planus ( slp ) was ( 0.06% ) , and olp and slp was ( 0.13% ) [ figure 1 ] . lesions mimicking oral and skin lp but not confirmed as lp through biopsy were 0.1% . prevalence of oral , skin , oral and skin lichen planus in the study population among the three categories of lp ( olp , slp , and olp and slp ) , there was a strong female predominance ( p=0.000036 ) in olp [ table 1 ] . no significant differences between the genders were seen in slp , and olp and slp categories of lp . age wise distribution of lp ( oral , skin , and oral and skin lp ) is presented in table 2 . among the age groups , highest peak of prevalence of oral , skin , and oral and skin lp was observed in 2140 and 4160 year age groups ; 12 years was the lowest age of a patient with lp ( oral lp ) and 65 years was the highest in our study . intraorally , lp was seen predominantly affecting the buccal mucosa 45% , followed by gingiva 25% , tongue 15% , labial mucosa 10% , and soft palate 5% [ figure 2 ] . extraorally , wrist and forearm were affected most often 45% , followed by knees and thighs 35% , scalp 10% , and nails 10% [ figure 3 ] . male : female ratio of patients with oral , skin , and oral and skin lps age wise distribution of lichen planus oral , skin , and oral and skin lp intraoral site of involvement of lichen planus extraoral site of involvement of lichen planus lichen planus derives its name lichen as it looked like lichens growing on the rock and planus is for flat.[24 ] lp may involve various mucosal surfaces either independently or concurrently ( oral , skin , and oral and skin lesions ) . oral form may precede or accompany the skin lesions or it may be the only manifestation of the disease . prevalence of skin lp in general population is 0.91.2% and prevalence of oral lp is reported between 0.1% and 2.2% . in our present study , shklar and mc carthy have reported the following distribution of oral lesions : buccal mucosa 80% , tongue 65% , lips 20% , gingiva , floor of mouth , and palate less than 10% . any site in the oral cavity may be involved , but the buccal mucosa and gingiva are the most commonest sites . in our present study , lesions of oral lp were seen mostly on buccal mucosa 45% followed by gingiva 25% , tongue 15% , labial mucosa 10% , and soft palate 5% figure 2 . clinically , oral lp appear in two major forms erosive oral lp and nonerosive oral lp . erosive form appears as a central area of erythema with peripheral radiating white striae[13 ] [ figure 4 ] and nonerosive form or the reticular variety is characterized by the presence of wickham striae [ figure 5 ] . erosive variety is of great concern to clinician as well as to the patient as patient often experiences burning sensation in the involved areas ; moreover , erosive form has an higher potential for malignant transformation ( 0.3%3% ) . in our present study , patients who reported with a chief complaint of burning sensation , majority of them had erosive lp and malignant transformation rate was 1% . intraoral photograph of a patient showing erosive lichen planus in the buccal mucosa intraoral photograph of a patient showing reticular variety of lichen planus in the buccal mucosa this study shows an increased prevalence of oral lp in ( 40 - 60 year ) age group with a female predominance . lesions of oral lp especially the erosive variety need to be monitored carefully as it has a higher risk for transformation into squamous cell carcinoma .

diabetes is one of the most common disorders worldwide , and its prevalence is estimated to increase by 122% from 1995 to 2025 ( 1 ) . due to the increasing prevalence of diabetes , factors related to diabetes and its complications have become increasingly important ( 2 , 3 ) . appropriate care , including controlling blood sugar , physical activity , and a suitable diet , can prevent some of the complications typically experienced by diabetic patients ( 1 ) . people with diabetes are at a higher risk of developing urinary tract infections ( uti ) ( 4 - 6 ) , especially recurrences of uti , compared to people without diabetes ( 5 ) . there are several theories concerning this effect : 1 ) diabetes increases the risk of infections , of which uti are one of the most prevalent varieties ( 7 ) ; 2 ) diabetes causes abnormalities in the host s immune system , which leads to the patients being prone to uti ( 8) ; and 3 ) diabetic neuropathy causes a defect in urine discharge that results in uti ( 9 ) . some studies have shown that patients who consume oral blood glucose - lowering medications or insulin , people who have had diabetes for more than five years , and diabetic patients with retinopathy are particularly at risk of recurrent uti ( 5 , 7 ) . the risk factors for uti in diabetic patients are not well defined , although several risk factors have been identified : age , metabolic control , duration of morbidity , diabetic cystopathy , sexual activity , macroalbuminuria , presence of diabetic complications , and insulin therapy ( 7 , 10 ) . showed that the association between lower urinary tract symptoms ( luts ) and diabetes varies by gender , which indicates different mechanisms of association in men and women ( 11 ) . some studies have revealed that sexual intercourse is the most significant risk factor for symptomatic uti in type 1 diabetic patients , whereas asymptomatic bacteremia ( asb ) is the most significant risk factor in type 2 diabetic patients ( 10 ) . in addition , renko et al . showed that diabetic subjects who suffer from asb are at increased risk of albuminuria and uti ( 12 ) . although asb is associated with an increased risk of hospitalization in diabetic subjects , a study has suggested that its treatment does not reduce the risk of symptomatic uti ( 13 ) . some studies have revealed that blood glucose control is associated with acute uti ( 14 ) , while other studies failed to find a relationship between blood glucose control and acute uti ( 7 , 8 , 10 ) . however , stapleton showed that blood glucose control can affect diabetes complications and , therefore , it can indirectly control uti via its impact on bladder dysfunction ( 8) . due to the significant impact that uti have on diabetic patients quality of life , we decided to evaluate the effect of demographic factors and diabetes control on the incidence of uti in order to help decrease the prevalence of this problem . we conducted an inferential cross - sectional descriptive study at a diabetes center in khorramabad , lorestan province , iran , from october 2013 to january 2014 . the center used in this study was the largest diabetes center in the city , as well as the only governmental and referral diabetes center in the area . data concerning 233 patients with type ii diabetes who had been actively referred to the center were recorded in this study . some 55 patients were excluded because their laboratory data were not completely recorded in their charts . the diagnosis of uti was based on both the laboratory records in their charts and the patients symptoms such as dysuria and urgent or frequent urination . this study complies with the ethical standards of the helsinki declaration as revised in 2008 . the data collection sheet did not list the names of the participants and , therefore , their privacy was protected . the sample size was calculated as 233 subjects considering = 5% , a statistical power of 90% ( moderate effect size ) , d = 0.23 , and the following equation : considering the 18% drop up sample , we added 35 samples to the optimal sample size and so , in total , we considered 233 samples . the relationships between the demographic factors and the incidence of uti in diabetes patients were calculated using chi - square . we also calculated the relationships between different oral medications , as well as various laboratory factors , and the incidence of uti using chi - square and logistic regression . a p < 0.05 was considered to be statistically significant . the odds ratio and confidence interval were recorded . the clinical characteristics of the study population were calculated as either means sd or no . the sample size was calculated as 233 subjects considering = 5% , a statistical power of 90% ( moderate effect size ) , d = 0.23 , and the following equation : considering the 18% drop up sample , we added 35 samples to the optimal sample size and so , in total , we considered 233 samples . the relationships between the demographic factors and the incidence of uti in diabetes patients were calculated using chi - square . we also calculated the relationships between different oral medications , as well as various laboratory factors , and the incidence of uti using chi - square and logistic regression . a p < 0.05 was considered to be statistically significant . the odds ratio and confidence interval were recorded . the clinical characteristics of the study population were calculated as either means sd or no . the patients characteristics were collected based on variables that might be associated with diabetes and uti , including gender , literacy , marital status , occupation , diabetes duration , age , bmi , history of addiction , hospitalization history , diabetes medication , and the most recent measurement of hba1c ( table 1 ) . we began this study with 233 patients , of whom 55 patients were excluded . of the 178 diabetic patients ultimately included in our study , 120 patients had uti symptoms , while 58 patients did not show any uti symptoms . the mean sd age was 56.09 12.39 years and the mean sd duration of diabetes was 80.96 63.27 months . we calculated the relationship between different demographic factors and the incidence of uti in diabetic patients . table 1 shows that the incidence of uti increased significantly with increasing age ( p = 0.009 ) , with the highest prevalence being found among subjects older than 70-years - old . the chi - square test also revealed a strong relationship between gender , occupation , and type of diabetes medication and the incidence of uti . the prevalence of uti among females was higher than that among males , and the prevalence was higher among unemployed individuals than it was among those in other occupations . the prevalence of uti among patients who control their diabetes through diet and exercise was about 6.2% , while in patients who are prescribed oral medication it was about 32.2% and in patients who use insulin it was 46.7% . the prevalence of uti among patients who take both insulin and oral medication was reported to be 50% ; however , no significant relationship was found between the different types of oral medication and the prevalence of uti ( table 2 ) . we did not find any relationship between the incidence of uti in diabetic patients and other factors such as literacy , marital status , bmi , addiction , history of hospitalization , and level of hba1c ( table 1 ) . the serologic factors that are presented as mean sd in table 3 did not have a significant relationship with uti in diabetic patients . the patients characteristics were collected based on variables that might be associated with diabetes and uti , including gender , literacy , marital status , occupation , diabetes duration , age , bmi , history of addiction , hospitalization history , diabetes medication , and the most recent measurement of hba1c ( table 1 ) . we began this study with 233 patients , of whom 55 patients were excluded . of the 178 diabetic patients ultimately included in our study , 120 patients had uti symptoms , while 58 patients did not show any uti symptoms . the mean sd age was 56.09 12.39 years and the mean sd duration of diabetes was 80.96 63.27 months . we calculated the relationship between different demographic factors and the incidence of uti in diabetic patients . table 1 shows that the incidence of uti increased significantly with increasing age ( p = 0.009 ) , with the highest prevalence being found among subjects older than 70-years - old . the chi - square test also revealed a strong relationship between gender , occupation , and type of diabetes medication and the incidence of uti . the prevalence of uti among females was higher than that among males , and the prevalence was higher among unemployed individuals than it was among those in other occupations . the prevalence of uti among patients who control their diabetes through diet and exercise was about 6.2% , while in patients who are prescribed oral medication it was about 32.2% and in patients who use insulin it was 46.7% . the prevalence of uti among patients who take both insulin and oral medication was reported to be 50% ; however , no significant relationship was found between the different types of oral medication and the prevalence of uti ( table 2 ) . we did not find any relationship between the incidence of uti in diabetic patients and other factors such as literacy , marital status , bmi , addiction , history of hospitalization , and level of hba1c ( table 1 ) . the serologic factors that are presented as mean sd in table 3 did not have a significant relationship with uti in diabetic patients . people who have diabetes are several times more likely to suffer uti than people who do not have this problem ( 16 ) . the middle east is estimated to witness the largest increase in the prevalence of diabetes by 2030 . more than 1% of the iranian urban population over 20-years - old develops type 2 diabetes each year ( 17 ) . the overall findings of this study indicate that the age of diabetic patients has a significant relationship with their likelihood of suffering from uti . in other studies , such as those by kojaiibidgoli et al . and geerlings et al . age has been introduced as a risk factor for uti in diabetic patients ( 16 , 18 ) . however , other studies , including those by boroumand et al . and ishay et al . did not find a significant relationship between age and uti in diabetic patients ( 19 , 20 ) . furthermore , gaymans et al . revealed that age can be a risk factor for asb , although it was not correlated with the development of uti during follow - up ( 21 ) . the fact that a longer duration of diabetes and changes in the host s defense mechanisms were significant predictors of uti in diabetes patients could explain why a higher age is associated a higher incidence of uti in this study ( 22 , 23 ) . furthermore , we found that medical therapy for diabetics also has a significant relationship with uti ; however , there was no association between different types of oral medication and the incidence of uti . in a study by gorter et al . it was found that diabetic patients who take medications ( particularly insulin ) to reduce their blood glucose are at risk of recurrent uti ( 5 ) . a study by boyko et al . revealed that a significantly higher risk of asymptomatic uti is related to the type of medication , namely insulin treatment in all multivariable models and oral medication treatment in one model ( 7 ) . nevertheless , in a study by kojaiibidgoli et al . there was no significant relationship found between the type of diabetes treatment and uti ( 16 ) . similar to our study , some previous studies have shown that the duration of diabetes has no effect on the risk of acute uti ( 4 , 16 ) . in this regard , boyko et al . concluded that the risk of uti is similar in postmenopausal women with a history of diabetes spanning more than ten years and those who have had diabetes for less than ten years ( 24 ) . in contrast , in a randomized study boyko et al . showed that the risk of uti increases with an increasing duration of diabetes ( 7 ) . moreover , gorter et al . showed that women who suffer from diabetes for more than five years are at a high risk of recurrent uti ( 5 ) . in our study , we did not find any relationship between glycemic control and uti , which is similar to the findings of other studies ( 4 , 5 , 7 , 25 ) . no relationship was found between age and hba1c in diabetic patients with uti ( 10 ) . the results of boroumand et al s study on iranian women with type ii diabetes are consistent with the results of our study ( 20 ) . in a study by yismaw et al . showed that the patterns of uti in males and females are different , which may indicate different mechanisms as well as anatomical differences between the urinary tracts of the two genders ( 11 ) . a study by muller et al . revealed that the risk of recurrent uti increased in women with type i diabetes ( 14 ) . we did not find any relationship between bmi and the incidence of uti ; however , in a survey by geerlings et al . a lower bmi was found to be a risk factor for asb in those with type ii diabetes ( 25 ) . it is noteworthy that this relationship might be accidental , since the or was near to 1 ( or = 0.96 ) and , besides , in the multivariate analysis bmi was not found to be a risk factor ( 25 ) . we also found a significant relationship between the patients occupation and the incidence of uti . although we could not identify similar findings in the literature , this may be due to the fact that the association between the incidence of uti in diabetic patients and the patients occupations has not been previously investigated . moreover , we have evaluated the association between some other factors , including literacy , marital status , addiction , and history of hospitalization , with the incidence of uti in diabetic patients , which is another area that has been overlooked in previous studies . the results of our study did not indicate any significant association between these factors and uti . hence , in order to verify these results , it is recommended that researchers should also consider these factors in their future studies . there are various limitations to this study in terms of what it covers and what it leaves out . the lack of data in some patients records ( i.e. , occupation , literacy , etc . ) should also be mentioned as a weak point of our study . yet , there are some strengths of our study that deserve to be mentioned . in particular , to the best of our knowledge , this is the first study to examine the management of diabetes and uti prevalence that has been conducted in iran .

androgenic alopecia ( aga ) is the most common hair loss condition in men and women with significant negative impact on their social and psychological wellbeing . aga , a hereditary androgen - dependent disorder , is characterized by a progressive process that causes a gradual conversion of terminal hair into miniaturized hair defined by various patterns . aga developing before 36 years of age and reaching at least stage 3 of hamilton - norwood classification is termed as early onset aga . in many studies , aga has been shown to be associated with several diseases , such as coronary heart disease , insulin resistance ( ir ) , hypertension , abnormal serum lipid profiles , obesity , prostate cancer , benign prostatic hyperplasia , and some environmental factors , such as smoking . however , controversies also exist regarding the association of those diseases and environmental factors with aga . recently , the term metabolic syndrome ( mets ) , came into common usage , and its importance regarding increased risk of developing cardiovascular disease ( cvd ) and diabetes is being emphasized more and more . coronary artery disease ( cad ) is a major cause of death and disability worldwide . advanced obstructive cad can exist in patients with minimal or no symptoms and can progress rapidly , so early detection is extremely important . until now , few studies have reported on the association between aga , mets , and cad . the aim of this study was to analyze the association of the presence of mets and carotid artery atherosclerosis with early onset aga ( age from 20 to 35 years ) . this age group was selected because mets and coronary disease often does not occur in this age group and also , a risk factor in these persons may be present but are in apparent . so , early detection of the risk factor is useful for early intervention to decrease the incidence of mets and cad . patients were selected from dermatology department , government medical college , district amritsar , india from january 2013 to june 2014 . the study was carried out on 100 male patients presenting with aga between the age of 18 and 35 years and on 100 age - and - gender matched controls ( for other minor skin problems ) after obtaining informed witnessed consent . this study was assessed and approved by the ethics committee and review board of the institution . patients suffering from another type of alopecia and alopecia associated with other dermatological conditions were excluded . the inclusion and exclusion criteria for the controls were the same as for the cases except that the control patients were not suffering from aga . the degree of hair loss was assessed and classified according to the hamilton and norwood classification . after obtaining written consent , a detailed history regarding age of onset , history of smoking , cvd and consumption of drugs ( antidiabetic agents , lipid - lowering , and antihypertensive agents ) , family history of aga and cvd , hypertension , diabetes mellitus was obtained and every patient was subjected to thorough general physical , systemic , and dermatological examination . the severity of alopecia was assessed , and indices of obesity such as weight and height were recorded to calculate the body mass index ( bmi ) . in addition , we recorded waist circumference ( wc ) , waist to hip ratio ( whr ) , and blood pressure . we determined serum levels of glucose , triglycerides ( tgs ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , total cholesterol , c - reactive protein ( crp ) , and erythrocyte sedimentation rate ( esr ) . carotid ultrasound scans were performed to study the presence of atheromatous plaques of the common carotid arteries , carotid bulb , and internal carotid arteries . the diagnosis mets was based on criteria defined by the national cholesterol education program adult treatment panel iii ( ncep atp iii , 2001 ) , which were modified to include the world health organization 's proposed wc cut - off points for asians . subjects were required to have three or more of the following : ( i ) wc > 90 cm in men and > 80 cm in women , ( ii ) serum tg 150 mg / dl ( 1.70 mmol / l ) , ( iii ) hdl - c levels < 40 mg dl ( < 1.04 mmol / l ) in men and < 50 mg / dl ) ( < 1.70 mmol / l ) in women , ( iv ) impaired fasting glucose of 110 mgdl ( 6.11 mmol / l ) or use of medication for hyperglycaemia , and ( v ) blood pressure 13085 mmhg or use of medication for hypertension . anova test applied for determining the significance of the difference between the cases and the controls for a different variable . p < 0.05 were considered significant and p < 0.01 were taken as highly significant . we studied 100 male patients with early onset aga , 12% with type i , 25% with type ii , 29% with type iii , and 12% with type iv alopecia . type v was present in 7% patients , type vi in 9% , whereas only 6% patients had most severe type vii alopecia according to the hamilton and norwood classification . the mean ( standard deviation ) age for both groups was similar , namely , 27.03 5.36 years in the cases and 26.22 5.10 years in the control ( p = 0.478 ) . a positive family history of aga , hypertension , and dm was found to be significantly more frequent in study cases than in the controls ( p < 0.05 ) [ figure 1 ] . family history in study group and control group in our study , we found statistically highly significant difference in bmi ( 25.03 4.35 kg / m vs. 22.34 3.41 kg / m , p < 0.05 ) and statistically significant difference in wc ( 87.8 11.28 cm , p < 0.05 ) in study group patients as compared to patients in control group ; however , the difference in whr was statistically not significant ( 0.93 0.06 vs. 0.90 0.06 , p > 0.05 ) [ table 1 ] . anthropometric and specific biochemical profile of study group and control group there were statistically significant differences in mean values of serum tgs ( p < 0.05 ) , serum cholesterol ( p < 0.001 ) , systolic blood pressure ( p < 0.05 ) , diastolic blood pressure ( p < 0.05 ) , fasting glucose concentration ( p < 0.05 ) , and very - ldl ( p < 0.05 ) . however , no significant differences were observed in the mean values of hdl - c ( p > 0.05 ) and ldl - c ( p > 0.05 ) [ table 1 ] . of 100 aga patients , 22 ( 22% ) patients fulfilled 3 or more of the criteria for mets compared with 8 ( 8% ) of the controls , which was statistically significant ( p < 0.05 ) [ table 2 ] . however , there was no relationship between severity of aga and mets [ table 3 ] . the differences between the cases and controls for all the parameters that make up the mets are shown in [ table 4 ] . comparative proportions of mets and caa plaque in aga and control groups comparative proportions of mets in various grades of aga abnormal components of mets in study and control group the atheromatous plaque was seen in 2% of cases however no atheromatous plaque was found in controls patients ( p = 0.155 ) [ table 2 ] . there were no significant differences in mean values of esr ( 9.94 6.66 vs. 8.55 1.91 , > 0.05 ) and crp ( 14.08 18.70 vs. 11.29 11.54 , p > 0.05 ) among study and control group . however crp levels were increased in 48 ( 48% ) patients in study group while in control group , levels were increased in 52 ( 52% ) patients ( p = 0.207 ) [ table 1 ] . there was statistically highly significant difference in the mean value for serum testosterone ( 21.98 23.84 vs. 5.41 2.52 , p < 0.001 ) among study and control group [ table 1 ] . the results of this study confirm that male aga is associated with a greater prevalence of cardiovascular risk factors included in the mets and with an increase in risk of carotid atherosclerosis . most of the studies have focused on the analysis of coronary disease ( acute myocardial infarction or cardiac death ) but only few authors have analyzed the association with cardiovascular risk factors , such as criteria for mets or the presence of carotid atheroma . we found a very significant association between the presence of aga and family history of this condition . in current study and as reported in similar studies , family history of aga and hypertension and diabetes mellitus were significantly high in study patients than controls . androgen receptor gene which is x - linked recessive has been mentioned to be the cardinal prerequisite for balding , but other genes are also involved . there was a highly significant difference in bmi values of aga patients and the controls in the present study [ table 1 ] . . found out statistically significant difference while arias - santiago et al . and ceydal et al . could not find statistically significant difference in bmi values of aga cases and controls . in the present study , on comparing two groups , wc measurements of the aga group were found to be higher than those of the control group ( 82.23 9.89 cm ) . however , the difference between whr values of the two groups was not found to be significant ( p > 0.05 ) [ table 1 ] . . found statistically significant difference in wc of cases and controls ( p = 0.032 ) . in contrast , ceydal . could not find statistically significant difference between wcs of aga patients and controls . the abdominal fat tissue is associated with serious metabolic disorders such as hypertension , hyperinsulinemia , increased tg , glucose intolerance , and diabetes mellitus . some studies have quoted abdominal fat tissue as an independent risk factor for cad calculated by measuring the waist - hip proportion . the international diabetes federation has emphasized the strong correlation between ir and abdominal obesity and has suggested that this criterion be made compulsory for mets diagnosis . there was significant difference in levels of total cholesterol and tgs among cases and controls in the present study but statistically no significant difference in levels of hdl - cholesterol , ldl - c was observed [ tables 1 and 4 ] in 1997 , sasmaz et al . also found meaningfully higher levels of serum tg and lipoprotein a in the aga group . found higher ldl - c and tgs in men with aga in patients who had undergone coronary artery bypass graft or percutaneous transluminal coronary angioplasty . . found out statistically significant higher tg and total cholesterol / hdl - c ratio , whereas hdl - cholesterol was lower in the study group ( p < 0.01 ) . in a study by arias - santiago et al . men with aga showed significant higher tgs , total cholesterol values , and ldl - c values . in contrast , guzzo et al . randomly compared lipid profile , but found no statistically meaningful differences in lipid indices between two groups . the elevated lipid values in aga patients may contribute , alongside other mechanisms , to the development of cvd . a meta - analysis by shepherd et al . in 2003 stated that a 1 mmol / l increase in the tg value increases the possibility of cad by 30% in men and by 69% in women . studied that both high tg values and low hdl - c values were associated with the transition from atheroma to atherothrombosis , and therefore , control of both these cardiovascular risk factors is essential in patients with subclinical disease . blood sugar values were significantly higher in the study cases ( 96.21 30.31 mg / dl vs. 91.860 19.12 mg / dl ; p < 0.05 ) [ tables 1 , 4 , and figure 2 ] . abnormal fasting glucose levels more than 100 mg / dl were present in 23% of the patients ; however , in the control group , it was present in 12% patients only [ table 4 ] . one of the most notable observations in the study by hirsso et al . was that 21% of their patients with aga and 12% of the control group had diabetes . abnormal biomedical parameters in study and control group mean systolic and diastolic blood pressure values were significantly higher in our cases . hypertension was present in 34 ( 34% ) patients in the study group as compared to 15 ( 15% ) patients in the control group , and this result was statistically significant ( p < 0.05 ) [ tables 1 and 4 ] hirsso et al . also found higher blood pressure values in patients with aga than in a control group ( 65% vs. 45% ) , as well as a greater frequency of diabetes and hyperinsulinemia . however , a study published in 2007 by hirsso et al . did not find statistically significant differences for systolic or diastolic blood pressure levels in patients aged < 35 years . the explanations offered for this association was that the androgens involved in the pathogenesis of aga bind to vascular receptors and also cause hyperaldosteronism both which in turn cause hypertension . the prevalence rate of mets as compared by ncep atp iii criteria in the study group was 22% and in the control group was 8% . mets was significantly associated with aga ( p < 0.05 ) in this study [ table 2 ] but there was no specific trend found in the prevalence rate of mets among the different degrees of the aga patient group [ table 3 ] . the association between cvd and mets is well documented . recent studies show that people who meet atp iii criteria are 2.59 - 3.5 times more likely to have a cardiovascular event during the next 10 years . the prevalence of mets reported for the general population ranges from 11.7% to 30% in the population of brazil , with a mean value of 20% in some studies published in spain . arias - santiago et al . who found statistically significant ( p < 0.05 ) . mets in 25% patients in the aga group and 10.4% in the control group according to the ncep atp iii 2001 diagnostic criteria . su and chen reported a significant association between aga and mets in 670 taiwanese men in a population - based cross - sectional survey with adjustment for age , family history , and smoking . however , hirsso et al . reported high - sensitivity crp values in patients aged < 35 years who had moderate or severe alopecia . chronic inflammation has been shown to play a key role in the presence of ir , endothelial dysfunction , and cvd . the hormone study performed in our patients revealed statistically significant higher levels of serum testosterone in the study group than in the control group ( 21.98 23.84 ng / dl vs. 5.41 2.52 ng / dl , p < 0.001 ) [ table 1 ] . in 1972 , it was first suggested that aga may be a risk factor for cvd when cotton et al . the second key area analyzed , in our study was the presence of carotid atheromatous plaques , as studied by doppler ultrasound . the association between aga and the presence of atheromatous plaque could be explained by a greater sensitivity of androgens in the scalp and in vascular smooth muscle , thus favoring miniaturization of the hair follicles in one case and the presence of atheromatous plaque in the other . many studies assume that pathogenesis was similar for coronary atherosclerosis and carotid atherosclerosis and , therefore , that the presence of carotid atheroma also predicts coronary disease . in our study , an atheromatous plaque was present in 2% of cases in contrast , no plaque was found in patients of control group , however , this result was not statistically significant [ table 2 ] . most studies on this topic do not analyze the presence of carotid atherosclerosis as a predisposing factor for cvd . instead , they have directly analyzed cardiovascular events , generally myocardial infarction , although they restrict their analysis to patients who have survived coronary disease ; therefore , they missed data on patients who die and did not take into account asymptomatic individuals with coronary disease . a study by shahar et al . concluded that male pattern baldness is not an important risk factor for myocardial infarction or asymptomatic atherosclerosis . they also did not find statistically significant differences in carotid intima - media thickness according to the degree of alopecia , despite studying a large number of patients . on the other hand , found an association between severe androgenetic alopecia and a higher carotid intima - media thickness , although they did not analyze the prevalence of atheromatous plaque either . the atheromatous plaque is an easier parameter to measure than carotid intima - media thickness and is very useful when evaluating overall cardiovascular risk in our patients . the approach is noninvasive , reliable , reproducible , and inexpensive ; therefore , it is the technique of choice for the detection of subclinical atherosclerosis and makes it possible to establish a classification that goes beyond common risk factors . young men with aga should be made aware of an increased risk of ir and its consequences later in life for diseases such as dyslipidemia , hypertension , and diabetes . aga patients with higher bmi or abdominal obesity ( suggested by higher wc ) should be advised regarding lifestyle modifications for weight reduction . it is suggested that since patients with aga have higher frequency of mets and a tendency to develop carotid atheromatous plaques , so all the patients of early onset aga should be screened for cardiovascular abnormalities by mets criteria and carotid ultrasound studies for early detection of atheromatous plaques .

oral contraceptive - induced menstrual migraine ( ocmm ) is a menstrual migraine subtype mainly caused by estrogen withdrawal occurring during the week of pill suspension [ 1 , 2 ] . ocmm is a poorly studied , but particularly severe and disabling form of menstrual migraine ; it is codified by the ichdii classification as estrogen - withdrawal headache ( code 8.4.3 ) . evidence - based guidelines recommend treating moderate or severe migraine , including menstrual migraine , with migraine - specific agents . among these , triptans are considered to provide a greater clinical benefit and a more favorable adverse event profile than other anti - migraine drugs . frovatriptan is a second - generation triptan , with a distinct pharmacokinetic profile . its high selectivity for the cerebral vasculature , long elimination half life and high persistence of therapeutic action may be useful in preventing menstrual migraine , but also for treating acute attacks , as shown by several randomized trials [ 47 ] . although frovatriptan is currently among the triptans recommended for prevention of menstrual migraine [ 810 ] , its efficacy in ocmm has had limited investigation . in the present retrospective analysis , we report on a pooled individual data analysis of three randomized double - blind studies [ 1214 ] performed to verify whether frovatriptan may be effective in the acute management of an ocmm attack and whether its efficacy may differ from that of other triptans . information on the study design for the three original studies is reported in the main publications [ 1214 ] . in brief , the studies recruited subjects of both genders , aged 1865 years , with a current history of migraine with or without aura , according to the international headache society definition , and with at least one , but no more than six migraine attacks per month for 6 months prior to entering the study . in the present retrospective analysis , only the group of women with ocmm was taken into consideration . ocmm was defined according to ihs criteria , as an estrogen - withdrawal headache , namely a migraine without aura developing within 5 days after cessation of estrogen use and resolving within 3 days . patients taking all types of combined oral contraceptives , except those entailing a withdrawal period of less than 5 days , were considered eligible for inclusion in the present retrospective analysis . the studies had a multicenter , randomized , double blind , cross - over design . each patient received frovatriptan 2.5 mg or rizatriptan 10 mg in the first study , frovatriptan 2.5 mg or zolmitriptan 2.5 mg in the second study , and frovatriptan 2.5 mg or almotriptan 12.5 mg in the third study , in a randomized sequence . after treating 13 episodes of migraine in no more than 3 months with the first treatment , the patient switched to the other treatment and was asked to treat a maximum of three episodes of migraine in no more than 3 months with the second treatment . the study design foresaw three visits and each patient s participation time in the study had not to exceed 6 months from randomization . subjects having no migraine episodes during one of the two observation periods were excluded from the study . blindness ensured by the overencapsulation technique , i.e. , by inserting study drug tablets in capsules . the present pooled analysis was carried out in all women with ocmm , who treated at least one episode of ocmm with both medications in each study . the following endpoints were evaluated : ( a ) the proportion of pain - free episodes at 2 and 24 h ( absence of migraine episodes 2 and 24 h after intake of one dose of study drug ) ; ( b ) the proportion of pain - relief episodes at 2 and 24 h ( a decrease in migraine intensity from severe or moderate to mild or none 2 and 24 h after the intake of one study drug dose ) ; ( c ) relapse within 24 and 48 h ( defined as an episode of migraine occurring within 24 and 48 h from the previous episode , after a period without migraine ) . continuous variables were summarized by computing average values and standard deviations ( sd ) , while categorical variables by computing the absolute value and the frequency ( as percentage ) . study endpoints were compared between groups by a student s t test ( continuous variables ) or by a test ( categorical variables ) . information on the study design for the three original studies is reported in the main publications [ 1214 ] . in brief , the studies recruited subjects of both genders , aged 1865 years , with a current history of migraine with or without aura , according to the international headache society definition , and with at least one , but no more than six migraine attacks per month for 6 months prior to entering the study . in the present retrospective analysis , only the group of women with ocmm was taken into consideration . ocmm was defined according to ihs criteria , as an estrogen - withdrawal headache , namely a migraine without aura developing within 5 days after cessation of estrogen use and resolving within 3 days . patients taking all types of combined oral contraceptives , except those entailing a withdrawal period of less than 5 days , were considered eligible for inclusion in the present retrospective analysis . the studies had a multicenter , randomized , double blind , cross - over design . each patient received frovatriptan 2.5 mg or rizatriptan 10 mg in the first study , frovatriptan 2.5 mg or zolmitriptan 2.5 mg in the second study , and frovatriptan 2.5 mg or almotriptan 12.5 mg in the third study , in a randomized sequence . after treating 13 episodes of migraine in no more than 3 months with the first treatment , the patient switched to the other treatment and was asked to treat a maximum of three episodes of migraine in no more than 3 months with the second treatment . the study design foresaw three visits and each patient s participation time in the study had not to exceed 6 months from randomization . subjects having no migraine episodes during one of the two observation periods were excluded from the study . blindness ensured by the overencapsulation technique , i.e. , by inserting study drug tablets in capsules . the present pooled analysis was carried out in all women with ocmm , who treated at least one episode of ocmm with both medications in each study . the following endpoints were evaluated : ( a ) the proportion of pain - free episodes at 2 and 24 h ( absence of migraine episodes 2 and 24 h after intake of one dose of study drug ) ; ( b ) the proportion of pain - relief episodes at 2 and 24 h ( a decrease in migraine intensity from severe or moderate to mild or none 2 and 24 h after the intake of one study drug dose ) ; ( c ) relapse within 24 and 48 h ( defined as an episode of migraine occurring within 24 and 48 h from the previous episode , after a period without migraine ) . continuous variables were summarized by computing average values and standard deviations ( sd ) , while categorical variables by computing the absolute value and the frequency ( as percentage ) . study endpoints were compared between groups by a student s t test ( continuous variables ) or by a test ( categorical variables ) . the main study population consisted of 346 subjects , of which 280 ( 81 % ) were women and 224 were in the fertile age . a total of 35 out of the 224 eligible women ( 16 % ) were taking oral contraceptives , treated at least one episode of ocmm with both medications and were thus included in the present analysis . a flow diagram of participants throughout the study 1.fig . 1flow diagram of the patients throughout the study flow diagram of the patients throughout the study demographic and clinical baseline data of the 35 women with ocmm attacks and of the 189 women with non - ocmm attacks are reported in table 1 . no statistically significant differences were observed between the two subgroups of women for any of the explored variables.table 1baseline demographic and clinical data of the 35 women with oral contraceptive - induced menstrual migraine ( ocmm ) and of the 189 women with non - ocmm of the intention - to - treat populationwomen with ocmm ( n = 35)women with non - ocmm ( n = 189)p valueage ( years , mean sd)35 934 7nsheight ( cm , mean sd)163 5164 6nsweight ( kg , mean sd)59 1059 10nsage at onset of migraine ( years , mean sd)18 716 6nsmigraine attack duration > 2 days ( n , % ) 7 ( 20)45 ( 24)nsmidas score ( mean sd)22 2023 15nsno use of triptans in the previous 3 months ( n , % ) 16 ( 46)79 ( 42)nsmigraine attacks with aura ( n , % ) 4 ( 10)29 ( 10)nsdata are shown as mean ( sd ) , or absolute ( n ) and relative frequency ( % ) . p value refers to the statistical significance of the between - group difference baseline demographic and clinical data of the 35 women with oral contraceptive - induced menstrual migraine ( ocmm ) and of the 189 women with non - ocmm of the intention - to - treat population data are shown as mean ( sd ) , or absolute ( n ) and relative frequency ( % ) . p value refers to the statistical significance of the between - group difference a total of 144 out of the overall 1,289 attacks occurring in the 224 women in fertile age were classified as ocmm : 73 of the 144 attacks ( 51 % ) were treated with frovatriptan and 71 ( 49 % ) with comparators ( 8 women treated with rizatriptan , 12 with zolmitriptan and 15 with almotriptan ) . as shown in fig . 2 , the proportion of pain free and pain - relief episodes at 2 h were not significantly ( p = ns ) different between frovatriptan ( 25 and 51 % ) and the comparators ( 28 and 48 % , respectively ) . at 24 h , a larger proportion of women treated with frovatriptan were pain free ( 71 vs. 60 % comparators , p < 0.05 ) and had pain relief ( 83 vs. 76 % comparators , p = ns).fig . 2main study endpoints in the group of women with oral contraceptive - induced menstrual migraine ( ocmm ) treated with frovatriptan ( open bars ) and the comparators ( grey bars ) . data are reported as relative ( % ) frequencies for pain free ( pf ) at 2 and 24 h , for pain relief ( pr ) at 2 and 24 h , and for relapses at 24 and 48 h. the p values on top of the bars refer to the level of the statistical significance of the difference between the two study drugs main study endpoints in the group of women with oral contraceptive - induced menstrual migraine ( ocmm ) treated with frovatriptan ( open bars ) and the comparators ( grey bars ) . data are reported as relative ( % ) frequencies for pain free ( pf ) at 2 and 24 h , for pain relief ( pr ) at 2 and 24 h , and for relapses at 24 and 48 h. the p values on top of the bars refer to the level of the statistical significance of the difference between the two study drugs finally , the rate of relapses at 24 and 48 h was significantly ( p < 0.05 ) lower with frovatriptan ( 17 and 21 % ) than with the comparators ( 27 and 31 % ) ( fig . 2 ) . as shown in numerous randomized trials , frovatriptan represents a well known and effective option for acute [ 46 , 11 ] or prophylactic treatment [ 1517 ] of menstrual migraine . the present study , based on a pooled analysis of three double - blind , randomized , direct comparative , cross - over studies [ 1214 ] , demonstrates that frovatriptan is clinically effective also for the acute treatment of ocmm , a particular menstrual migraine subtype . according to our results , the clinical efficacy of frovatriptan at 2 h was similar to that of the other triptans , with the overall pain - free rates of approximately 25 % and of pain - relief rates of approximately 50 % . as expected , such proportions progressively increased during the period of observation , reaching larger values at 24 h under frovatriptan . in particular , this drug showed a more sustained relieving effect on migraine than the other triptans , with lower headache relapses after 24 h and even more so after 48 h and such findings might be explained by differences in the pharmacokinetics of frovatriptan when compared with the other triptans [ 7 , 18 ] . very poor evidence is available on the efficacy of triptans in ocmm and no previous direct comparison studies of various triptans have ever been published . in a pilot , open label , uncontrolled study , 20 women treating an ocmm acute attack with frovatriptan 2.5 mg displayed pain - free and pain - relief rates of 10 and 55 % at 2 h , and of 60 and 75 % at 24 h . migraine relapsed within 24 h in 36 % of patients . in a cross - over , randomized study , 46 % of the women taking 50 mg of oral sumatriptan achieved pain relief at 2 h and 18 % were pain free , with almost comparable results observed with the suppository formulation of sumatriptan . an additional interesting finding of our retrospective analysis is that immediate and delayed efficacy of frovatriptan in the subgroup of ocmm is superimposable to that previously observed in the whole subgroup of women with menstrual migraine . despite the interesting results however , to our knowledge , no comparative prospective studies of triptans in ocmm are yet available or have been planned . our results might be helpful to stimulate the design and implementation of larger direct comparative randomized clinical trials evaluating triptan efficacy in such a specific subtype of menstrual migraine . in conclusion , our analysis of individual data of double - blind , randomized , cross - over trials , suggests that frovatriptan and other widely employed triptans share a similar efficacy in the immediate treatment of acute attack of ocmm . however , frovatriptan due to its sustained antimigraine effect , may be more suitable for the management of ocmm than other triptans .

lichen planus is a chronic inflammatory mucocutaneous disease of unknown etiology that frequently involves the oral mucosa . the disease affects skin and mucosa in prevalence estimated between 0.5 and 2.0% of the population with predilection for females and a mean age at onset in the fourth or fifth decade 1 . about half of the patients with skin lesions have oral lesions , whereas about 25% present with oral lesions alone 2 . the buccal mucosa , tongue and gingival are the most common sites , whereas palatal lesions are uncommon 3 . they are usually symmetrical and bilateral lesions or multiple lesions may occur in the mouth 4 . olp can appear clinically in at least six forms that can occur separately or simultaneously : reticular , papular , plaque , atrophic , erosive and bullous 2 . the reticular , papular , plaque - like forms are usually painless and appear clinically as white keratotic lesions . the erosive , atrophic and bullous forms are often associated with a burning sensation and in many cases can cause severe pain 5 . the possible malignant transformation of olp is a subject of controversial discussion in the literature . while some studies support a premalignant nature of olp 6,7 , others argue against the validity of this statement 8,9 . it has been reported that the actual overall frequency of olp malignant transformation varies between 0.3 and 12.5% 6,9 . analysis of the published data is difficult because of differences in diagnostic criteria , time to follow - up and information on exposure to known oral carcinogens 10 . whether cancer development is part of the natural evolution of olp lesions or is due to extrinsic factors is unknown 11 . however , the association between chronic inflammation with a variety of epithelial malignancies has been recognized 12 . there is now enough evidence to suggest that the inflammatory cells and cytokines found in peri - tumoural stroma can contribute to tumor development and progression rather than mount an effective host antitumor response 12 . the human dna mismatch repair ( hmmr ) system plays an important role in reducing mutation and maintaining genomic stability . it corrects base - base mismatches and short insertions / deletions generated as a consequence of dna replication errors and homologous recombination 13 . mmr genes , including hmsh2 , hmlh1 , hmsh3 , hmps1 , hpms2 and gtbp / hmsh6 are very important in distinguishing and repairing misparing and slippage errors in dna synthesis . the mismatch is detected by two complexes , the hmsh2-gtbp / hmsh6 and the hmsh2-hmsh3 heterodimers after interaction with hmlh1-hpms2 heterodimers , an endonuclease is activated and incises the newly synthesized dna strand that contains the mutation 14 . immunohistochemical study on non - polyposis colorectal cancer demonstrated no expression of hmsh2 gene in the tumoral cells 15 . with regard to sporadic forms of tumors , reduced expression of hmsh2 have been reported in a distinct subset of oral and neck squamous cell 16 , colorectal 17 , endometrial 18 , ovarian 19 and urinary bladder carcinomas 20 . as hmsh2 alterations have been reported in oral squamous cell carcinoma and there are evidences suggesting the association between oral lichen planus and squamous cell carcinoma , in this study , we aim to investigate the immunolocalization of hmsh2 protein in oral lichen planus compared to oral normal mucosa epithelium . the material and methods have been approved by the ethical committee of universidade federal de minas gerais . twenty - six samples of olp diagnosed on clinical and histological criteria 21 were retrieved from the files of the oral pathology service , universidade federal de minas gerais . twelve olp ( six females and six males ; age range from 27 to 61 years ) were reticular clinical subtypes and 14 were atrophic / erosive olp ( eight females and six males ; age range from 19 to 65 years ) . ten samples of oral normal mucosa were included in the control group ( six males and four females ; age range from 8 to 67 years ) . the hmsh2 protein staining was performed by the lsab kit ( dako , carpinteria , usa ) . endogenous peroxidase was blocked by incubating sections in 6% ( v / v ) h2o2/methanol . slides were placed in 10 mm citrate buffer ( ph 6.0 ) and heated to 96c in a steamer for 30 minutes and incubated with the primary antibody for 18 h at room temperature . the primary serum used were anti - hmsh2 ( clone g219 - 1129 , pharmingen , san diego , usa ) diluted 1:100 in 20 mmol / l tris - hcl buffer ( ph 7.4 ) containing 0.9% nacl . after washing in the tris - hcl buffer , sections were incubated for 30 min at room temperature with biotinylated multi - link swine anti - goat , mouse and rabbit immunoglobulin . sections were washed and incubated for 30 min at room temperature with prediluted streptavidin - peroxidase conjugated . negative controls consisted of omission of the primary or the secondary antibody or primary incubation in the presence of non - immunized rabbit serum instead of the primary antibody . epithelial cells were considered to be positive if there was any staining of the nucleus , regardless of staining intensity . cell counting was performed in six high - power microscopic fields ( 400x , standard 20 , zeiss ) , which comprised almost all of the olp epithelium . the count was performed by two investigators without knowledge of whether the samples were control or experimental . the total number of basal and suprabasal epithelial cells along the six microscopic fields and the number of basal and suprabasal cells stained to hmsh2 protein were assessed . as percentage does not conform normal distribution , the mann - whitney test was used for statistical analysis . the material and methods have been approved by the ethical committee of universidade federal de minas gerais . twenty - six samples of olp diagnosed on clinical and histological criteria 21 were retrieved from the files of the oral pathology service , universidade federal de minas gerais . twelve olp ( six females and six males ; age range from 27 to 61 years ) were reticular clinical subtypes and 14 were atrophic / erosive olp ( eight females and six males ; age range from 19 to 65 years ) . ten samples of oral normal mucosa were included in the control group ( six males and four females ; age range from 8 to 67 years ) . the hmsh2 protein staining was performed by the lsab kit ( dako , carpinteria , usa ) . endogenous peroxidase was blocked by incubating sections in 6% ( v / v ) h2o2/methanol . slides were placed in 10 mm citrate buffer ( ph 6.0 ) and heated to 96c in a steamer for 30 minutes and incubated with the primary antibody for 18 h at room temperature . the primary serum used were anti - hmsh2 ( clone g219 - 1129 , pharmingen , san diego , usa ) diluted 1:100 in 20 mmol / l tris - hcl buffer ( ph 7.4 ) containing 0.9% nacl . after washing in the tris - hcl buffer , sections were incubated for 30 min at room temperature with biotinylated multi - link swine anti - goat , mouse and rabbit immunoglobulin . sections were washed and incubated for 30 min at room temperature with prediluted streptavidin - peroxidase conjugated . negative controls consisted of omission of the primary or the secondary antibody or primary incubation in the presence of non - immunized rabbit serum instead of the primary antibody . epithelial cells were considered to be positive if there was any staining of the nucleus , regardless of staining intensity . cell counting was performed in six high - power microscopic fields ( 400x , standard 20 , zeiss ) , which comprised almost all of the olp epithelium . the count was performed by two investigators without knowledge of whether the samples were control or experimental . the total number of basal and suprabasal epithelial cells along the six microscopic fields and the number of basal and suprabasal cells stained to hmsh2 protein were assessed . does not conform normal distribution , the mann - whitney test was used for statistical analysis . the hmsh2 immunoreativity was detected mainly in basal and intermediate epithelial layers ( fig . 1 and 2 ) . the mean percentage of positive epithelial cells to hmsh2 in olp and oral normal mucosa were listed in table 1 . the percentage of positive cells in reticular and atrophic / erosive subtypes of olp was significantly decreased compared to oral normal mucosa ( table 1 ) . several conditions in oral pathology have been characterized as premalignant and many factors have been considered to promote the development of carcinoma in the oral cavity . most studies have shown that patients with olp develop oral cancer at an increased rate as compared with the general population 4,7,11 . many articles are found in the literature suggesting an association of lichen planus to oral squamous cell carcinoma , but several of them suffer from poor documentation . the most common errors resulting in misleading conclusions are : the initial diagnostic errors , the inclusion of cases that had a known history of exposure to recognized carcinogens , and the report of cases of lichenoid dysplasia as malignant transformation of lichen planus . despite differences in experimental designs , the majority of studies have reported a rate of malignant transformation of olp between 0.5 and 2% over five - year period 23 . histopathologically it is characterized by a sub - epithelial lymphocytic infiltrate , disruption of epithelial basement membrane , degeneration of basal keratinocytes , hyperkeratinization and acanthosis 24 . the mechanism of basal cell damage is related to a cell - mediated immune process involving langerhans cells , t lymphocytes and macrophages 5 . the majority of olp - related t - cells are activated cytotoxic cd8 t - cells , which are known to trigger keratinocyte apoptosis via tnf - alpha release 25 . the association between chronic inflammation with a variety of epithelial malignancies has been recognized . examples of well known association between inflammation and cancer risk include , inflammatory bowel diseases and colorectal cancer , atrophic gastritis and gastric cancer , gall bladder cancer related to chronic cholecyistitis , and esophageal carcinoma after reflux esophagitis 12 . there is now enough evidence to suggest that in some instances inflammatory cells and cytokines found in peritumoral stroma can more likely contribute to tumor development and progression than to mount an effective host antitumoral response 12 . recent studies have focused on the stromal inflammatory environment before the malignant lesions appear , and have underlined its active role during the transition from normal tissue to in situ and invasive carcinoma 26 . stromal cells and their products , in association with insoluble intracellular matrix components , can act as an oncogenic agent , causing the disruption of homeostatic regulation of adjacent cells , such as tissue architecture , cell death and proliferation , and leading to the development of solid neoplasia , such as breast , colon and prostate carcinoma 27 . additional studies are necessary to establish whether olp could be classified among these chronic disorders . many inflammatory factors known to be related with cancer initiation , progression , and invasion are also expressed by olp - related chronic inflammatory infiltrate , and thus may contribute to olp malignant transformation 24 . hmsh2 is especially expressed in human cells that are undergoing rapid renewal : in the lower - two thirds of gastrointestinal glands 15 , in the more primitive testicular germ cells 28 , in the transitional epithelial cells of the bladder 20 , and in the basal cell line of epidermis and oral mucosa 29 . inactivation of hmsh2 , one of the most commonly hmmr genes , is a described alternative pathway in cancer development and progression . the resulting genetic instability is characterized by somatic mutations in repetitive sequences of tumor dna and is termed microsatellite instability 13 . although described in a variety of tumors 17 - 20 , hmmr deficiency has been studied predominantly in colorectal carcinomas . a distinct proportion of sporadic ( and almost all hereditary ) non - polyposis colorectal cancers exhibit hmsh2 gene alterations . these tumors show high frequency of microsatellite instability and are immunohistochemically characterized by the lack of hmsh2 protein expression 15 29 reported that 5% of the cases of oral squamous cell carcinoma show diminished expression of hmsh2 protein . in the present study , we demonstrated decreased expression of hmsh2 protein in reticular and atrophic / erosive olp compared to oral normal mucosa . da silva fonseca & do carmo 30 showed that olp kerationocytes present higher pcna index and agnor numbers than epithelial cells from normal mucosa , which suggests increased cellular proliferation in olp . as dna damage is more prone to occur during the cell cycle , it can be assumed that a higher index of cellular proliferation in olp would require an increased immunolocalization of hmsh2 . therefore , the reduced expression of hmsh2 protein could make the epithelium of olp more susceptible to dna mutation , making it prone to oral squamous cell carcinoma development . positive labelling for hmsh2 protein in basal and intermediate epithelial layers of reticular subtype of oral lichen planus patient ( streptavidin - biotin amplified system , x 400 ) . increased labelling of hmsh2 protein in basal and intermediate epithelial layers of normal oral mucosa ( streptavidin - biotin amplified system , x 400 ) . percentage of positive cells to hmsh2 proteins in reticular and atrophic / erosive subtypes of oral lichen planus ( olp ) compared to oral normal mucosa .

cerebral venous and sinus thrombosis is a rare condition accounting for < 1% of all strokes.7 ) its annual incidence is estimated at three to four cases per one million members of the population , most often affecting young adults and children.19 ) in adults , the peak incidence is in their third decade , and approximately 75% of adult patients are female.5)7 ) thrombotic risk factors or direct causes are identified in approximately 85% of patients with cerebral sinus thrombosis.19 ) superior sagittal sinus thrombosis ( ssst ) is associated with a variety of hypercoagulable , states including crohn 's disease , diabetes insipidus , direct injury to the sinus , iatrogenic causes , malignancy , nephrotic syndrome , oral contraceptives , paroxysmal nocturnal hemoglobinuria , polycythemia , pregnancy , puerperium , systemic lupus erythematosus , and ulcerative colitis.1)19 ) coagulation abnormalities are commonly seen in patients with hyperthyroidism.6)9)18 ) however , the association between hyperthyroidism and cerebral venous thrombosis is poorly understood . to investigate the possible association between these two clinical conditions , we report on a case of a 31-year - old male patient with a six - year history of hyperthyroidism who developed ssst . a 31-year - old male patient was admitted to our institute with seizure , headache , and mental deterioration . during the recent six years he did not undergo a regular follow - up and had poor compliance to the medication . one day before visiting our institution , the patient was admitted at a local hospital for transient weakness of both lower extremities . the patient underwent treatment for thyrotoxic periodic paralysis or thyroid crisis , but became progressively stuporous and experienced seizure attacks several times . findings on immediate brain computerized tomography ( ct ) showed multiple hemorrhages in the subcortical area of both middle frontal gyrus ( fig . after two hours , the patient was transferred to our institute and still exhibited a stuporous mentality with no motor weakness . findings on brain ct showed a slight increase of hemorrhage and ct angiography showed no abnormalities of the major cerebral artery system ( fig . laboratory results showed hyperthyroidism with the following values : 176.2 ng / dl of t3 ( normal range , 80 - 200 ng / dl ) , 3.84 ng / dl of high free t4 ( normal range , 0.89 - 1.76 ng / dl ) , and less than 0.01 iu / ml of thyroid - stimulating hormone ( normal range , 0.35 - 5.5 iu / ml ) . hematologic and coagulation parameters , including platelet count , prothrombin time , activated partial thromboplastin time , fibrinogen , anti - thrombin ill , fibrin degradation products , d - dimer , lupus anticoagulant , protein c activity , and protein s activity were all normal . two days after the initial visit , the patient became nearly alert and fully oriented . cerebral digital subtraction angiography ( dsa ) was performed in order to determine the causes of the non - traumatic cerebral hemorrhage . cross - sectional echocardiography showed a normal left ventricular size and function with no evidence of intra - cardiac thrombus . following the diagnosis of ssst , the patient received anticoagulation therapy with warfarin 5 mg qd and avoided systemic heparinization on account of the aggravating hemorrhage . four weeks after treatment , follow - up cerebral dsa showed a recanalization of the superior sagittal sinus ( fig . 4a , b ) , and laboratory findings indicated a normal free t4 level of 0.81 ng / dl . the patient had no recurrence of neurological symptoms and maintained a euthyroid state for the next nine months . ssst is an uncommon cerebrovascular accident ; due to a broad spectrum of symptoms and clinical courses , a diagnosis is still frequently overlooked or delayed.7 ) the average delay from the onset of symptoms to the diagnosis is seven days.8 ) in addition , approximately 15% of cases of ssst are considered to be idiopathic.19 ) although coagulation disturbances are reported in hyperthyroidism , an association between hyperthyroidism and cerebral venous thrombosis has not been well established . schutta et al.16 ) reported on a case of a 34-year - old male patient with hyperthyroidism and inherited plasminogen deficiency . siegert et al.17 ) documented on two patients ( a 24-year - old male and a 32-year - old female ) with ssst during the thyrotoxic phase of grave 's disease . dai et al.4 ) demonstrated on a 39-year - old male patient with undiagnosed hyperthyroidism who developed ssst . the patient had a high free t4 level , hot thyroid up - take , and rapid atrial fibrillation . in our patient , along with previous reports , it can be hypothesized that hyperthyroidism is an independent risk factor for cerebral venous thrombosis . thrombotic events in thyrotoxic patients are in danger of systemic and cerebral arterial infarctions and have been directly attributed to atrial fibrillation , suggesting emboli as the causative agent.2)11)13 ) in our case , however , normal sinus rhythm was observed on the electrocardiography . in addition , findings on echocardiography showed normal left ventricular size and function with no evidence of intra - cardiac thrombus . development of ssst occurs due to multi - factorial causes , commonly including three factors : stasis of the blood stream , abnormalities of the vessel wall , and hypercoagulability . these three factors are involved in promoting venous thrombosis.17 ) large thyrotoxic goiters may cause stasis of venous blood flow from the cerebral circulation through local obstruction by itself or large blood flow of the thyroid gland.3)10 ) however , our patient did not present with a goiter . and no other causes of vessel wall abnormality , such as vascular inflammation and direct injury to veins , were detected . therefore , we suppose that hypercoagulability was the main cause of ssst in our patient with hyperthyroidism . to date , numerous studies have indicated an association of coagulation disturbances with hyperthyroidism . several authors have reported that the intrinsic clotting system , particularly factor viii , was markedly affected by hyperthyroidism.6)9)15)18 ) high adrenergic tone and hyper - metabolic state of hyperthyroidism resulted in factor viii hyperactivity , thus increasing the likelihood of hypercoagulation.6)14)18 ) although our patient exhibited a normal range of factor viii activity ( 135% , normal range , 60 to 140% ) , he showed high activity of factors ix and xi ( > 160% for both ) . these results are consistent with hypercoagulation through activation of the intrinsic clotting pathway , factor xi in particular.12)20 ) in this regard , based on our case report , it is probable that activation of intrinsic clotting factors by hyperthyroidism might be the factor responsible for the occurrence of ssst . abnormal activity of the clotting factor is recovered to normal after reaching the euthyroid state ; therefore , anti - thyroid therapy constitutes an important part of the treatment.6)9)15)18 ) we suggest that , because of the hypercoagulability , patients with hyperthyroidism may be predisposed to development of ssst . consideration of the possibility of development of cerebral venous thrombosis in patients with hyperthyroidism who present with neurological symptoms is mandatory . conversely , hyperthyroidism as the thrombotic factor in a patient with cerebral venous thrombosis who is not at risk of developing cerebrovascular events must also be considered .

myofascial release has been commonly regarded as a therapeutic , post - exercise technique aimed towards repair and recovery ( 1 , 2 , 18 , 22 ) . more recently , myofascial release has been regarded as a performance enhancing , pre - exercise technique within the athletic population ( 19 , 24 ) . this current pre - exercise myofascial release technique has been seen in the form of total - body foam rolling . this is a technique of self - myofascial release in which the targeted musculature is rolled and compressed utilizing a foam rolling device ( 6 , 13 , 24 ) . the trend has emerged and is highly regarded within the strength and conditioning field . before the emergence of myofascial release as a pre - exercise technique , rehabilitation practitioners frequently explored the technique in alleviating pain and aiding in the recovery of physical activity . pain and fatigue are often associated with particular trigger point tissue damage ( 18 ) . one of the more commonly researched therapeutic approaches to pain and recovery has been trigger point soft tissue massage therapies ( i.e. , myofascial release techniques ) . myofascial release research has shown to be effective in pain alleviation due to a series of physiological responses ( 1 , 2 ) . the most common of these responses is an increase in the dilation of the arterial system ( 22 ) . other common responses associated with myofascial release include restoration of soft - tissue , increased nitrogen dioxide ( no2 ) , and improved vascular plasticity ( 22 ) . this rehabilitation has been particular to somatic system dysfunction and somatic system disorders ( 9 , 26 ) . to improve these somatic deficits , physicians commonly prescribe the compressive form of self - myofascial release technique of foam rolling . compression has previously demonstrated recovery capabilities ; therefore , the compressive nature of foam rolling has also been researched for its recovery capabilities ( 16 ) . an acute bout of foam rolling self - myofascial release following physical activity has demonstrated improved recovery in multiple cardiovascular variables including heart rate variability and diastolic blood pressure ( 3 ) . until recently , foam rolling self - myofascial release has been used as a post - performance recovery and rehabilitation therapy . with the evolution of the strength and conditioning field , foam rolling self - myofascial release has emerged as an additional component to an athlete s warm - up . in the strength and conditioning field , a diverse range of warm - up techniques recent strength and conditioning research has demonstrated that static stretching during the warm - up decreases force production and muscular performance ( 11 , 28 ) . although foam rolling in adjunct with static stretching has demonstrated slight improvements in physical performance , it is not ideal for strength and conditioning coaches ( 24 ) . it s not ideal because it has been demonstrated and well documented that an active dynamic warm - up improves many aspects of athletic performance such as speed , balance and power ( 5 , 23 , 28 ) . athletic gains in speed , balance , and power can directly translate to agility improvements as well ( 25 ) . therefore , strength and conditioning practitioners use a dynamic warm - up as a method to potentially improve an athlete s performance . as recently discussed , self - myofascial release in the form of foam rolling has demonstrated multiple positive therapeutic effects ( e.g. , vascular plasticity and soft tissue restoration ) on performance and recovery ( 1 , 2 , 3 , 9 , 16 , 26 ) . the existing research has suggested an increase of myogenic and endothelial dilation , as well as an increase in no2 as a response to foam rolling self - myofascial release ( 22 ) . as a result , many strength and conditioning coaches now incorporate foam rolling self - myofascial release in adjunct with dynamic warm - ups to improve overall performance ; however , there is little research to support beneficial effects . therefore , the purpose of the current study was to determine if an acute bout of foam rolling self - myofascial release in addition to a dynamic warm - up will improve performance . based on the current research , it was hypothesized that an acute bout of foam rolling in adjunct with a dynamic warm - up will improve performance testing when compared to an acute dynamic warm - up without foam rolling . the study was a counterbalanced , crossover within - subjects design in which subjects participated in a control condition that consisted of a standard 5-minute general warm - up followed by a 5-minute dynamic warm - up ( dyn ) and an experimental condition that consisted of the same standard 5-minute general warm - up followed by the same 5-minute dynamic warm - up with the addition of a bout of total body foam rolling ( smr ) . measures of non - fatiguing exercise performance , agility , muscular strength , and speed were administered following each warm - up . eleven physically active , athletic healthy males ( table 1 ) agreed to participate in the study . the population included subjects whom previously competed or currently compete in professional , collegiate division i , and collegiate division ii athletics . the spectrum of sports includes football , baseball , soccer , and track - n - field . also , subjects were asked to refrain from physical activity , alcohol , and caffeine 24 hours prior to testing . subjects read and signed an informed consent form prior to participation in the study . following consent , subjects reported to the nova southeastern exercise and sports science laboratory for all testing . subjects were measured for physical characteristics including body weight , height , and body composition . seven - site skin fold measurements were taken using lange skin fold calipers ( beta technology , inc . , santa cruz , california ) . the seven sites were marked and included the thigh , abdomen , suprailiac , midaxillary , chest , triceps , and subscapular skin fold ( 15 ) . two measurements were taken at each individual site to improve accuracy . if measurements varied by 2 mm , a third measurement was taken . body density was calculated from the sum of the seven skin fold sites and then entered in to the jackson and pollock equation to extrapolate body fat percent ( 4 ) . bmi was also calculated as weight ( kg ) divided by height squared ( m ) . subjects participated in two separate experimental trial conditions ( dyn , smr ) separated by a 7-day recovery period . the experimental trial condition dyn began with a 5-minute general warm - up in which subjects jogged at a self - selected pace for 1000 meters . following the general warm - up , subjects were instructed through a variety of mobility and full range of motion dynamic warm - up techniques that included arm circles , body weight squats , and body weight squat jumps . flow maneuvers were followed with sprinting high knees , sprinting butt kickers , alternating lunge jumps , alternating log jumps , scapular push - ups , thoracic rotations , and clapping push - ups . each technique followed a 2 10 scheme indicating each technique was performed for two sets of ten ( repetitions or meters ) in the same order by all subjects . following dyn , the subjects were tested on a battery of performance tests that included a flexibility and power measures ( sit - and - reach , vertical jump , and standing long jump ) , an agility measure ( 18.3 m pro - agility test ) , a maximum strength measure ( indirect 1-rm bench press ) , and finally a sprint measure ( 37 m sprint ) ( 4 ) . the experimental trial condition smr also began with the 5-minute general warm - up in which subjects jogged at the same self - selected pace for 1000 meters . following the general warm - up , subjects were instructed through a variety of self - myofascial release techniques utilizing a conventional foam roller ( black molded foam roller - 6 12 round , perform better , cranston , ri ) . the conventional foam roller has previously proved effective in its ability to cover the greatest amount of muscular surface area ( 6 ) . the rolling progression ( figure 1 ) targeted the thoracic / lumbar regions ( erector spinae , multifidis ) , the gluteal region ( gluteus maximus , gluteus medius , gluteus minimus ) , the hamstring region ( semitendinosus , semimembranosus , biceps femoris ) , the calf region ( gastrocnemius , soleus ) from the supine body position . the progression continued with the quadriceps / flexor region , ( rectus femoris , sartorius , psoas major , iliacus ) and finally the pectoral region ( pectoralis major , pectoralis minor ) from the prone body position . each group of muscles was rolled over their entire surface area , and was applied at 5 strokes per 30 seconds . , subjects were then instructed through the same variety of mobility and full range of motion dynamic warm - up techniques as dyn . these again included a 2 10 scheme of arm circles , body weight squats , and body weight squat jumps . flow maneuvers were followed with sprinting high knees , sprinting butt kickers , alternating lunge jumps , alternating log jumps , scapular push - ups , thoracic rotations , and clapping push - ups . following the warm - up , the same battery of performance tests including flexibility and power measures , an agility measure , a maximum strength measure , and a sprint measure all sit - and - reach measures were recorded using a standard sit - and - reach box ( baseline evaluation instruments , white plains , ny ) . the subjects sat shoeless with their feet placed 30 cm apart , and touching the standard box . the subjects leaned forward slowly reaching as far as possible while keeping their hands adjacent with one another . the best of three trials following an initial guided trial was recorded to indicate hamstring and lower back flexibility . as a measure of maximum muscular power ( high - speed strength ) , the subjects performed the vertical jump using a commercial vertec device ( sports imports , columbus , oh ) . after using the stack of adjustable horizontal vanes to determine the subjects flat - footed standing touch height , the stack of vanes was raised to an estimated height so that the athlete was capable of reaching the lowest set of vanes , but incapable of reaching the highest vane . the subject was then informed that the best of three trials would be recorded as highest point in jump ( the difference in flat - footed standing touch height and highest point in jump was used as the vertical jump measure ) . the subject began each trial with a countermovement in which both flexion of the hips and knees occurred . following the countermovement , the athlete then generated muscular power while extending at both the knees and hips reaching as high as they could while in air with their dominant hand . the subject tapped the set of vanes ; the highest vane tapped indicated the highest point in the jump as another measure of maximum muscular power ( high - speed strength ) , the subjects performed the standing long jump . the subjects began each trial with both feet behind a designated starting line . once in place a mark was placed at the subject s heel , and a measurement was made with a tape measure . it is important to note that if the subject did not stick their landing , the trial was repeated . the best of three trials was recorded as longest jump . as a measure of agility a subject began the test in a three - point stance , while straddling a center line . the foot of the subject must come in contact with the line ( monitored by a line judge ) . once contact was made , the subject then turned and sprinted to a line 9.1 m to the right ( 4.6 m to the right of the center line ) and again makes contact with the foot . once contact is made with the right line , the subject then turned and sprinted to the left another 4.6 m through the center line . the best time of two trials was recorded as agility speed . as a measure of muscular strength ( low - speed strength ) the procedure started with a subject completing a warm - up set at 60% of estimated 1-rm for 10 repetitions followed by an additional warm - up set at 80% of estimated 1-rm for 3 repetitions . after a three minute rest , the subjects performed a rep out max at 90% of estimated 1-rm . utilizing the adam s equation [ kg / ( 1(0.02 number of repetitions ) ) ] , an indirect 1-rm bench press value was determined ( 12 ) . as a measure of speed , the subjects performed the 37 m sprint . following two build - up practice runs at submaximal speeds , the subjects were measured for speed twice . the test started with the subject in a three - point stance with the entire body positioned behind the starting line . means and measures of variability were calculated for all subject and performance data . following shapiro - wilk normality testing , paired samples t - tests were utilized to determine if there were any significant differences on performance variables ( sit - and - reach [ cm ] , vertical jump [ cm ] , standing long jump [ cm],18.3 m pro - agility [ sec ] , indirect 1-rm bench press [ kg ] , and 37 m sprint [ sec ] ) between conditions ( dyn vs. smr ) . all statistical analyses were performed using spss for windows ( version 17.0 , spss inc . , eleven physically active , athletic healthy males ( table 1 ) agreed to participate in the study . the population included subjects whom previously competed or currently compete in professional , collegiate division i , and collegiate division ii athletics . the spectrum of sports includes football , baseball , soccer , and track - n - field . also , subjects were asked to refrain from physical activity , alcohol , and caffeine 24 hours prior to testing . subjects read and signed an informed consent form prior to participation in the study . following consent , subjects reported to the nova southeastern exercise and sports science laboratory for all testing . subjects were measured for physical characteristics including body weight , height , and body composition . seven - site skin fold measurements were taken using lange skin fold calipers ( beta technology , inc . , the seven sites were marked and included the thigh , abdomen , suprailiac , midaxillary , chest , triceps , and subscapular skin fold ( 15 ) . two measurements were taken at each individual site to improve accuracy . if measurements varied by 2 mm , a third measurement was taken . body density was calculated from the sum of the seven skin fold sites and then entered in to the jackson and pollock equation to extrapolate body fat percent ( 4 ) . bmi was also calculated as weight ( kg ) divided by height squared ( m ) . subjects participated in two separate experimental trial conditions ( dyn , smr ) separated by a 7-day recovery period . the experimental trial condition dyn began with a 5-minute general warm - up in which subjects jogged at a self - selected pace for 1000 meters . following the general warm - up , subjects were instructed through a variety of mobility and full range of motion dynamic warm - up techniques that included arm circles , body weight squats , and body weight squat jumps . flow maneuvers were followed with sprinting high knees , sprinting butt kickers , alternating lunge jumps , alternating log jumps , scapular push - ups , thoracic rotations , and clapping push - ups . each technique followed a 2 10 scheme indicating each technique was performed for two sets of ten ( repetitions or meters ) in the same order by all subjects . following dyn , the subjects were tested on a battery of performance tests that included a flexibility and power measures ( sit - and - reach , vertical jump , and standing long jump ) , an agility measure ( 18.3 m pro - agility test ) , a maximum strength measure ( indirect 1-rm bench press ) , and finally a sprint measure ( 37 m sprint ) ( 4 ) . the experimental trial condition smr also began with the 5-minute general warm - up in which subjects jogged at the same self - selected pace for 1000 meters . following the general warm - up , subjects were instructed through a variety of self - myofascial release techniques utilizing a conventional foam roller ( black molded foam roller - 6 12 round , perform better , cranston , ri ) . the conventional foam roller has previously proved effective in its ability to cover the greatest amount of muscular surface area ( 6 ) . the rolling progression ( figure 1 ) targeted the thoracic / lumbar regions ( erector spinae , multifidis ) , the gluteal region ( gluteus maximus , gluteus medius , gluteus minimus ) , the hamstring region ( semitendinosus , semimembranosus , biceps femoris ) , the calf region ( gastrocnemius , soleus ) from the supine body position . the progression continued with the quadriceps / flexor region , ( rectus femoris , sartorius , psoas major , iliacus ) and finally the pectoral region ( pectoralis major , pectoralis minor ) from the prone body position . each group of muscles was rolled over their entire surface area , and was applied at 5 strokes per 30 seconds . following the self - myofascial release techniques , subjects were then instructed through the same variety of mobility and full range of motion dynamic warm - up techniques as dyn . these again included a 2 10 scheme of arm circles , body weight squats , and body weight squat jumps . flow maneuvers were followed with sprinting high knees , sprinting butt kickers , alternating lunge jumps , alternating log jumps , scapular push - ups , thoracic rotations , and clapping push - ups . following the warm - up , the same battery of performance tests including flexibility and power measures , an agility measure , a maximum strength measure , and a sprint measure all sit - and - reach measures were recorded using a standard sit - and - reach box ( baseline evaluation instruments , white plains , ny ) . the subjects sat shoeless with their feet placed 30 cm apart , and touching the standard box . the subjects leaned forward slowly reaching as far as possible while keeping their hands adjacent with one another . the best of three trials following an initial guided trial was recorded to indicate hamstring and lower back flexibility . as a measure of maximum muscular power ( high - speed strength ) , the subjects performed the vertical jump using a commercial vertec device ( sports imports , columbus , oh ) . after using the stack of adjustable horizontal vanes to determine the subjects flat - footed standing touch height , the stack of vanes was raised to an estimated height so that the athlete was capable of reaching the lowest set of vanes , but incapable of reaching the highest vane . the subject was then informed that the best of three trials would be recorded as highest point in jump ( the difference in flat - footed standing touch height and highest point in jump was used as the vertical jump measure ) . the subject began each trial with a countermovement in which both flexion of the hips and knees occurred . following the countermovement , the athlete then generated muscular power while extending at both the knees and hips reaching as high as they could while in air with their dominant hand . the subject tapped the set of vanes ; the highest vane tapped indicated the highest point in the jump as another measure of maximum muscular power ( high - speed strength ) , the subjects performed the standing long jump . the subjects began each trial with both feet behind a designated starting line . once in place a mark was placed at the subject s heel , and a measurement was made with a tape measure . it is important to note that if the subject did not stick their landing , the trial was repeated . the best of three trials was recorded as longest jump . as a measure of agility a subject began the test in a three - point stance , while straddling a center line . the foot of the subject must come in contact with the line ( monitored by a line judge ) . once contact was made , the subject then turned and sprinted to a line 9.1 m to the right ( 4.6 m to the right of the center line ) and again makes contact with the foot . once contact is made with the right line , the subject then turned and sprinted to the left another 4.6 m through the center line . the best time of two trials was recorded as agility speed . as a measure of muscular strength ( low - speed strength ) , the procedure started with a subject completing a warm - up set at 60% of estimated 1-rm for 10 repetitions followed by an additional warm - up set at 80% of estimated 1-rm for 3 repetitions . after a three minute rest , the subjects performed a rep out max at 90% of estimated 1-rm . utilizing the adam s equation [ kg / ( 1(0.02 number of repetitions ) ) ] , an indirect 1-rm bench press value was determined ( 12 ) . as a measure of speed , following two build - up practice runs at submaximal speeds , the subjects were measured for speed twice . the test started with the subject in a three - point stance with the entire body positioned behind the starting line . means and measures of variability were calculated for all subject and performance data . following shapiro - wilk normality testing , paired samples t - tests were utilized to determine if there were any significant differences on performance variables ( sit - and - reach [ cm ] , vertical jump [ cm ] , standing long jump [ cm],18.3 m pro - agility [ sec ] , indirect 1-rm bench press [ kg ] , and 37 m sprint [ sec ] ) between conditions ( dyn vs. smr ) . all statistical analyses were performed using spss for windows ( version 17.0 , spss inc . , the data indicated differences in maximum muscular power and revealed that there were significantly greater performance scores for both the vertical jump ( p = 0.012 ) and the standing long jump ( p = 0.007 ) after the smr warm - up , relative to the dyn warm - up protocol . the analysis also revealed significantly greater performance scores for the 18.3 m pro - agility test ( p = 0.001 ) and the 37 m sprint ( p = 0.002 ) following smr . finally , the data demonstrated a significantly greater indirect 1-rm bench press ( p = 0.024 ) following smr . there was no difference in sit and reach performance scores between dyn and smr conditions ( p = 0.833 ) it was hypothesized that an acute bout of foam rolling in adjunct with a dynamic warm - up will improve performance when compared to an acute dynamic warm - up without foam rolling . recent trends in strength and conditioning have incorporated the use of foam rolling in addition to a traditional dynamic warm - up ; however , there has been a lack of evidence supporting this trend . consequently , the purpose was to investigate this topic by comparing smr to dyn in regards to acute performance . this study is the first to compare smr to dyn ; however , it is not the first to investigate foam rolling . the current results suggest that a warm - up combined with a series of foam rolling techniques has the potential to improve power , speed and agility performance test results . recently , it was determined that an acute bout of foam rolling is an effective method of increasing range of motion , particularly to the knee joint ( 19 ) . it was also recently studied that direct application of rolling techniques on the hamstrings promoted improved sit - and - reach results without impairing performance ( 27 ) . contrary to previous findings ( 27 ) , the results suggest that a warm - up with the addition of certain foam rolling exercises was unsuccessful at improving flexibility as measured via the standard sit - and - reach . the physiological effects of arterial dilation ( 22 ) may explain these differences , as the other studies only applied direct tissue rolling prior to testing . therefore , an increase of blood flow to the targeted muscle group would be even more advantageous when compared to the full body rolling application in the current study . smr is a full body warm - up technique , and blood flow may be circulated and distributed differently to other targeted regions . this can be achieved by improving many aspects of power production including force and velocity . improving the ability to generate force at a rapid pace has been established as a requisite ability for all athletic performance and ability ( 8) . techniques of power production have been measured through a series of performance tests including the vertical jump or the standing long jump . the most common of these to be tested in performance research is the vertical jump ( 7 , 10 , 16 , 17 , 20 , 23 , 28 ) . because of the nonfatiguing manner of the tests , both were tested as indicators of horizontal and vertical power performance . recently , it was determined that smr was not responsible for improvements in vertical lower body power and agility when compared to planking ( 13 ) . contrary to these previous findings , the results revealed that smr did improve lower body power performance in the vertical jump , broad jump , and pro - agility when compared to dyn . this can be explained in part to the physiological improvements of movement and fiber pattern recruitment associated with myofascial release . therefore , in the case of improving power production , smr may have increased recruitment patterning or firing rate associated with the neural stimulation associated with foam rolling . the ability of increasing strength , especially when compared to body mass , has been established as a major component to performance . previous literature has suggested that smr warm - up can maintain muscle performance ( 19 ) . the data differs in it suggests smr is successful warm - up for improving muscle performance when compared the dyn . this may be in part to the sample population , as the sample population used in the particular study was highly trained and familiar with all testing procedures . the data demonstrated increases in acute measures of the indirect 1-rm bench press values and the 37-m sprint times as a result of smr compared to dyn . while this was the first study to assess the effect of foam rolling and non - foam rolling warm - up , it is not without limitations . to avoid injury , a warm - up for each condition therefore , the present study lacks a true control condition , that is , a condition that is completed with no warm - up . it may be beneficial in the future to include a greater sample size ; however , significance was still meant utilizing within - subjects analysis . in conclusion , an acute warm - up bout of foam rolling in addition to a dynamic warm - up improved performance testing results when compared to an acute dynamic warm - up without foam rolling . in terms of the testing battery , the inclusion of foam rolling improved power , agility , strength , and speed when compared to the absence of foam rolling . therefore , the inclusion of foam rolling with a dynamic warm - up may be a beneficial method to improve physical performance . future studies are currently underway monitoring weight , rolling techniques and performance . often times , increasing body mass is associated with a decrease in athletic performance ( 14 ) . although , it is unknown , the data suggests that implementing smr prior to training could contest this performance deficit . in fact , the combination of weight gain with smr could result in overall increased athletic performance .

severe hyperkalemia during orthotopic liver transplantation , is very dangerous , and needs vigilant monitoring of serum potassium and acute management of the hyperkalemia . the causes of hyperkalemia during different stages are ; 1 ) extracellular shift in exchange for h during severe metabolic acidosis in an - hepatic phase and reperfusion of the graft liver , and 2 ) exogenous potassium due to blood transfusion or entry of the preservative fluid university of wisconsin ( uw ) solution into systemic circulation during reperfusion of the graft liver . however , this morbid hyperkalemia is more common in the early reperfusion phase than at other times during liver transplantation . although hyperkalemic episodes occurring immediately after reperfusion of new transplanted liver are most frequent and substantial , hyperkalemia in other phases during orthotopic liver transplantation is also hazardous and serious . for a short duration ( about 3 - 5 min ) after reperfusion of the graft liver , patients usually develop hyperkalemia . the main sources of this hyperkalemia are preservative fluid ( uw solution ) , which contains high concentration of potassium , and severe acidosis following reperfusion , which can mobilize intracellular potassium from all of the tissues . however , hyperkalemia before reperfusion during liver transplantation anesthesia is not common . the two independent risk factors for pre - reperfusion hyperkalemia during liver transplantation are high baseline potassium concentration and red blood cell ( rbc ) transfusion . an insulin protocol , in which separated doses of the regular insulin is administered together with blood transfusion in patients with high baseline k , has been used to prevent hyperkalemia due to blood transfusion . herein in we present a case that developed hyperkalemia without blood transfusion during pre - anhepatic phase of liver transplantation . the patient was a 49-year - old male who underwent liver transplantation for a meld score 22 cryptogenic cirrhosis , which had been complicated by bleeding from esophageal varices , ascites , and spontaneous bacterial peritonitis . the patient s baseline lab and paraclinic data prior to anesthesia are shown in table 1 . baseline laboratory and paraclinical data of a 49-year - old man , who presented severe hyperkalemia during liver transplantation anaesthesia was induced using thiopental , morphine , fentanyl and midazolam . normal saline and albumen 25% were administrated to maintain a central venous pressure of about 10 cm h2o . calcium gluconate and sodium bicarbonate were used to correct low ca levels and metabolic acidosis ( base excess -4 ) , respectively . we monitored cardiovascular functions using electrocardiogram , arterial pressure via a radial artery catheter , and central venous pressure via a catheter inserted into the right internal jugular vein . urine output was more than 500 ml during 3 hours of hepatectomy , and surgical bleeding was less than 400 ml ; therefore , no blood transfusion was performed . metabolic parameters including serum potassium was checked by serial measurements of arterial blood gases ( abg ) as needed ( table 2 ) . mmol / l , and after 40 minutes of anesthesia and surgery it was in normal range . thirty minutes after the ligation a tall t wave and bradycardia were noted on ekg monitoring . hyperkalemia was managed by 10 ml calcium gluconate 10% , 50 ml dextrose 50% , and 25 u regular insulin . near the end of hepatectomy phase , k increased to 7.8 mmol / l ; therefore , the operation was stopped , and patient received 20 ml calcium gluconate 10% , 1150 ml nahco3 , and 20 u to 210 u regular insulin . this led to a decrease of serum potassium to 4.09 mmol / l without episode of hypoglycemia . the hepatectomy was then done , and the second phase of the operation was followed . metabolic parameters during anesthesia of a 49-year - old male , who presented severe hyperkalemia during liver transplantation severe hyperkalemia frequently occurs immediately after revascularization during orthotropic liver transplantation . there is , however , only one report on severe pre - anhepatic hyperkalemia in living - related liver transplantation . the changes in metabolic and hemodynamic parameters in different phases of the liver transplantations surgery vary significantly . one of such parameters is serum potassium that may increase dramatically during any phase of the operation . identification of risk factors for hyperkalemia during phases ( pre - reperfusion , early post - reperfusion and late post - reperfusion ) of orthotropic liver transplantation are very important . moreover , risk factors in one phase may not apply to others . using multivariate logistic regression analyses , a previous study determined independent risk factors for development of hyperkalemia in three phases of orthotropic liver transplantation . the study showed that the incidence of pre - reperfusion hyperkalemia was less than post - reperfusion one , and higher baseline serum potassium and red blood cell transfusion were independent risk factors for the development hyperkalemia in the pre - reperfusion phase . the study suggested that since higher baseline potassium and red blood cell transfusion were two predictors of pre - anhepatic hyperkalemia , insulin should be administered intravenously as soon as the transfusion begins in patients with a baseline potassium above 4.0 mmol / l ( 12 iu of regular insulin for each unit of red blood cells ) . the amount of bleeding during hepatectomy was less than 400 cc in the present case ; therefore , no blood transfusion or administration of regular insulin was performed . although baseline potassium was 4 mmol / l and urine output was above 500 ml during three hours of hepatectomy , ligation of hepatic artery gradually increased potassium reaching 7.8 mmol / l near the end of hepatectomy . therefore , ligation of hepatic artery may have acted as a stress causing ischemia , which resulted in the flow of potassium from liver into systemic circulation and subsequent hyperkalemia . the effect of insulin on serum potassium occurs within seconds after insulin administration . in a person with normal liver , majority ( 70% ) of potassium uptake by insulin patients with end stage liver disease due to liver cirrhosis have potassium intolerance , which means that potassium uptake response to insulin is very sluggish and unusual dose of insulin is required . moreover , cirrhotic patients have abnormal cellular glucose uptake and metabolism due to marked insulin resistance . the lack of hypoglycaemia might also be due to the administration of methylprednisolon ( 15 mg / kg ) for immune suppression therapy . the changes in serum potassium in the present case suggest that it is necessary to take care of the changes of serum potassium concentration not only in post - reperfusion but also in pre - anhepatic stage during liver transplantation .

human saliva is a biological fluid with myriad of biological functions important for the maintenance of oral and general health . it is a plasma ultra - filtrate containing proteins either synthesized in situ from blood or in the salivary glands 1 . major salivary glands consisting of submandibular glands , sublingual glands , and parotid glands lie at the vicinity of the oral cavity whereas other minor exocrine glands lie beneath the oral mucosa 2 . several roles of saliva in the oral cavity include lubrication and binding , solubilisation of dry food , oral hygiene , and initiation of starch digestion 3 . apart from water as its major constituent , whole saliva also contains bacterial and exfoliated cells , electrolytes , glycoproteins , enzymes , and antibacterial compounds . likewise , minute amounts of gingival crevicular fluid coexist with saliva in the gingival crevice surrounding the teeth 4 . changes in saliva quality and quantity are indicative of the wellness of the patient 5 . human saliva , as a mirror of oral and systemic health , provides valuable information because it contains biomarkers specific for the unique physiologic aspects of periodontal and systemic diseases . proteomic markers from immunoglobulins to bone remodelling proteins were previously discovered in existing periodontal diseases 6 . in one study , salivary epidermal growth factor was found to be significantly raised in women with active and nonactive breast cancer compared to healthy women 7 . compared to blood , saliva has been clinically shown to produce more accurate , inexpensive , and convenient results . the diagnostic potential of this fluid has been studied in many laboratories in order to find its advantages over other biological fluids and potential biomarkers in numerous diseases . unlike plasma its noninvasive approach renders this biological fluid an effective alternative to blood and urine testing in monitoring patient 's health condition 8 . whole saliva can be easily collected by drooling , spitting , or swabbing into a designated tube as opposed to invasive blood collection procedure . these methods of obtaining saliva pose minimal risk of contracting deadly pathogens to the healthcare professionals . plus , sufficient quantities of saliva can be easily obtained for analysis by a practitioner even with modest training . for diagnostic and research purposes , , ultrasal-2 from oasis diagnostics , orasure oral specimen collection device from orasure technologies , inc . , certus collection device from alere , and saliva collection system from greiner bio - one . in one study , budget estimates for both blood and saliva collections were developed by a group of scientists incorporating personnel expenses and corresponding collection methods into the calculation . when comparing both budget estimates , these advantages have attracted many researchers to study and identify potential salivary biomarkers with unparalleled opportunities for clinical applications . as illustrated in figure 1 , human salivary proteomes exhibit promising potential as biomarkers to predict the onset of acute myocardial infarction ( ami ) , or heart attack . on a global scale this pathologic event occurs as a result of acute myocardial ischemia when there is evidence of myocardial necrosis 10 . according to world health organization 11 , this disease is one of the major forms of cardiovascular disease ( cvd ) in which its prevalence constitutes approximately 48% of all deaths in 2008 , affecting more than 17 million patients around the globe . other ami complications may include severe cardiac disability upon survival of the onset 12 . since ami is the leading cause of death worldwide , recent salivary findings and progress pertaining to this condition will be discussed to elucidate the promising use of saliva as a diagnostic fluid in acute cardiac care among a very high - risk group of type-2 diabetes mellitus ( t2 dm ) , as well as relevant methods employed . current challenges in saliva collection will be highlighted for further improvement on the quality of salivary biomarkers discovered in future . biomarkers can be indicative of a specific physiological or pathological state of a biological fluid including blood and saliva 13 . many researchers worldwide have been studying the use of biomarkers due to its ability to monitor susceptibility , progression , and resolution of diseases , health condition , and treatment outcome . despite the variability of the term biomarker used in diverse aspects of its applications , the most comprehensible definition of biomarker is cellular , biochemical , molecular , or genetic alterations by which a normal , abnormal , or simple biological process can be recognized or monitored 14 . any molecular species which demonstrates significant variation in concentration , as compared to those of control subjects , is a potential biomarker . therefore , if expression of interleukin-1 receptor antagonist is significantly enhanced compared to the normal group , the protein can be potentially targeted as an early biomarker to indicate risks of tobacco - related diseases 15 . saliva contains biomarkers derived from serum , gingival crevicular fluid , and mucosal transudate which are useful in multiplexed assays that are being developed as point - of - care devices , rapid tests , or in more standardized formats for centralized clinical laboratory operations 16 . the prospect of salivary diagnosis appeals to many scientists around the world for disease prognosis and diagnosis . current discoveries on potential salivary biomarkers encompass various systemic diseases such as autoimmune diseases , bone turnover markers , cardiovascular markers , dental caries and periodontal diseases , diseases of the adrenal cortex , drug level monitoring , forensic evidence , genetic disorders , infections , malignancy , occupational and environmental medicine , psychological research , and renal diseases 17 . in oral squamous cell carcinoma , ni et al . 18 acquired several potential salivary biomarkers corresponding to early detection and evaluation of aggressiveness and occurrence of the cancer based on recent researches . as such , further salivary investigation can yield more comprehensive information about this fluid especially in the field of health medicine . apart from genomics and metabolomics , salivary proteomics 19 has demonstrated a great potential and has been widely utilised as a means to identify candidate biomarkers and possible immunochemistry markers for various illnesses predominantly infectious and neoplastic diseases 20 . in peptide identification , mass spectrometry ( ms)-based proteomic techniques have been commonly applied including two - dimensional gel electrophoresis - mass spectrometry ( 2-de / ms ) , liquid chromatography tandem mass spectrometry ( lc - ms / ms ) , matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof / ms ) , and surface - enhanced laser desorption / ionization time - of - flight mass spectrometry ( seldi - tof / ms ) 21 . by employing one of ms - based proteomic techniques , chan et al . 22 detected significant altered abundance of polymeric immunoglobulin receptor , plastin-2 , actin related protein 3 , leukocyte elastase inhibitor , carbonic anhydrases 6 , immunoglobulin j , and interleukin-1 receptor antagonist among t2 dm patients with periodontitis compared to their control t2 dm cohorts 23 , 24 detected increased abundance and structural microheterogeneity of haptoglobin beta chains in the saliva of patients with oral squamous cell carcinoma which were also subsequently observed in the saliva of habitual betel quid chewers . many other salivary biomarkers with diagnostic potential were already characterised in oncology , head and neck carcinoma , breast and gastric cancers , salivary gland function and disease , sjgren syndrome , systemic sclerosis , dental and gingival pathology , systemic , psychiatric , and neurological diseases using ms - based proteomics 25 . it is possible that efficiency and accuracy of these proteomic technologies will turn salivary diagnostics into a clinical reality . the prospect of utilizing saliva samples in the diagnosis of ami is appealing to a large group of scientists due to its noninvasive and economical nature . extensive biomarker research on cvd has elucidated various proteins associated with this disease . since approximately 27% of the whole saliva proteins resemble those found in plasma 26 , similar proteins present in both saliva and plasma will be very useful to facilitate monitoring of both disease progression and therapeutic treatments among these patients . in association with ami , some studies on plasma proteins revealed significant biomarkers involved in myocardial injury , myocardial stress , inflammation , neuroendocrine activation , atherosclerotic process , platelet activation , plaque instability , endothelial dysfunction , oxidative stress , and myocardial stretch . out of all these proteins , natriuretic peptides , c - reactive protein ( crp ) , creatine kinase ( ck ) , and cardiac troponin were included as commonly used cardiac biomarkers in acute cardiac care 27 . in a clinical setting , a kit for measuring human salivary crp , another recent cutting - edge technology is oral fluid nanosensor test ( ofnaset ) which provides portable , cheap , accurate , definitive , and quantitative results . besides its intended use in oral cancer , this particular alternative can possibly benefit the point - of - care multiplex detection of salivary biomarkers among ami patients 28 . given the above - mentioned clinical benefits of saliva collection , a patient 's salivary proteome should be very useful in determining heart condition of the patient in order to predict ami . several studies have demonstrated salivary biomarkers associated with ami , as shown in table 1 . by comparing these salivary biomarkers with those in plasma , as elaborated by kossaify et al . 27 , crp , ck , cd40 ligand , cardiac troponin i , cardiac troponin t , some families of interleukin ( il ) , tumour necrosis factor alpha , matrix metalloproteinase ( mmp ) , and myeloperoxidase share similarities with plasma biomarkers which also play significant roles in inflammation and plaque instability ( figure 2 ) . although these discoveries may enlighten the diagnostic utility of salivary proteomes as biomarkers in relation to cvd , none of the salivary biomarkers listed above have been verified to predict the onset of ami . all of these studies , as tabulated in table 1 , were conducted retrospectively after the incidence . on the other hand , prospective studies should be able to alternatively facilitate the researchers to find predictive ami biomarkers . in future , these newly detected salivary biomarkers will conceivably provide an early molecular diagnosis and eventually increase the survival rate of cardiovascular patients as opposed to that of plasma . however , more validation needs to be carried out in order to find robust and discriminatory biomarkers for this disease . this is validated when heart disease and stroke constitute no less than 70 percent of mortality among people with this metabolic disorder 39 , rendering the t2 dm patients to fall into a high - risk group to be later inflicted by ami . t2 dm is initially preceded by insulin resistance and hyperinsulinemia , both of which are related with cardiovascular risk factors such as obesity , hypertension , lipid abnormalities , and endothelial dysfunction 41 . consequently , when the heart loses the ability to pump blood effectively , congestive heart failure occurs which is often contributory to the later onset of ami . 42 discovered that the risk of adults with t2 dm to suffer from cvd is two to four times higher than that of adults without the disorder . between 2010 and 2030 , the number of t2 dm cases is projected to increase by 69% in developing countries and by 20% in developed countries 43 . an upsurge projection of the number of t2 dm adults in developing countries is strongly associated with adoption of western lifestyle due to overconsumption of inexpensive , high - calorie food , and sedentary routine . the resulting potent obesity rapidly escalates the percentages of diabetic incidence in these developing nations which are already stricken under the burden of communicable diseases such as lower respiratory tract infections , acquired immune deficiency syndrome ( aids ) , diarrheal diseases , and others 44 . to date , practitioners in the u.s . have recommended diverse cardiac tests to diagnose coronary heart disease despite the limitation that no individual test is sufficiently reliable 45 . the tests may incorporate electrocardiogram , stress testing , echocardiography , chest x - ray , blood tests , electron - beam computed tomography , coronary angiography , and cardiac catherization . thus far , none of these tests are adequately relevant to the prediction of infarction in the majority of individuals tested , even if their biochemical results are also supplemented to foresee the cardiac event . likewise , in developing countries , the lofty costs of highly - trained practicioners , hospitalizations , and these tests are inherently unfeasible 46 . for this reason , prospective patients from these regions are more likely to forgo the testing process and leave themselves undetected until reaching the chronic stage . due to the reliability and inexpensive nature of saliva collection , salivary diagnosis can be easily aimed at these high - risk patients residing in developing regions . consequently , this method is expected to revamp the current health system and increase the availability of accurate diagnostics in remote and impoverished areas . in a proteomic research of salivary biomarkers , 47 identified 487 unique proteins correlated with t2 dm of which 33% of these had not been previously reported in human saliva . sixty - five proteins were markedly expressed between control and t2 dm samples predominantly involved in regulating metabolism and immune response pathways . even though extensive studies have been conducted by scientists around the globe pertaining to respective diseases of t2 dm and ami , it is still paramount for the practitioners to be able to predict the onset of ami among t2 dm patients due to its strong propensity . a review conducted by syed ikmal et al . suggested ykl-40 , alpha - hydroxybutyrate , soluble cd36 , leptin , resistin , interleukin-18 , retinol binding protein-4 , and chemerin , some of which may be present in saliva , could act as predictors of cardiovascular events in diabetic patients . practically , a t2 dm patient diagnosed with an enhanced level of any clotting proteins or inflammatory markers in saliva should be regularly monitored for an early intervention prior to the onset of ami . in future , the generated database of these biomarkers will provide an instructive reference to assess the susceptibility of t2 dm patients to heart condition , hence reducing morbidity and mortality associated with ami and improving the quality of life of the patients predominantly from developing nations . this is validated when heart disease and stroke constitute no less than 70 percent of mortality among people with this metabolic disorder 39 , rendering the t2 dm patients to fall into a high - risk group to be later inflicted by ami . t2 dm is initially preceded by insulin resistance and hyperinsulinemia , both of which are related with cardiovascular risk factors such as obesity , hypertension , lipid abnormalities , and endothelial dysfunction 41 . consequently , when the heart loses the ability to pump blood effectively , congestive heart failure occurs which is often contributory to the later onset of ami . 42 discovered that the risk of adults with t2 dm to suffer from cvd is two to four times higher than that of adults without the disorder . between 2010 and 2030 , the number of t2 dm cases is projected to increase by 69% in developing countries and by 20% in developed countries 43 . an upsurge projection of the number of t2 dm adults in developing countries is strongly associated with adoption of western lifestyle due to overconsumption of inexpensive , high - calorie food , and sedentary routine . the resulting potent obesity rapidly escalates the percentages of diabetic incidence in these developing nations which are already stricken under the burden of communicable diseases such as lower respiratory tract infections , acquired immune deficiency syndrome ( aids ) , diarrheal diseases , and others 44 . to date , practitioners in the u.s . have recommended diverse cardiac tests to diagnose coronary heart disease despite the limitation that no individual test is sufficiently reliable 45 . the tests may incorporate electrocardiogram , stress testing , echocardiography , chest x - ray , blood tests , electron - beam computed tomography , coronary angiography , and cardiac catherization . thus far , none of these tests are adequately relevant to the prediction of infarction in the majority of individuals tested , even if their biochemical results are also supplemented to foresee the cardiac event . likewise , in developing countries , the lofty costs of highly - trained practicioners , hospitalizations , and these tests are inherently unfeasible 46 . for this reason , prospective patients from these regions are more likely to forgo the testing process and leave themselves undetected until reaching the chronic stage . due to the reliability and inexpensive nature of saliva collection , salivary diagnosis can be easily aimed at these high - risk patients residing in developing regions . consequently , this method is expected to revamp the current health system and increase the availability of accurate diagnostics in remote and impoverished areas . in a proteomic research of salivary biomarkers , 47 identified 487 unique proteins correlated with t2 dm of which 33% of these had not been previously reported in human saliva . sixty - five proteins were markedly expressed between control and t2 dm samples predominantly involved in regulating metabolism and immune response pathways . even though extensive studies have been conducted by scientists around the globe pertaining to respective diseases of t2 dm and ami , it is still paramount for the practitioners to be able to predict the onset of ami among t2 dm patients due to its strong propensity . a review conducted by syed ikmal et al . suggested ykl-40 , alpha - hydroxybutyrate , soluble cd36 , leptin , resistin , interleukin-18 , retinol binding protein-4 , and chemerin , some of which may be present in saliva , could act as predictors of cardiovascular events in diabetic patients . practically , a t2 dm patient diagnosed with an enhanced level of any clotting proteins or inflammatory markers in saliva should be regularly monitored for an early intervention prior to the onset of ami . in future , the generated database of these biomarkers will provide an instructive reference to assess the susceptibility of t2 dm patients to heart condition , hence reducing morbidity and mortality associated with ami and improving the quality of life of the patients predominantly from developing nations . saliva diagnostic tests can be potentially used within a wide range of clinical applications including population - based screening programs , confirmatory diagnosis , risk stratification , prognosis determination , and therapy response monitoring . nevertheless , decreasing the number of false - negative and false - positive test result outcomes should be invariably taken into consideration in order to maintain the quality of patient diagnosis and therapy 49 . in certain cases , some salivary proteomes may be spuriously discovered as biomarkers within a large pool of samples . one reason is certain peptides may have been subjected to modifications resulting to polymorphic isoforms and post translational modifications 50 . for instance , genetic polymorphisms in ca6 gene influence the expression and catalytic activity of human salivary carbonic anhydrase vi 51 . protein degradation in whole saliva also plays a role in the variety of peptides observed . takehara and her co - workers 52 proposed that the n - terminal region of mucin 7 was particularly prone to proteolytic degradation due to varying biological states of the human body . 53 proved that many salivary glycoproteins were associated with age , gender , and immunity . it is also noteworthy that even though passive drooling , paraffin gum , and salivette collection methods cover whole saliva proteome , specific proteins observed are dependent on the collection approach 54 . in addition , older age is often associated with a number of cases related to functional limitations , heart failure , prior coronary disease , and renal insufficiency 55 . as a result , for example , geriatric patients are prone to xerostomia upon medications with anticholinergic properties , dehydration , diabetes , and radiotherapy for head and neck cancer 56 . some alternative sample collection approaches have been evaluated such as passive drool , filter paper , and microsponges but each individual approach has its own advantages and disadvantages in terms of sufficient sample recovery 57 . since there is no concrete resolution , the researcher has to be able to decide the best technique in accordance to the nature of research . it is also important to keep in mind that upon saliva collection , subsequent centrifugation for removing precipitated mucins and cellular contaminants will eliminate some proteins of interest . despite incorporating accurate choice of collection system for easy quantification of volume with ample sample recovery , other essential standardisations of some preanalytical variables should always include precise and standardised collection schedules corresponding to individual hydration state , body posture , lighting , smoking status , circadian and circannual cycles , medications , food and visual stimulation , size of salivary glands , body weight , salivary flow index , physical exercise , alcohol intake , systemic diseases , nutrition , nausea , age , gender , and prevention of sample contamination with blood from oral mucosa and periodontal lesions 58 . even though potential salivary biomarkers related to ami were already discovered , other tests should be able to verify the presence of these proteins before its further applications . in clinical studies , biomarkers must be robust , distinguishable , and validated , particularly with regard to which biomarker is strongly linked to disease onset and progression . to achieve this goal , whole proteome - wide application and target biomarker discovery such as ms - based proteomics approaches will be able to provide an avenue due its higher sensitivity and accuracy in identifying and quantifying proteins than immunological assays 59 . there has been growing interest in diagnosis based on analysis of saliva due its simple and non - invasive collection procedure . since saliva collection is inherently painless , practitioners will be able to diagnose and monitor the patients ' wellbeing more frequently while approaching the sensitivity and specificity of a blood test . plus , the collection is also cheap and safe for both the practitioner and patient . with that , patients especially from developing countries can be diagnosed with minimal costs but great precision . in summary , human salivary proteomes exhibit promising potential as biomarkers to predict the onset of ami particularly among its high - risk group of t2 dm . comprehensive analysis of human salivary proteome through various methods is indeed necessary in order to find the most reproducible markers for later utility in acute cardiac care . it is projected that salivary diagnostics will be conclusive such that fewer diagnostic biomarkers can be determined with immediate results , thus greatly improving the life quality of patients . in the long run , more available saliva collection kits developed by manufacturing companies will be able to aid and eventually provide solid biomarker findings in future . m.aa.r . conceived the idea , performed literature search , and wrote the paper . z.h.a.r . revised the paper from dental aspect . w.a.w.a . revised the paper from clinical aspect .

vitamin k is a fat - soluble vitamin existing mostly in nature and comprises vitamin k1 ( phylloquinone ) , contained mostly in deep green vegetables , and vitamin k2 ( menaquinone ) , found mainly in meats , dairy , fish , and fermented foods . the classic role of vitamin k is as a cofactor for the enzyme gamma - glutamyl carboxylase , which converts glutamate residues to gamma - carboxy glutamate residues on vitamin k - dependent proteins such as coagulation factors ii , vii , ix , and x ; proteins c and s ; osteocalcin ; and matrix gla protein . in recent years , considerable evidence has reported that vitamin k has anticancer , anti - inflammatory , and metabolic disease preventive properties in addition to blood coagulation activities and bone health effects . however , until now optimal vitamin k intake was established as an adequate intake ( ai ) based only on the hepatic requirement for blood clotting factor synthesis . the us institute of medicine has established sufficient daily intakes of vitamin k of 120 g for men and 90 g for women , and the uk recommends vitamin k intakes of 1 g per kg body weight for adults and 2 g per kg body weight for children . the ai of vitamin k is 75 g for adult men and 65 g for adult women in korea . warfarin is a vitamin k antagonist widely prescribed as an oral anticoagulant medicine for the prevention and treatment of ischemic stroke , deep vein thrombosis , cardiovascular disease , and other thrombotic diseases . warfarin - based anticoagulation therapies seek to maintain a patient 's international normalized ratio ( inr ) within an appropriate treatment range between 2.0 and 3.0 . if one 's inr increases , his or her thrombosis risk decreases , but the risk of bleeding and other complications could increase . on the other hand , if inr decreases , one 's bleeding risk decreases but thrombosis risk increases . therefore , maintenance of appropriate inr levels is important for people in taking warfarin , and assessments of dietary vitamin k intake are critical to stabilize anticoagulation therapy . various dietary assessment tools can be used in clinical practice and research to estimate vitamin k intake . the food record method has been widely used ; however , it demands a great deal of time and expense to analyze data . food frequency questionnaires ( ffq ) are cost - effective and reliable methods of estimating usual dietary intakes in relation to health concerns . some ffqs have been used to assess dietary vitamin k intake , but there is no tool to assess vitamin k intake in korean individuals . due to differences in dietary habits according to population , ethnically specific ffqs could be used in nutrition surveys . each questionnaire should be validated and compared to a gold standard diet analysis technique for the specific population being studied . thus , the purpose of this study was to validate an ffq specifically developed to assess vitamin k intake in korean adults . data from the fifth korean national health and nutrition examination survey ( knhanes v ) of 12,546 adults aged 1964 years were utilized to develop the vitamin k - specific ffq . dietary intake was assessed by the 24-hour dietary recall method , and vitamin k contents were analyzed using the 475 food databases of computer aided nutritional analysis program ( korean nutrition society , seoul , korea ) and 76 united states department of agriculture food databases . to select vitamin k food source items to be included in the ffq , a contribution analysis was used to determine the percent of vitamin k intake contributed by each food item . food items with larger variations in vitamin k intakes between individuals were analyzed via regression using daily vitamin k intake as the dependent variable and vitamin k intakes from each food as the independent variables for each research participant . the regression analyses also included foods with the accumulated total of model explanation power of 90% from the stepwise regression analysis . the rank of twenty - five food items that contributed cumulatively up to 90% of the total vitamin k intake were selected to develop the ffq . the present study was approved by the clinical test deliberation commission of institutional review board ( irb ) , wonkwang university ( wkirb-201308-sb-003 , wkirb-201406-sb-038 ) , and written informed consent was obtained from each subject . this research was performed from september to december 2013 and from may to june 2014 . a total of 54 participants recruited voluntarily for the validity and reliability analyses study were free from diseases and aged between 19 and 64 years . for validity verification of the developed ffq , we collected 5-day food records ( including one weekend day ) ( 5dr ) . on the first visit to the subjects , a trained dietitian recorded the first day food record through face - to - face interviews using the 24-hour recall method . participants were instructed to record their normal meals and to exclude dining - out , parties , or other special event meals from the food journals . a total of 6 subjects were excluded from the final validation analysis , as they had incomplete food records . for reliability verification of the ffq , we compared each participant 's first and second ffq sets within an interval of about 6 months . during ffq administration , subjects were asked about the quantity and frequency of consumed food items . the reference quantities of food items were set based on one serving size of the dietary reference intakes for koreans ( kdris ) , and relevant photographs were presented to identify serving size . for intake amounts , three different levels of portion size , such as half , same , and 1.5-times larger than the reference amount , were presented as options . each food frequency was categorized into 9 items : hardly eat it , once a month , 23 times a month , once a week , 23 times a week , 46 times a week , once a day , two times a day , and three times a day . vitamin k intake from each food item was computed based on the portion size , frequency of consumption , and amount per serving . results are reported as means ( sd ) or as percentages and 95% confidence intervals ( ci ) . vitamin k intake data were log - transformed because they were not normally distributed from both 5dr and ffq . to examine the validity and reliability of the ffq , the student 's paired t - test and pearson 's correlation were performed on vitamin k intakes in the ffq versus the dietary records and ffq1 versus ffq2 , respectively . we calculated an adjusted de - attenuated correlation coefficient between ffq and 5dr to reduce random error in intra - individual variability . a cross - classification analysis was performed to compare the categorization of vitamin k intake into quartiles by the food records and ffq . the overall percentages of subjects classified into the same , adjacent , or extreme quartiles were determined , and the coincidence levels between the two methods were calculated by cohen 's kappa coefficient . a bland - altman plot was used to assess measurement error of the log - transformed vitamin k intake levels between the 5dr and ffq . all analyses were carried out using spss 19.0 for windows ( spss , chicago , ca , usa ) and the statistical significance level was set at p < 0.05 . data from the fifth korean national health and nutrition examination survey ( knhanes v ) of 12,546 adults aged 1964 years were utilized to develop the vitamin k - specific ffq . dietary intake was assessed by the 24-hour dietary recall method , and vitamin k contents were analyzed using the 475 food databases of computer aided nutritional analysis program ( korean nutrition society , seoul , korea ) and 76 united states department of agriculture food databases . to select vitamin k food source items to be included in the ffq , a contribution analysis was used to determine the percent of vitamin k intake contributed by each food item . food items with larger variations in vitamin k intakes between individuals were analyzed via regression using daily vitamin k intake as the dependent variable and vitamin k intakes from each food as the independent variables for each research participant . the regression analyses also included foods with the accumulated total of model explanation power of 90% from the stepwise regression analysis . the rank of twenty - five food items that contributed cumulatively up to 90% of the total vitamin k intake were selected to develop the ffq . the present study was approved by the clinical test deliberation commission of institutional review board ( irb ) , wonkwang university ( wkirb-201308-sb-003 , wkirb-201406-sb-038 ) , and written informed consent was obtained from each subject . this research was performed from september to december 2013 and from may to june 2014 . a total of 54 participants recruited voluntarily for the validity and reliability analyses study were free from diseases and aged between 19 and 64 years . for validity verification of the developed ffq , we collected 5-day food records ( including one weekend day ) ( 5dr ) . on the first visit to the subjects , a trained dietitian recorded the first day food record through face - to - face interviews using the 24-hour recall method . participants were instructed to record their normal meals and to exclude dining - out , parties , or other special event meals from the food journals . a total of 6 subjects were excluded from the final validation analysis , as they had incomplete food records . for reliability verification of the ffq , we compared each participant 's first and second ffq sets within an interval of about 6 months . during ffq administration , subjects were asked about the quantity and frequency of consumed food items . the reference quantities of food items were set based on one serving size of the dietary reference intakes for koreans ( kdris ) , and relevant photographs were presented to identify serving size . for intake amounts , three different levels of portion size , such as half , same , and 1.5-times larger than the reference amount , were presented as options . each food frequency was categorized into 9 items : hardly eat it , once a month , 23 times a month , once a week , 23 times a week , 46 times a week , once a day , two times a day , and three times a day . vitamin k intake from each food item was computed based on the portion size , frequency of consumption , and amount per serving . results are reported as means ( sd ) or as percentages and 95% confidence intervals ( ci ) . vitamin k intake data were log - transformed because they were not normally distributed from both 5dr and ffq . to examine the validity and reliability of the ffq , the student 's paired t - test and pearson 's correlation were performed on vitamin k intakes in the ffq versus the dietary records and ffq1 versus ffq2 , respectively . we calculated an adjusted de - attenuated correlation coefficient between ffq and 5dr to reduce random error in intra - individual variability . a cross - classification analysis was performed to compare the categorization of vitamin k intake into quartiles by the food records and ffq . the overall percentages of subjects classified into the same , adjacent , or extreme quartiles were determined , and the coincidence levels between the two methods were calculated by cohen 's kappa coefficient . a bland - altman plot was used to assess measurement error of the log - transformed vitamin k intake levels between the 5dr and ffq . all analyses were carried out using spss 19.0 for windows ( spss , chicago , ca , usa ) and the statistical significance level was set at p < 0.05 . the list of major vitamin k contributing food sources extracted from knhanes v to develop the vitamin k - specific ffq is shown in table 1 . foods with the highest contribution to total vitamin k intake were spinach ( 15.00% ) , followed by kimchi ( 10.61% ) , radish leaves ( 7.20% ) , sesame leaves ( 5.89% ) , and laver ( 4.38% ) ( data not shown ) . women and men with a mean age ( sd ) of 35.43 years ( 13.46 years ) participated in the study . of the 54 subjects who agreed to participate in this study , 48 ( 88% ) subjects completed 5drs . mean , sd , and median daily vitamin k intakes for ffq1 and ffq2 and the 5dr are shown in table 2 . comparing ffq1 to ffq2 , vitamin k intakes were 226.69 172.65 g / day and 194.51 152.49 g / day , respectively , with no significant difference between the results of the two questionnaires . there were no significant differences in vitamin k intakes derived from the ffq1and 5dr ( p > 0.05 ) . table 3 describes the correlation of vitamin k intake between the ffq and 5dr ( validity ) and the two ffqs ( reliability ) . the pearson 's correlation coefficient between the ffq and 5dr was 0.652 , and the calorie - adjusted de - attenuated correlation was 0.740 , indicating a strong correlation . the correlation between ffq1 and ffq2 was 0.560 , representing a statistically significant difference ( p < 0.001 ) . the coincidence or proximity ratio of vitamin k intake between the ffq and the 5dr was 83.6% , and the discordance ratio was 6.0% . following the categorization of vitamin k intake from ffq1 and ffq2 into quartiles , and the coincidence or proximity ratio of vitamin k intake was 83.4 , the discordance ratio was 5.6 , and cohen 's kappa coefficient was 0.333 . indicated significant correlation between ffq1 and 5dr , p < 0.001 . indicated significant correlation between ffq1 and ffq2 , p < 0.001 . the coincidence of vitamin k intakes estimated under the log transformation based on the ffq and the 5dr was analyzed with a bland - altman plot , and the outcomes are described in figure 1 . the log transformed mean difference between 5dr and ffq methods was 0.002 , and limits of agreement being from -0.20 to 0.20 . bland - altman plot of the difference in vitamin k intake as assessed by ffq and the 5dr ( n = 48 ) , values are log - transformed . the present study was performed to develop a ffq specifically designed to assess dietary vitamin k intake among the general population in korean adults . a 25-item ffq , which reflects the food intake patterns of koreans , was developed and tested for validity and reliability . our findings showed that a total of 25 food items contributes to 90% of vitamin k intake in korean adults and vegetables account for a considerable percentage of vitamin k intake , and this phenomenon is likely due to the food sources and eating habits of korean people . in previous studies , the vitamin k intakes analysis by ffq were significantly higher than those of 5dr and the difference of intakes between two methods were about 93 g / day146 g / day . in our study , mean vitamin k intakes from the ffq and 5dr were not significantly different ; this result represents an improvement over previous studies in which the difference in vitamin k intake between the ffq and the diet record method was relatively high . a generally acceptable correlation coefficient between an ffq and the gold standard method is between 0.5 and 0.7 . the correlation coefficient obtained from vitamin k intakes assessed by our ffq and 5dr ( r = 0.652 ) was comparable with results of the previous validation studies , where vitamin k intake from ffqs was moderately correlated to that from food records ( r = 0.530.83 ) . therefore , this ffq can be considered for estimating an individual 's actual vitamin k intake . generally , vitamin k intakes measured by the ffq were higher than those estimated by food records . a possible reason for this discrepancy is that subjects might have included vitamin k - rich food items when responding to the ffq but did not eat those foods during the diet recording periods . another reason might be that the answers to the ffq are fixed as multiple frequencies at presented intervals . in clinical settings , the efficacy of an ffq would be dependent on good relative agreement in a cross - classification of nutrient intake . our ffq showed a favorable ability to classify individuals into quartiles of vitamin k intake , with 83.6% of subjects being correctly classified into the same or adjacent quartiles compared to 5dr , and only 6.0% of subjects being grossly misclassified . the cohen 's kappa was 0.519 , which was similar to that in an earlier trial . a high degree of cross - classification coincidence was found between the two methods in our study . developed vitamin k - specific ffq consists of only 25 items , and then it takes only 10 to 15 minutes to complete . therefore , the ffq can be used as a clinical tool for the rapid assessment of vitamin k for dietary intervention . from a reproducibility perspective , the questionnaire should be administered at two points with reasonable test interval to the same group of people . however , only less than 50% of reported papers regarding the ffq development had been performed the test - retest reliability . when study design was set , the reasonable time interval between repeated test performances should be contemplated . if the interval was short , the memory of a subject may affect his or her responses on the second ffq , and if the interval was long , the subject 's dietary pattern may have changed thereby reducing objective measurements of reliability . in our study , due to relatively short ffq ( 25 items ) , the time interval between ffq1 and ffq2 was set at 6 months to limit errors . our study showed good reproducibility of the ffq , with a pearson 's correlation coefficient of 0.560 . cross - classification also showed a fair agreement , because 83.4% of subjects were classified into the same or adjacent quartile of intake for vitamin k. the present study has several potential weaknesses . first , the utility of the ffq in the general population must be considered due to the selection of subjects with similar ethnic backgrounds as well as the relatively small sample size , which limit generalizability . second , the selection of the reference method that is mostly close to gold standard value is the key component of the validation process . it was suggested that recording more than three days is proper for a reference method in validation study for vitamin intake assessment . in our study , 5dr were used as reference method to fulfill a sufficient length to reliably assess vitamin k intake by considering intra- and inter - personal variation . however , a large number of recording periods may result in lowering accuracy and changes in eating habits . to increase the validity of the results , further studies may require the inclusion of biomarker reference methods , such as serum vitamin k and percentage of uncarboxylated osteocalcin levels . the vitamin k - specific ffq presented in this study showed good relative correlation with dietary records , and therefore , it is a valid tool for assessing individuals ' vitamin k intakes . this new ffq represents a substantially lower burden to subjects than food records and will be fruitfully used as a practical tool in the research field and disease prevention settings . the vitamin k - specific ffq could also be clinically useful to identify adults with the greatest need of nutritional counseling , such as anticoagulation therapy patients with uncontrolled vitamin k intake or those at the highest risk of vitamin k inadequacy .

ehlers danlos syndrome ( eds ) is a group of inherited connective tissue disorders caused by a defect in collagen synthesis . the clinical presentation varies widely based on the type of eds.1 joints , skin and blood vessels are typically affected , the latter being the problem in the case presented in this paper . some types of eds lead to fragile blood vessels resulting in cystic medial necrosis with a high tendency to aneurysms at any location of the vascular system.2 we report an interdisciplinary treatment approach in a middle - aged man with a continuously growing giant extracranial aneurysm of the right internal carotid artery ( ica ) caused by type iv eds . a 42-year - old man with eds type iv was admitted to our department with a rapidly progressive cervical pulsatile and painful swelling on the right side ( figure 1 ) . apart from the swelling , a neurological examination also showed right - sided palsy of the hypoglossal nerve . the patient 's history indicated that , in 1999 , direct revascularization using a vessel graft to treat an extracranial giant aneurysm of the left ica had failed due to technical problems in performing an adequate anastomosis causing left hemispheric ischemia . neurological impairment such as right - sided hemiparesis or aphasia was not seen at the current presentation . ( a ) frontal and ( b ) lateral views showing a large pulsatile swelling on the right side of the neck in a patient with ehlers danlos syndrome with a giant extracranial internal carotid artery aneurysm . cerebral digital subtraction angiography ( dsa ) showed a giant aneurysm of the right ica ( c1 segment ) measuring 1077 cm , extending from the carotid bifurcation to the base of the skull ( figure 2 ) . a largely dilated basal artery and left posterior communicating branch indicating compensatory supply to the left hemisphere was also demonstrated . a doppler - controlled balloon occlusion test of the right ica revealed a moderate insufficiency of the middle cerebral artery ( mca ) territory and additional collateralization of the right hemisphere via the ophthalmic branches . mri demonstrated ischemic lesions of the left hemisphere as a result of the previous occlusion of the left ica . angiographic study prior to treatment showing the contrast in the giant extracranial internal carotid artery aneurysm measuring 1077 cm . the radiological findings were then thoroughly discussed in an interdisciplinary conference including neuroradiological , neurological and neurosurgical colleagues in order to achieve a low - risk and adequate treatment of the growing aneurysm of the right ica . the agreed strategy consisted of securing right cerebral perfusion via flow redirection through a double - barrel extracranial intracranial bypass followed by surgical trapping of the aneurysm . the double - barrel bypass was successfully placed from the superficial temporal artery to two m4 cortical branches of the right mca ( figure 3 ) . , a temporary ica occlusion test was conducted using an eclipse balloon in order to confirm sufficient contrast perfusion of the right hemisphere through the previously placed tandem bypass ( figure 4 ) . the occlusion test showed good flow through the bypass and additional ophthalmic collateral filling , so we were able to proceed with the permanent elimination of the aneurysm from the circulation . surgical trapping of the aneurysm failed due to the risk of sacrificing the external carotid artery . two separate sessions were needed to occlude both the distal and proximal segments of the giant aneurysm . a stent was then placed in the right external carotid artery to secure its patency and , from there , stent - supported coiling was performed to occlude the proximal segment ( figure 6 ) . secure proximal placement of the coils was not possible in a single - catheter technique owing to the high flow turbulence within the aneurysm sac . by using a double - catheter method , two coils could be placed simultaneously directly outside the stent wall facing the proximal segment of the extracranial ica . immediately after the procedure the patient had a transient ischemic attack with latent left - sided hemiparesis which regressed within 48 h. ischemic lesions were excluded in post - procedural non - enhancement ct scans . the bulging skin collapsed instantly , reducing the large visible swelling on the patient 's neck . the patient recovered rapidly from the individual procedures without any additional impairment and was discharged in an unaltered clinical / neurological condition . angiographic study demonstrating patent superficial temporal artery angiographic study showing coiling of the distal segment of the right internal carotid artery ( lateral view ) . follow - up at 2 months revealed a good clinical result without any deficits with slight recurrence of the swelling on the right side of the neck . ct and ct angiography showed a hypodense non - enhancing filling of the remaining aneurysm sac . density measurements indicated the filling to be serous fluid which was safely drained via ct - assisted puncture . again the swelling collapsed immediately , leaving the patient with a good cosmetic result ( figure 7 ) . the patient died a year later due to a ruptured aneurysm of the splenic artery . photograph showing reduction of the swelling on the right side of the neck after drainage of the residual hematoma at clinical follow - up . cerebral digital subtraction angiography ( dsa ) showed a giant aneurysm of the right ica ( c1 segment ) measuring 1077 cm , extending from the carotid bifurcation to the base of the skull ( figure 2 ) . a largely dilated basal artery and left posterior communicating branch indicating compensatory supply to the left hemisphere was also demonstrated . a doppler - controlled balloon occlusion test of the right ica revealed a moderate insufficiency of the middle cerebral artery ( mca ) territory and additional collateralization of the right hemisphere via the ophthalmic branches . mri demonstrated ischemic lesions of the left hemisphere as a result of the previous occlusion of the left ica . angiographic study prior to treatment showing the contrast in the giant extracranial internal carotid artery aneurysm measuring 1077 cm . the radiological findings were then thoroughly discussed in an interdisciplinary conference including neuroradiological , neurological and neurosurgical colleagues in order to achieve a low - risk and adequate treatment of the growing aneurysm of the right ica . the agreed strategy consisted of securing right cerebral perfusion via flow redirection through a double - barrel extracranial intracranial bypass followed by surgical trapping of the aneurysm . the double - barrel bypass was successfully placed from the superficial temporal artery to two m4 cortical branches of the right mca ( figure 3 ) . , a temporary ica occlusion test was conducted using an eclipse balloon in order to confirm sufficient contrast perfusion of the right hemisphere through the previously placed tandem bypass ( figure 4 ) . the occlusion test showed good flow through the bypass and additional ophthalmic collateral filling , so we were able to proceed with the permanent elimination of the aneurysm from the circulation . surgical trapping of the aneurysm failed due to the risk of sacrificing the external carotid artery . two separate sessions were needed to occlude both the distal and proximal segments of the giant aneurysm . the distal segment was successfully occluded with platinum spiral coils ( figure 5 ) . a stent was then placed in the right external carotid artery to secure its patency and , from there , stent - supported coiling was performed to occlude the proximal segment ( figure 6 ) . secure proximal placement of the coils was not possible in a single - catheter technique owing to the high flow turbulence within the aneurysm sac . by using a double - catheter method , two coils could be placed simultaneously directly outside the stent wall facing the proximal segment of the extracranial ica . immediately after the procedure the patient had a transient ischemic attack with latent left - sided hemiparesis which regressed within 48 h. ischemic lesions were excluded in post - procedural non - enhancement ct scans . the bulging skin collapsed instantly , reducing the large visible swelling on the patient 's neck . the patient recovered rapidly from the individual procedures without any additional impairment and was discharged in an unaltered clinical / neurological condition . angiographic study demonstrating patent superficial temporal artery ( a ) internal carotid artery ( ica ) occlusion test during cerebral angiography . ( b ) anteroposterior view of patent contrast via extracranial intracranial bypass during temporary ica occlusion . angiographic study showing coiling of the distal segment of the right internal carotid artery ( lateral view ) . follow - up at 2 months revealed a good clinical result without any deficits with slight recurrence of the swelling on the right side of the neck . ct and ct angiography showed a hypodense non - enhancing filling of the remaining aneurysm sac . density measurements indicated the filling to be serous fluid which was safely drained via ct - assisted puncture . again the swelling collapsed immediately , leaving the patient with a good cosmetic result ( figure 7 ) . the patient died a year later due to a ruptured aneurysm of the splenic artery . photograph showing reduction of the swelling on the right side of the neck after drainage of the residual hematoma at clinical follow - up . extracranial giant ica aneurysms are rare lesions and treatment is often accompanied by a high risk of morbidity and mortality.3 4 surgical resection with reconstruction is considered to be the best therapeutic option for these extracranial vascular pathologies.4 5 in the case presented here , surgical treatment had been attempted for a similar lesion of the left eica but failed due to the dimensions of the lesion and led to ischemic stroke . an alternative treatment method for the abovementioned lesion of the right eica in this patient was therefore sought . a well - accepted approach for giant and complex intracranial ica aneurysms is the combination of flow redirection followed by definite exclusion of the aneurysm from the circulation.68 a transcranial doppler - controlled balloon occlusion test performed during the initial diagnostic angiography revealed moderate insufficiency of the right mca territory , so a sta mca bypass was seen as adequate in this case . after interdisciplinary consultation , we decided to apply this combined approach to this extracranial ica aneurysm in order to prevent further ischemic events . exclusion from the circulation can be achieved either through surgical trapping or by endovascular occlusion.6 9 10 both methods are reported to be equally acceptable . extracranial intracranial bypass and aneurysm exclusion can be carried out as a single - stage or two - stage procedure.11 if aneurysm exclusion is performed as surgical trapping , the single - stage procedure under a single anesthesia is the more common protocol.11 to avoid the increased risk of postoperative bleeding , a two - stage procedure with anticoagulation after the endovascular occlusion of the aneurysm should be considered . given the fact that the patient had already had an ischemic stroke of the left hemisphere due to ica ligation after failed surgery , we decided to apply the aforementioned standard methods for intracranial ica aneurysms to this giant aneurysm of the right eica . at first we chose a single - stage strategy in which we planned to place an extracranial trapping failed again due to the extreme dimensions of the aneurysm at the bifurcation , making it impossible to trap the aneurysm and not the external ica . as mentioned above , endovascular occlusion is a well - accepted treatment method in combination with bypass for intracranial aneurysms which has not previously been used in a patient with eds with an extracranial ica aneurysm . occlusion was successfully performed in two separate endovascular procedures ( distal coiling followed by proximal stent - assisted coiling ) . there are few reports of the use of this method for the treatment of extracranial ica aneurysms in the literature , and this is the first report of its use in a patient with type iv eds . a combination of flow redirection using cerebral bypass surgery and endovascular occlusion is a rational treatment method for intracranial aneurysms and also seems to be a valid option for high - risk extracranial giant ica aneurysms , even in a patient with eds .

the philippine archipelago , consisting of about 7107 islands , is a center of endemicity and biodiversity where a large number of endemic plants have been reported to exhibit medicinal properties . previous ethnobotanical studies of medicinal plants in this country were conducted in the archipelagos of batanes and visayas and the southern island of mindanao . there are limited ethnobotanical studies conducted in luzon , the largest island in the philippines . an online survey of ethnobotanical studies conducted in the philippines showed that up to date , no documentations have been done on the anecdotal therapeutic uses of plants in pangasinan , a province which is situated in the central eastern part of luzon . pangasinan is one of the most populated provinces in the philippine archipelago , with a population of about 3 million as of the 2011 census and a total area of 5369 km . it is located in the northern ilocos region of the island of luzon [ figure 1 ] . the predominant climate is a wet season from june to october followed by a dry season from november to may . the average monthly temperature is 27.91c with the highest occurring in may and the lowest in january . about 95% of the people embrace christianity with minorities belonging mainly to islam , hinduism , and buddhism . comprising the province are six political districts which have different dialects , cultures , and traditions , including the self - care use of medicinal plants which have been passed to them from previous generations . map of the philippines showing the province of pangasinan and the three surveyed areas the asthma weeds plant , euphorbia hirta , also known as chamaesyce hirta ( l. ) millsp . and identified by its vernacular names tawa - tawa , is a hairy herb grown in open grasslands , roadsides , and pathways and with a pantropic distribution . considered this indigenous plant to be the most popular folkloric treatment for dengue in the philippines . demonstrated how the lyophilized leaf decoction of e. hirta augmented platelet count in thrombocytopenic rats . practitioners of traditional medicines believe that tawa - tawa leaf decoction can reverse viral infection and prevent the fever from moving into critical stages . water was effective in increasing platelet count and improving the symptoms in dengue patients . in a cohort study , paredes et al . reported that the beneficial effect of e. hirta in dengue patients depends on the degree of changes in platelet levels . despite these developments , there is no concrete evidence that reveals the effectiveness of this plant in humans infected with the dengue virus . since little information is known about the purported therapeutic claim of e. hirta against dengue and other illnesses , this study seeked to assess , quantify , qualify and compare the ethnobotanical uses of e. hirta from three selected indigenous communities in the province of pangasinan . three different indigenous areas in pangasinan [ figure 1 ] where e. hirta was widely propagated were selected as sampling sites on the basis of the existence of traditional herbalists ( herbolarios ) who depend on medicinal plants to treat illnesses due to the absence of modern health care facilities , clinics and pharmacies as well as the lack of access to electricity . most plants were seen growing wild in places adjacent to homes , making collection possible , instead of cultivation . it must be considered that the therapeutic uses of plants and the synthesis of bioactive constituents they contain are most optimized when these plants grow more slowly , particularly in the wild . the indigenous communities surveyed are : the island municipality of anda ( surveyed on april 2015 ) at the northwestern tip of pangasinan , which is characterized by shallow white sand coastal lines with lush forested and semi - forested vegetations that spread on plains that are traversed by streams . the locals speak bolinao , a minority dialect , and ilocano and thrives on farming and deep sea fishing;mt . balungao ( surveyed on april 2015 ) , a dormant volcano at the southeastern boundary of pangasinan with nueva ecija with secondary dipterocap forests , hot springs and a mossy peak . the locals who speak mostly ilocano depend on fruit crops , farming , and logging ; and , mt . colisao municipal park ( surveyed on may and june 2015 ) in the municipality of san fabian , described by the shortage of water supply and the presence of denuded hilly forests brought about by illegal mining and logging , and a variety of both wild and cultivated plants . the island municipality of anda ( surveyed on april 2015 ) at the northwestern tip of pangasinan , which is characterized by shallow white sand coastal lines with lush forested and semi - forested vegetations that spread on plains that are traversed by streams . the locals speak bolinao , a minority dialect , and ilocano and thrives on farming and deep sea fishing ; mt . balungao ( surveyed on april 2015 ) , a dormant volcano at the southeastern boundary of pangasinan with nueva ecija with secondary dipterocap forests , hot springs and a mossy peak . the locals who speak mostly ilocano depend on fruit crops , farming , and logging ; and , mt . colisao municipal park ( surveyed on may and june 2015 ) in the municipality of san fabian , described by the shortage of water supply and the presence of denuded hilly forests brought about by illegal mining and logging , and a variety of both wild and cultivated plants . the survey , data gathering and analysis was conducted from april to june of 2015 . the specimens in the field were pressed in between newspapers and treated with denatured alcohol . in the laboratory , the specimens were soaked in 100 ml of 95% ethanol - phenol ( 60:40 ) and subsequently oven - dried . herbarium specimens and photographs of the plants in their natural habitats were submitted to the philippine national herbarium for authentication . each specimen of e. hirta collected from the three sampling sites surveyed , together with their photographs in their natural habits , was shown to each respondent . a validated semi - structured questionnaire was used to determine the number of use report for each specimen collected . the questionnaire and the objectives of the study were relayed to each respondent , in either the pangasinense or ilocano dialect , whichever was appropriate . information on the manner by which plant parts were prepared into dosage forms before administration was obtained per use report corresponding to symptoms of dengue . use reports pertaining to symptoms of dengue were properly documented and tallied while use reports pertaining to other symptoms were tallied only . for each of the 3 indigenous areas surveyed , an interview of municipal health officers in the poblacion or town centers of the municipalities of anda , balungao , and san fabian was also conducted to know the prevalence of patients infected with dengue from january to march 2015 . to ensure no issues on the infringement of biodiversity rights arise , the local government in all areas surveyed , in collaboration with the department of environment and natural resources , are consulted on the details of the sampling and collection of plant materials and the interview of local respondents on the use reports of these plants . the major uses agreement ( mua ) was determined as the ratio between the number of informants that independently cited e. hirta for a single use report pertaining to symptoms of dengue and the total number of informants [ n = 76 , table 1 ] that mentioned the plant for any use reports . a correction factor ( cf ) was applied to calculate the corrected mua ( cmua ) , given by the formula : cmua = mua the cf is the ratio between the number of informants citing e. hirta for any use report pertaining to a symptom of dengue and the highest number of informants citing the most employed medicinal plant . in this study , vernonia cinerea l. was the most employed medicinal plant with 57 out of 82 informants . tally of use reports of e. hirta for any symptoms in three indigenous communities in pangasinan the fidelity level ( fl ) of e. hirta in each of the 3 areas surveyed was computed , thus : % fl = ip / iu100 , where ip is the number of informants who independently suggested a use report pertaining to symptoms of dengue and iu is the number of informants who mentioned the same plant for any use report [ n = 76 , table 1 ] . three different indigenous areas in pangasinan [ figure 1 ] where e. hirta was widely propagated were selected as sampling sites on the basis of the existence of traditional herbalists ( herbolarios ) who depend on medicinal plants to treat illnesses due to the absence of modern health care facilities , clinics and pharmacies as well as the lack of access to electricity . most plants were seen growing wild in places adjacent to homes , making collection possible , instead of cultivation . it must be considered that the therapeutic uses of plants and the synthesis of bioactive constituents they contain are most optimized when these plants grow more slowly , particularly in the wild . the indigenous communities surveyed are : the island municipality of anda ( surveyed on april 2015 ) at the northwestern tip of pangasinan , which is characterized by shallow white sand coastal lines with lush forested and semi - forested vegetations that spread on plains that are traversed by streams . the locals speak bolinao , a minority dialect , and ilocano and thrives on farming and deep sea fishing;mt . balungao ( surveyed on april 2015 ) , a dormant volcano at the southeastern boundary of pangasinan with nueva ecija with secondary dipterocap forests , hot springs and a mossy peak . the locals who speak mostly ilocano depend on fruit crops , farming , and logging ; and , mt . colisao municipal park ( surveyed on may and june 2015 ) in the municipality of san fabian , described by the shortage of water supply and the presence of denuded hilly forests brought about by illegal mining and logging , and a variety of both wild and cultivated plants . the island municipality of anda ( surveyed on april 2015 ) at the northwestern tip of pangasinan , which is characterized by shallow white sand coastal lines with lush forested and semi - forested vegetations that spread on plains that are traversed by streams . the locals speak bolinao , a minority dialect , and ilocano and thrives on farming and deep sea fishing ; mt . balungao ( surveyed on april 2015 ) , a dormant volcano at the southeastern boundary of pangasinan with nueva ecija with secondary dipterocap forests , hot springs and a mossy peak . the locals who speak mostly ilocano depend on fruit crops , farming , and logging ; and , mt . colisao municipal park ( surveyed on may and june 2015 ) in the municipality of san fabian , described by the shortage of water supply and the presence of denuded hilly forests brought about by illegal mining and logging , and a variety of both wild and cultivated plants . the survey , data gathering and analysis was conducted from april to june of 2015 . the specimens in the field were pressed in between newspapers and treated with denatured alcohol . in the laboratory , the specimens were soaked in 100 ml of 95% ethanol - phenol ( 60:40 ) and subsequently oven - dried . herbarium specimens and photographs of the plants in their natural habitats were submitted to the philippine national herbarium for authentication . each specimen of e. hirta collected from the three sampling sites surveyed , together with their photographs in their natural habits , was shown to each respondent . a validated semi - structured questionnaire was used to determine the number of use report for each specimen collected . the questionnaire and the objectives of the study were relayed to each respondent , in either the pangasinense or ilocano dialect , whichever was appropriate . information on the manner by which plant parts were prepared into dosage forms before administration was obtained per use report corresponding to symptoms of dengue . use reports pertaining to symptoms of dengue were properly documented and tallied while use reports pertaining to other symptoms were tallied only . for each of the 3 indigenous areas surveyed , at least 20 respondents aged 20 - 80 were randomly selected . an interview of municipal health officers in the poblacion or town centers of the municipalities of anda , balungao , and san fabian was also conducted to know the prevalence of patients infected with dengue from january to march 2015 . to ensure no issues on the infringement of biodiversity rights arise , the local government in all areas surveyed , in collaboration with the department of environment and natural resources , are consulted on the details of the sampling and collection of plant materials and the interview of local respondents on the use reports of these plants . the major uses agreement ( mua ) was determined as the ratio between the number of informants that independently cited e. hirta for a single use report pertaining to symptoms of dengue and the total number of informants [ n = 76 , table 1 ] that mentioned the plant for any use reports . a correction factor ( cf ) was applied to calculate the corrected mua ( cmua ) , given by the formula : cmua = mua the cf is the ratio between the number of informants citing e. hirta for any use report pertaining to a symptom of dengue and the highest number of informants citing the most employed medicinal plant . in this study , vernonia cinerea l. was the most employed medicinal plant with 57 out of 82 informants . tally of use reports of e. hirta for any symptoms in three indigenous communities in pangasinan the fidelity level ( fl ) of e. hirta in each of the 3 areas surveyed was computed , thus : % fl = ip / iu100 , where ip is the number of informants who independently suggested a use report pertaining to symptoms of dengue and iu is the number of informants who mentioned the same plant for any use report [ n = 76 , table 1 ] . tables 2 - 4 show the demographic data of the 82 informants in this study according to sex , age group , their distributions among the three indigenous areas surveyed and educational attainment . distribution of informants according to area surveyed demographic data according to age group and sex educational attainment of informants the questionnaires were evenly distributed to each of the 3 sampling sites surveyed , but more allocations were given to female respondents because females are perceived to have higher knowledge over males in the traditional self - care uses of medicinal plants , a tradition that could have been passed to them from previous generations . in this study , significantly higher number of use reports was generated by female respondents compared to their male counterparts ( p < 0.0001 , chi - square ) . the majority of use reports pertaining to symptoms of dengue were given by respondents aged 60 - 80 , a significantly higher number as compared to younger counterparts ( 227 vs. 79 , p < 0.0001 , chi - square ) . this is because the respondents within this age bracket are more experienced than younger informants in the self - care uses of medicinal plants for dengue based on anecdotal information that is consistent with the rapid rise in the epidemics of this disease for the previous years . the use of medicinal plants also increased with increasing age to imply that the high dependence of these three indigenous communities on plants to treat illnesses is still consistent with time . elementary and secondary schools were found to have important roles in the dissemination of information on the self - care uses of medicinal plants in the three areas surveyed . respondents who finished primary and secondary schoolings generated higher use reports related to dengue symptoms ( p < 0.001 , chi - square ) . interviews with teachers in these areas reveal that the study of medicinal plants on the scientific and anecdotal points of view has been incorporated in elementary and high school science curriculums . there is a low number of respondents with higher educational attainment because most of the informants surveyed have lower income compared to inhabitants in more populated municipalities , in addition to the great distances of colleges which are mostly located in pangasinan city propers findings showing similarities to the observations of abe and ohtani among ivatans in batan island north of luzon . table 1 shows the number of informants interviewed and the frequency of use reports relative to any symptom in the 3 sampling sites surveyed . colisao cited e. hirta for at least 5 symptoms of dengue as well as 7 other symptoms . in mt . balungao , 4 respondents cited the same plant for use in 7 symptoms of dengue in addition to 10 other symptoms . the relatively high number of use reports for any symptoms generated in anda island ( n = 197 ; p < 0.001 ) can be due to a larger population that is dependent on medicinal plants in treating diseases , the higher density and diversity of plants available for use as medicinal agents and the high prevalence of acute diseases , including dengue . furthermore , the absence of modern health facilities , clinics , hospitals and pharmacies in anda island may explain for the high dependence of its residents on medicinal plants . an interview of municipal health officers within the vicinities of the 3 sampling sites surveyed shows a high prevalence of dengue infections in anda island from january to march of 2015 , with 36 reported cases , including 2 casualties that affected mostly children ages 2 years and above . within this period , 17 and 15 cases of dengue the high prevalence of dengue infection in the 3 sampling sites surveyed can be due to the lack of proper hygiene and sanitation in possible breeding places of mosquito vectors . from january 1 to september 5 , 2015 , the department of health reported 78,808 cases of dengue in the entire country , a 16.5% increase from the same time period last year , with about 8.2% accounted for the ilocos region ( region 1 ) including pangasinan which was declared under a state of dengue outbreak at the time of survey . table 5 tallies and compares the number of use reports generated as symptoms of dengue in the 3 samplings sites surveyed and their cmuas and fls . prevalence of dengue symptoms in three indigenous communities in the province of pangasinan the cmua reflects the relative importance of e. hirta in the 3 communities surveyed for a given symptom . cmua of at least 35% were obtained for cardinal symptoms of dengue related to blood thinning episodes ( i.e. nose bleeding , skin blisters and mouth bleeding ) . the febrile symptoms fever , headache , and joint pains have relatively high cmuas as well . low cmuas ( i.e. 2 - 4% ) were obtained for symptoms in the recovery phase , namely , skin rashes , itching , and diarrhea partly because inhabitants do not follow - up or consult with respondent herbalists after the febrile phase . symptoms such as hypotension , capillary fragility , and tachycardia have not been reported because the respondents are not familiar with the use of manual apparatus such as sphygmomanometers and torniquets . it should be noted that the symptoms of dengue enumerated in table 5 may be symptoms of other diseases as well , such as bacterial and viral infections and certain inflammatory conditions . a high fl is reflected when e. hirta is the most preferred medicinal plants to treat a particular symptom while a low fl is obtained when the plant is indicated for several symptoms and other diseases . relatively high fl values were obtained for similar symptoms with high cmuas such as the bleeding episodes as well as symptoms observed in the febrile phase . consistently , lower fls were reflected for symptoms with lower cmuas . from the 76 use reports related to dengue symptoms , about 132 corresponding dosage forms of e. hirta are compounded extemporaneously as shown in table 6 . dosage preparation of euphorbia hirta compounded according to plant part about 111 dosage preparations were administered internally by mouth while the rest were applied topically to affected areas . topical dosage forms ( i.e. paste , poultice , cataplasm , rubifacient , or emollient ) are applied to the skin and the forehead for headache , fever , joint pain , muscle pain , stomachache , skin rashes and itching while preparations for internal use ( i.e. infusion and decoction ) are indicated for symptoms related to bleeding of the skin , nose , oral cavites and the gastrointestinal tract . colisao recommended the use of at least 3 preparations for a single use report or symptom as well as the use of both internal and external preparations for a single symptom . several ethnomedicinal studies have also reported that leaves are the most frequently used part because of their remarkable identity and accessibility in addition to the fact that most biosynthesis of therapeutically active constituents occur in leaves [ 10 - 12 ] . on the other hand , the use of leaf sap and latex was founded from the belief that these exudates from plants belonging to the family euphorbiaceae possess antibacterial and antiviral properties and finds great application as antiseptics in the treatment of wounds . the respondents did not described how cataplasms , poultices , paste , emollients and rubefacients are compounded although the issue of hygiene in their preparation are important , particularly if these dosage forms are to be applied externally to infected sites . tables 2 - 4 show the demographic data of the 82 informants in this study according to sex , age group , their distributions among the three indigenous areas surveyed and educational attainment . distribution of informants according to area surveyed demographic data according to age group and sex educational attainment of informants the questionnaires were evenly distributed to each of the 3 sampling sites surveyed , but more allocations were given to female respondents because females are perceived to have higher knowledge over males in the traditional self - care uses of medicinal plants , a tradition that could have been passed to them from previous generations . in this study , significantly higher number of use reports was generated by female respondents compared to their male counterparts ( p < 0.0001 , chi - square ) . the majority of use reports pertaining to symptoms of dengue were given by respondents aged 60 - 80 , a significantly higher number as compared to younger counterparts ( 227 vs. 79 , p < 0.0001 , chi - square ) . this is because the respondents within this age bracket are more experienced than younger informants in the self - care uses of medicinal plants for dengue based on anecdotal information that is consistent with the rapid rise in the epidemics of this disease for the previous years . the use of medicinal plants also increased with increasing age to imply that the high dependence of these three indigenous communities on plants to treat illnesses is still consistent with time . elementary and secondary schools were found to have important roles in the dissemination of information on the self - care uses of medicinal plants in the three areas surveyed . respondents who finished primary and secondary schoolings generated higher use reports related to dengue symptoms ( p < 0.001 , chi - square ) . interviews with teachers in these areas reveal that the study of medicinal plants on the scientific and anecdotal points of view has been incorporated in elementary and high school science curriculums . there is a low number of respondents with higher educational attainment because most of the informants surveyed have lower income compared to inhabitants in more populated municipalities , in addition to the great distances of colleges which are mostly located in pangasinan city propers findings showing similarities to the observations of abe and ohtani among ivatans in batan island north of luzon . table 1 shows the number of informants interviewed and the frequency of use reports relative to any symptom in the 3 sampling sites surveyed . colisao cited e. hirta for at least 5 symptoms of dengue as well as 7 other symptoms . in mt . balungao , 4 respondents cited the same plant for use in 7 symptoms of dengue in addition to 10 other symptoms . the relatively high number of use reports for any symptoms generated in anda island ( n = 197 ; p < 0.001 ) can be due to a larger population that is dependent on medicinal plants in treating diseases , the higher density and diversity of plants available for use as medicinal agents and the high prevalence of acute diseases , including dengue . furthermore , the absence of modern health facilities , clinics , hospitals and pharmacies in anda island may explain for the high dependence of its residents on medicinal plants . an interview of municipal health officers within the vicinities of the 3 sampling sites surveyed shows a high prevalence of dengue infections in anda island from january to march of 2015 , with 36 reported cases , including 2 casualties that affected mostly children ages 2 years and above . within this period , 17 and 15 cases of dengue the high prevalence of dengue infection in the 3 sampling sites surveyed can be due to the lack of proper hygiene and sanitation in possible breeding places of mosquito vectors . from january 1 to september 5 , 2015 , the department of health reported 78,808 cases of dengue in the entire country , a 16.5% increase from the same time period last year , with about 8.2% accounted for the ilocos region ( region 1 ) including pangasinan which was declared under a state of dengue outbreak at the time of survey . table 5 tallies and compares the number of use reports generated as symptoms of dengue in the 3 samplings sites surveyed and their cmuas and fls . prevalence of dengue symptoms in three indigenous communities in the province of pangasinan the cmua reflects the relative importance of e. hirta in the 3 communities surveyed for a given symptom . cmua of at least 35% were obtained for cardinal symptoms of dengue related to blood thinning episodes ( i.e. nose bleeding , skin blisters and mouth bleeding ) . the febrile symptoms fever , headache , and joint pains have relatively high cmuas as well . low cmuas ( i.e. 2 - 4% ) were obtained for symptoms in the recovery phase , namely , skin rashes , itching , and diarrhea partly because inhabitants do not follow - up or consult with respondent herbalists after the febrile phase . symptoms such as hypotension , capillary fragility , and tachycardia have not been reported because the respondents are not familiar with the use of manual apparatus such as sphygmomanometers and torniquets . it should be noted that the symptoms of dengue enumerated in table 5 may be symptoms of other diseases as well , such as bacterial and viral infections and certain inflammatory conditions . a high fl is reflected when e. hirta is the most preferred medicinal plants to treat a particular symptom while a low fl is obtained when the plant is indicated for several symptoms and other diseases . relatively high fl values were obtained for similar symptoms with high cmuas such as the bleeding episodes as well as symptoms observed in the febrile phase . from the 76 use reports related to dengue symptoms , about 132 corresponding dosage forms of e. hirta are compounded extemporaneously as shown in table 6 . dosage preparation of euphorbia hirta compounded according to plant part about 111 dosage preparations were administered internally by mouth while the rest were applied topically to affected areas . topical dosage forms ( i.e. paste , poultice , cataplasm , rubifacient , or emollient ) are applied to the skin and the forehead for headache , fever , joint pain , muscle pain , stomachache , skin rashes and itching while preparations for internal use ( i.e. infusion and decoction ) are indicated for symptoms related to bleeding of the skin , nose , oral cavites and the gastrointestinal tract . colisao recommended the use of at least 3 preparations for a single use report or symptom as well as the use of both internal and external preparations for a single symptom . several ethnomedicinal studies have also reported that leaves are the most frequently used part because of their remarkable identity and accessibility in addition to the fact that most biosynthesis of therapeutically active constituents occur in leaves [ 10 - 12 ] . on the other hand , the use of leaf sap and latex was founded from the belief that these exudates from plants belonging to the family euphorbiaceae possess antibacterial and antiviral properties and finds great application as antiseptics in the treatment of wounds . the respondents did not described how cataplasms , poultices , paste , emollients and rubefacients are compounded although the issue of hygiene in their preparation are important , particularly if these dosage forms are to be applied externally to infected sites . the global incidence of dengue has grown dramatically in recent decades , particularly in urban areas in countries with tropical climates , and becoming a leading cause of death among children . since there is no specific cure against dengue , treatment modalities include supportive measures to address specific symptoms . in rural areas , such as the three indigenous communities surveyed in this study , limited access to modern health care facilities the lack of vector control against dengue - carrying mosquitoes in these areas further complicates the situation . the most commonly - used plants in the folkloric treatment of dengue in the philippines include e. hirta and carica papaya . although the people of pangasinan have a long history of the traditional uses of medicinal plants , no ethnobotanical studies have been undertaken to document the traditional knowledge on their self - care uses of these plants . documentation of the traditional uses of e. hirta against dengue indicates the relative folkloric importance of the plants in the three surveyed indigenous communities in pangasinan where dengue continues to be an epidemic . the current supportive therapies for hospitalized dengue patients include hydration and blood transfusion as well as treatment with acetaminophen since most non - steroidal anti - inflammatory drugs are contra - indicated due to their anti - platelet augmenting properties . since e. hirta was found in this study to be effective against most symptoms of dengue in the initial , febrile and recovery stages , the findings warrant the development of the plant into dosage forms that can be utilized in clinical trials aimed at ensuring the efficacy and safety of e. hirta in the supportive therapy of dengue . this study was able to document the anecdotal and traditional self - care uses of e. hirta in the treatment of dengue in three indigenous communities in pangasinan according to demography , relative importance , and fl .

among the different human cell types , janus kinase ( jak)/signal transducer and activator of transcription ( stat ) signaling cascades transduce signals of more than 50 cytokines and peptide hormones . a subset of these growth factors such as prolactin ( prl ) , growth hormone ( gh ) , leukemia inhibitory factor ( lif ) , and oncostatin m ( osm ) are crucial for mammary epithelial cell proliferation , differentiation , and apoptosis . the binding of these ligands to their respective receptors induces a conformational change that autoactivates the receptor - associated janus tyrosine kinases through transphosphorylation of tyrosine residues . active jaks then phosphorylate critical tyrosines on the cytoplasmic portion of the receptor , thereby creating docking sites for the src homology 2 ( sh2)-domains of signal transducers and activators of transcription ( stats ) that are subsequently phosphorylated by jaks at conserved tyrosine residues . activated stats form stable homo- or heterodimers through reciprocal sh2-phosphotyrosine interactions and translocate to the nucleus where they function as latent transcription factors by binding to conserved recognition sites.[24 ] the mammalian family of janus kinases is composed of four members : jak1 , jak2 , jak3 , and the tyrosine kinase 2 ( tyk2 ) . among these , jak1 and jak2 are expressed in many tissues , including normal and neoplastic mammary epithelial cells . in contrast , the expression of jak3 and tyk2 is mostly limited to hematopoietic cells , and these kinases have not been subject to intensive studies in breast cancer . in normal mammary epithelial cells , jak2 is known to mediate signals through single - chain receptors , in particular , the prl - r and gh - r . jak1 , on the other hand , is suggested to function together with jak2 in signal transduction of gp130 receptor ligands such as lif and osm . however , it has yet to be determined using gene deletion models that jak1 and jak2 are each necessary or sufficient to relay signals through these particular multi - chain receptors in the mammary gland . as described later , various hormones and cytokines such as prl and lif can initiate a completely different response ( e.g. , cell survival versus apoptosis ) in epithelial subtypes , and therefore , jak1 and jak2 have multifaceted functions at particular stages of mammary gland development . depending on the cell type and ligand - receptor complex , each jak activates one or more of the seven members of the stat family : stat14 , stat5a , stat5b , and stat6 . with the exception of stat2 , all other stats are expressed , albeit at various levels , throughout mammary gland development . the expression of stat4 could not be detected in cultured mammary epithelial cells , and it was therefore concluded that this signal transducer might be predominantly activated in the mammary stroma . although the transcriptional activation of the genes encoding stat1 , stat3 , stat5a , stat5b , and stat6 may vary to some degree throughout the gestation cycle , the jak1/jak2-mediated phosphorylation of these signal transducers in response to specific growth factors is the key event for their activation and functionality . during postnatal mammary gland development , elevated levels of stat1 phosphorylation are found in the nulliparous and parous gland following postlactational remodeling . very recently , klover et al . reported the generation of a stat1 conditional knockout model , and surprisingly , the deletion of this particular signal transducer using mmtv - cre and wap - cre transgenics did not affect the functionality of the mammary gland in nulliparous or lactating females . in addition to stat1 , a basal activation of stat5a and stat5b occurs in response to prl signaling in luminal epithelial cells within the ductal compartment of nonpregnant women and female mice . the significant increase in the level of prl during pregnancy leads to a very substantial elevation in the expression and activation of stat5 and consequentially an upregulation of milk protein genes . stat5a , which has been originally identified as the mammary gland factor ( mgf ) , is more abundant compared to stat5b in the functionally differentiated mammary epithelium . similar to stat5 , stat6 becomes increasingly phosphorylated during pregnancy when a significant subset of mammary epithelial cells switches from the production of interleukin ( il)12a , tumor necrosis factor ( tnf) , and interferon gamma ( ifng ) to the secretion of il4 , il13 , and il5 in response to pregnancy hormones , including prl . this might also explain why the phosphorylation of stat1 , which is a main downstream effector of ifn signaling , is lower during this stage of mammogenesis . finally , the activation of stat3 within less than 24 hours following weaning the offspring heralds the beginning of the postlactational remodeling period . while lif may serve as the initial upstream ligand to activate this particular stat protein , recent work by tiffen et al . suggests that the subsequent stat3-mediated expression of osm is required as part of an autocrine loop for a sustained activation of this signal transducer . the stat3-mediated apoptosis of functionally differentiated epithelial cells and increase in the levels of phosphorylated stat1 concludes the precisely controlled activation of stats at distinct phases during normal mammary gland development . as discussed in the following sections , any disturbance in the state of equilibrium of these jak / stat pathways , in particular , jak2/stat5 and jak1/stat3 signaling , leads to developmental defects and contributes to mammary carcinogenesis . both stat5 proteins share an overall 96% amino acid similarity , but gene deletion studies in single knockout mice lacking either protein show that only the functional ablation of stat5a leads to impaired alveologenesis during pregnancy and lactation . this is likely a consequence of the dissimilar expression of the stat5a and stat5b loci in the mammary epithelium . following multiple gestation cycles , however , stat5a - deficient mammary epithelial cells are able to upregulate stat5b , which partially restores normal alveolar development and milk protein gene expression . in contrast to stat5a single knockout mice , the deletion of both stat5 genes causes a complete absence of alveolar cells , and transplant experiments as well as the examination of a stat5 conditional knockout model show that this phenotypic abnormality is the result of cell autonomous functions of stat5a and stat5b.[202210 ] besides activation of the jak / stat pathway , binding of prl to its receptor stimulates additional signal transducers such as src , mitogen activated protein ( map ) kinases , phosphatidylinositol 3-kinase ( pi3k ) , and protein kinase c ( pkc ) ( for citations please refer to wagner and rui ) . the striking phenotypic similarities between stat5 knockout mice and females that are deficient in prl or the prl receptor suggested that important biologically relevant functions of prl signaling during normal mammary gland development are mediated primarily through the jak / stat pathway . although there is a wealth of knowledge about the activation and functionality of stats , much less is known about the biological significance of jak1 and jak2 downstream of various growth factor receptors in the mammary gland . conventional gene deletion models of each of these two jaks die perinatally due to neurological or hematopoietic defects.[2528 ] studies using an orthotopic transplantation model of jak2-deficient embryonic mammary gland anlagen into wild - type recipient mice as well as the development and analysis of a jak2 conditional knockout model show that this kinase is required for the development of secretory alveolar cells . on a mechanistic level , the examination of mice conditionally deficient in jak2 clearly demonstrated that this kinase is the essential link between prl signaling and stat5 activation in the normal mammary gland . jak2-deficient mammary epithelial cells lack phosphorylated stat5 even after administration of extraphysiological levels of prl , and the functionality of this kinase is not compensated by jak1 or receptor tyrosine kinases such as erbb2 , as previously suggested . as discussed later , this has significant implications for the prevention of cancer in mice with enhanced prl autocrine signaling in their mammary glands . interestingly , while the ablation of jak2 had no effect on ductal elongation and branching morphogenesis , nulliparous mammary glands in jak2-deficient females were completely devoid of alveolar buds that usually reside at the terminal end of ducts , suggesting that this kinase is essential for the specification of alveolar progenitors prior to pregnancy . in line with this notion , a recent report by yamaji et al . demonstrated that deficiency in stat5 caused a reduction in the number of cd61 + luminal progenitors that are committed to the alveolar lineage and that normally multiply in response to prl signaling during pregnancy . this work also showed that exogenous expression of stat5a was sufficient to restore the numeric expansion and differentiation of luminal progenitors . given that the mammary gland develops normally in stat5b single knockout mice , the outcome of this stat5a rescue experiment might not be entirely surprising . on the other hand , if the proposed functional redundancy between both stat5 isoforms is correct , an identical expression of exogenous stat5b might also be able to restore , at least in part , the specification and proliferation of luminal alveolar progenitors in mice that conditionally lack the entire endogenous stat5a / b locus . the fact that jak2/stat5 signaling is critical for the genesis of alveolar progenitors may have significant implications for breast cancer prevention . this region is known as the terminal duct lobular unit ( tdlu ) , and it has been suggested that tdlus are the sites in the human breast where a subset of breast cancers originate . this may explain why , besides age and genetic susceptibility , the reproductive status of a woman is the strongest and most reliable risk factor for breast cancer . in support of these observations in humans , erbb2-induced mammary cancers in a mouse model arise predominantly from luminal alveolar progenitors , and the selective elimination of these cells from the developing mammary gland prevents mammary tumorigenesis in mice expressing wild - type erbb2 . there is epidemiological and molecular evidence that hormone - responsive breast epithelial cells exhibit an elevated susceptibility to neoplastic transformation , and recent findings suggest that these cells might also contribute to basal - type or hormone receptor - negative breast cancers . for example , loss of brca1 has been reported to be associated with an expansion of luminal progenitors in the normal human breast . similarly , studies in a brca1-deficient mouse model showed that the mammary - specific deletion of this tumor suppressor led to an accumulation of cd61 + luminal progenitors during pregnancy , which is precisely the cell type that is dependent on the jak2-mediated activation of stat5 . besides the encouraging results about the prevention of mammary cancer in the stat5- and jak2-deficient models as discussed later in this article , future studies might show whether the inhibition of jak2/stat5 signaling could be an effective strategy for the prevention of brac1-associated , hormone receptor negative breast cancers . conditional gene deletion models provide unique opportunities to study essential biological functions of a gene at various stages of mammary gland development beyond the arrested development of a conventional knockout . while the mmtv - cre - mediated deletion of jak2 or stat5 throughout the mammary epithelium revealed that this jak / stat pathway is essential for the genesis of alveolar progenitors , a selective wap - cre - induced ablation of jak2 or stat5 in the lactating mammary gland showed that these downstream mediators of prl signaling are equally important for the survival of functionally differentiated alveolar cells . following a normal lactation period , the mammary gland undergoes a series of molecular events that culminate in the programmed cell death of the vast majority of secretory epithelial cells . this involution process , which requires the downregulation of survival factors and the activation of pro - apoptotic signals , is characterized by a rapid switch between jak / stat signaling cascades . while the levels of circulating prl and therefore active stat5 decline , local growth factors such as lif and osm accumulate in the mammary gland in response to milk stasis and initiate the phosphorylation of stat3 . in support of this paradigm , deficiency in stat3 prolongs the functional competence of secretory epithelial cells and causes a delay in programmed cell death . on the mechanistic level , abell et al . p55. these subunits then downregulate the activity of the pi3k holoenzyme ( p85/p110 ) , and subsequently akt , which is a known survival pathway for functionally differentiated mammary epithelial cells . it has been repeatedly demonstrated that a gain - of - function of stat5 can extend the survival of differentiated mammary epithelial cells in the absence of elevated lactogenic hormones . in addition , a recent study by creamer et al . shows that expression of active stat5 results in a significant delay in postlactational remodeling despite the initiation of stat3-mediated pro - apoptotic signaling events and the transcriptional upregulation of p50 and p55. the extended survival of terminally differentiated cells during involution in this model might be a direct consequence of the stat5-mediated sustained expression of akt1 . active stat5 has been shown to bind to two or more consensus sequences within the akt1 locus in a growth factor - dependent manner , and it enhances the transcription of a unique akt1 mrna from a distinct promoter in vitro and in vivo . this novel transcript encodes for the full - length akt1 serine threonine kinase . in support of the proposed regulation of akt1 downstream of stat5 , all mammary - specific models that express active stat5 share striking phenotypic similarities with transgenic mice that express exogenous akt1 under regulation of the mmtv - ltr.[4345 ] it was also previously reported that expression of akt1 promotes the survival of the functionally differentiated mammary epithelium despite activation of stat3 . therefore , a gain - of - function of stat5 or elevated expression of akt1 is sufficient to override , at least in part , the functionality of stat3 . at this point , it is still unclear how this biological phenomenon is being accomplished if , as suggested , the shorter pi3k regulatory subunits p50 and p55 are sufficient to inhibit the p85/p110 pi3k holoenzyme from its subsequent activation of akt . in these efforts , it is necessary to verify the biological significance of the shorter pi3k subunits during mammogenesis using p50/p55 double knockout mice . also , the effects of active stat5 on the expression and assembly of the catalytically active pi3 kinase still need to be examined in more detail . as an integral part of a multifaceted signaling complex sakamoto et al . have shown that stat5 might directly modify the activity of the pi3 kinase in the mammary gland through binding to the sh2 domain - containing regulatory subunit p85. while the biological significance of this particular interaction needs to verified , it is evident that a gain - of - function of p110 or loss of phosphatase and tensin homolog ( pten ) recapitulates several of the phenotypic abnormalities associated with the overexpression of active stat5 . collectively , the phenotypic and molecular analyses of several genetically engineered models revealed that similar to a gain - of - function of the pi3 kinase and akt1 , a prolonged or constitutive activation of jak2/stat5 signaling facilitates evasion from apoptosis and self - sufficiency in growth signals , which are two important hallmarks of cancer . in line with this notion , it has been demonstrated that stat5a has oncogenic properties , but the exact mechanisms by which this stat protein promotes neoplastic transformation are still not entirely known . nonetheless , the proposed molecular interactions between jak / stat and pi3k / akt signaling [ figure 1 ] provides a model by which active stat5 may function as a potent modifier for pi3k signal transduction , i.e. one of the most commonly altered pathways in hereditary and sporadic forms of breast cancer . active stat5 modifies signaling through the pi3 kinase and akt1 by at least two distinct mechanisms in luminal epithelial cells , i.e. by binding to the regulatory subunit of the pi3 kinase and by enhancing the transcriptional activation of the akt1 gene from a mammary - specific promoter chromosomal translocations that result in oncogenic jak2 fusion gene products such as tel jak2 , bcr jak2 , and pcm1jak2 are mainly associated with the initiation and progression of myeloproliferative disorders and a subset of leukemias ( for references see ) . similar genetic events have not been detected in a significant portion of adenocarcinomas , including human breast cancers . these types of malignancies acquire active jaks through alternative mechanisms such as a ) epigenetic silencing of suppressors of jak / stat signaling , b ) aberrant increase in autocrine signaling , and to a lesser extent , c ) missense mutations within jaks . the recent discovery of constitutively activating mutations of jak2 ( e.g. jak ) in myeloproliferative disorders spurred a number of investigations into identifying similar somatic abnormalities within jaks in solid cancers . collectively , these studies showed that the prominent v617f mutation in jak2 does not contribute to the activation of stat5 and stat3 in adenocarcinomas . two recent reports describe a rare occurrence of missense mutations within the pseudokinase domain of jak1 in breast , lung , and hepatocellular carcinomas , but their biological relevance and contribution to carcinogenesis remain elusive . in contrast to the lack or low incidence of somatic mutations within jaks , the epigenetic silencing of suppressors of jak / stat signaling are more frequently observed in breast cancers . the main negative regulators of jaks and stats are protein tyrosine phosphatases ( ptps ) , suppressors of cytokine signaling ( socs ) , and protein inhibitors of activated stats ( pias ) . these proteins possess diverse functions as inhibitors , including the dephosphorylation or proteosomal degradation of jaks , and their association with stats to alter their localization , dna binding , and transcriptional activation . among the three families of jak / stat inhibitors , ptps and socs have been shown to be epigenetically silenced through dna methylation in a variety of human cancers . specifically , silencing of the socs1 gene has been reported in 9% of primary breast cancer cases . in addition , loss of caveolin-1 , a potent suppressor of jak2/stat5 signaling , contributes to neoplastic transformation and mammary tumorigenesis in humans and in genetically engineered mouse models . beside the suppression of negative regulators of jak / stat signaling , breast cancer cells initiate the synthesis of hormones and cytokines as part of autocrine signaling networks to overcome external growth factor dependence . specifically , prl , erythropoietin ( epo ) , and il-6 are frequently co - overexpressed with their respective receptors in breast cancer cells , and all of these cytokines are potent activators of jak / stat signaling cascades . epo has attracted considerable attention recently when the us food and drug administration warned against the use of this growth factor to increase hemoglobin levels during breast cancer treatment . administration of epo to late - stage breast cancer patients promoted disease progression , and it has also been reported recently that this cytokine may offset the treatment of er-positive and her2-positive breast cancers with tamoxifen and trastuzumab . this clearly demonstrates that the aberrantly expressed epo receptor plays an important functional role in cancer cell growth and survival , and this may not be entirely surprising since the intracellular signaling domains of the receptors for epo and prl are functionally identical . it has been recognized for some time that high circulating levels of prl are associated with an increased breast cancer risk in humans ( for references see ) . in support of this notion , elevated levels of prl in circulation in addition to the pituitary prl , breast cancer cells locally synthesize this growth factor and upregulate the expression of the prl receptor . the importance of this hormone as part of an autocrine loop for mammary carcinogenesis was experimentally verified in transgenic mice that express prl locally in the mammary epithelium . these animals develop luminal - type neoplasia with both estrogen receptor positive and negative lesions . although prl signals mainly through jak2 and stat5 in normal luminal breast epithelial cells , this hormone is capable of aberrantly activating stat3 in human breast cancer cell lines . a more recent study shows that prl activates jak1 in a jak2-dependent manner , and this finding may provide an underlying mechanism by which prl activates stat3 and map kinases that , as discussed later , seem to play a more important role than stat5 in breast cancer progression . collectively , these observations suggest that signaling networks undergo a substantial rewiring process during or following neoplastic transformation . the changes within the prl signaling network between normal and neoplastic epithelial cells exemplify another two - faced nature of jaks in their ability to activate additional stats and thereby contribute to different biological processes . depending on the expression of growth factors and their receptors , along with downstream changes in growth regulatory networks , the activation of individual stats may vary among diverse breast cancer subtypes or different stages of disease progression . there is increasing evidence that stat1 has tumor suppressive functions , and this signal transducer also plays a role in growth arrest and in pro - apoptotic signaling during chemotherapy . in addition , activation of stat1 and the concomitant upregulation of socs1 and interferon regulatory factor-1 ( irf-1 ) expression in human mammary carcinoma was reported to be a predictor of a good prognosis . similarly , expression of active stat5 , which was observed in approximately 76% of human breast tumors , positively correlated with tumor differentiation . a study by nevalainen et al . the examination of stat5 activation in more than 1100 primary breast cancers in that study revealed that the nuclear expression of stat5 was gradually lost during cancer progression and was absent in the vast majority of metastases . one mechanism that may facilitate the inactivation of stat5 in metastatic cells is the upregulation of the protein tyrosine phosphatase 1b ( ptp1b ) that targets active jak2 . in line with the notion that the functionality of stat5 is diminished in disease progression , sultan et al . reported that active stat5 promoted the differentiation of breast cancer cells and increased the cell surface levels of e - cadherin . collectively , the examination of stat5 expression and activation during human breast cancer progression , as well as studies in prl- and stat5-induced tumor models in rodents , revealed another side of the multifaceted nature of jak2/stat5 signaling in mammary carcinogenesis . while activation of stat5 and a simultaneous increase in cell survival may initially promote neoplastic transformation , a continuous activation of this signal transducer mediates the maintenance of a more differentiated and less metastatic tumor phenotype . a similar two - faced specifically , hutchinson et al . examined the effects of a gain - of - function of akt1 during mammary tumorigenesis . while the expression of active akt1 decreased the latency of erbb2-induced mammary tumorigenesis , the sustained upregulation of this kinase prevented cancer invasion and metastasis . as mentioned earlier , akt1 has recently been shown to be a transcriptional target of stat5 , and it is , therefore , tempting to speculate that stat5 exerts its suggested role as a metastasis suppressor through increased expression of akt1 . transgenic mice that allow expression of hyperactive and wild - type stat5 in a ligand - inducible manner are suitable experimental models to assess in vivo whether a ) stat5 can control the levels of akt1 in preneoplastic and cancerous lesions and b ) the ablation or continuous expression of stat5 in mammary tumors affects the metastatic dissemination of cancer cells . as discussed earlier , stat5 and stat3 are successively activated during lactation and involution in the normal mammary gland , and both stats seem to function in a similar sequential order during breast tumorigenesis . as the expression and activation of stat5 declines , the level of phosphorylated stat3 increases significantly in malignant breast cancer cells . a number of independent studies have reported that the majority of primary breast cancers ( about 5060% ) exhibit a constitutive activation of stat3 and that this particular signal transducer plays an important role in breast cancer cell growth and metastatic progression . a recent report by barbieri and colleagues shows that the constitutive activation of stat3 results in accelerated mammary tumorigenesis and an increase in the metastatic potential of erbb2 expressing cancer cells . in contrast , a knockout of stat3 prior to tumorigenesis shows that this signal transducer is not required for neoplastic transformation . however , the ablation of stat3 was sufficient to suppress the anchorage - independent growth of breast cancer cells and their ability to metastasize . therefore , targeting stat3 may not be a relevant strategy for cancer prevention , but the functional inhibition of this signal transducer as part of a targeted therapy for advanced breast cancers may prevent the metastatic dissemination of malignant cells . since transcription factors , which include stats , rely more extensively on protein protein and protein dna interactions for their functionality , they are less accessible biological targets and often referred to as un - druggable . nevertheless , a number of agents have been identified in screens that inhibit stats , and these compounds might be useful for the treatment of specific human malignancies . for example , a very recent report showed that the drug pimozide is a stat5 inhibitor that might be effective for the treatment of chronic myelogenous leukemia ( cml ) and potentially other myeloproliferative diseases . considering the multifaceted role of stat5 during breast cancer initiation and the suggested function of this transcription factor as a metastasis suppressor , targeting stat5 might be a suitable strategy for cancer prevention but not to treat late - stage breast cancers . in contrast , stat3 is an unlikely target for breast cancer prevention . since the inhibition of this particular stat is suggested to reduce cancer cell invasion and metastasis , stat3 inhibitors such as opb-31121 ( otsuka pharmaceutical development and commercialization , inc . , princeton , nj ) might be suitable for the treatment of invasive breast cancer , but their efficacy needs to be tested in a clinical setting . unlike transcription factors , protein kinases may become the most important group of drug targets besides g - protein - coupled receptors . it is therefore not surprising that more than a dozen studies are currently underway to test investigational jak inhibitors to treat hematopoietic malignancies and carcinomas.[9092 ] the selectivity of these new agents to specifically target their corresponding jaks still needs to be determined . due to differences in the mechanisms of jak activation , not all drugs that might effectively inhibit mutant jak2 in hematopoietic disorders are necessarily useful for the treatment of carcinomas that , as discussed earlier , utilize alternative modes of jak / stat activation . also , jak1 and jak2 are co - activated in solid cancers , and it needs to be determined whether they have therapeutically relevant redundant functions . if so , dual kinase inhibitors such as incb018424 from incyte , wilmington , de might be useful for the treatment of cancers where both kinases are functionally equivalent for cancer cell growth and survival . it is evident that the specificity and effectiveness of pharmacological agents for the targeted inhibition of jaks and stats need to be more carefully examined . in parallel , genetically engineered preclinical models are being employed to study the significance of individual signal transducers during mammary carcinogenesis , and these models may herald the usefulness of particular therapeutic strategies to prevent and treat breast cancer . in support of the notion that stat5 plays a key role during the onset and early progression of mammary cancer , it has been demonstrated that the functional ablation of stat5a is sufficient to delay the formation of t - antigen and tgf-induced mammary cancers . cyclin d1 is one of the downstream targets of stat5 that might link prl signaling to the cell cycle machinery , and it has been suggested that lack of this cell cycle regulator protects against the onset of erbb2- and myc - induced mammary tumorigenesis . while the outcome of these experiments might suggest that targeting stat5 and its downstream effectors is a suitable strategy for breast cancer prevention , it was premature to suggest that targeting these proteins is equally important for the treatment of established cancers . to more accurately assess whether specific signal transducers are appropriate therapeutic targets , it is necessary to delete a gene or ablate the function of its encoded protein in fully neoplastic cancer cells in vivo . the design and examination of such experimental models can lead to a completely different outcome and conclusion . two recent studies by sakamoto and colleagues show that the deletion of the jak2 gene ( and consequently , the inhibition of stat5 ) in the mammary epithelium prior to tumor onset completely protected female mice from developing mammary tumors in response to an overexpression of erbb2 and prl . the conditional knockout of this janus kinase from fully neoplastic cells , however , had no effect on tumor cell growth and survival in vitro or in vivo . collectively , these studies suggest that targeting jak2 might be an appropriate way to prevent mammary cancer , but inhibiting the function of this particular tyrosine kinase is unlikely a suitable strategy to treat established breast cancer . the findings by sakamoto and others about the multifaceted role of jak2/stat5 signaling in mammary tumorigenesis are in line with observations in human breast cancer specimens that the activation of stat5 is gradually lost during cancer progression . on the other hand , given the suggested importance of stat3 in late - stage cancers and the proposed joint function of jak1 and jak2 for the activation of this particular stat protein , future studies may show whether a combined inhibition of both jak1 and jak2 can halt the growth and prevent the metastatic dissemination of cancer cells in vivo . to develop such a complex model , it would be first necessary to establish a jak1 conditional knockout mouse since a conventional gene targeting approach for this kinase results in prenatal lethality . dr . kay - uwe wagner , eppley institute for research in cancer and allied diseases , university of nebraska medical center , 985950 nebraska medical center , drcii , rm . jeffrey w. schmidt , eppley institute for research in cancer and allied diseases , university of nebraska medical center , 985950 nebraska medical center , drcii , rm .

endothelial injury has been studied in septic and non - septic patients , both at risk for and with established acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) . several clinical studies have demonstrated that circulating levels of angiopoietin-2 ( ang-2 ) , which are associated with mortality , are elevated at the onset of critical illness or in those at risk for ali / ards . experimental studies have demonstrated that angiopoietin-1 ( ang-1 ) and ang-2 bind to endothelial tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 receptor ( tie2 receptor ) . ang-2 provokes pulmonary inflammation and vascular leakage , partly by preventing ang-1 from binding the tie2 receptor , thereby inhibiting the stabilizing effect of ang-1 . this suggests that ang-2 is not only a marker but also a direct mediator in the pathogenesis of vascular permeability and pulmonary injury and dysfunction in septic and non - septic patients [ 1820 ] . furthermore , the ratio of ang-2 concentration to ang-1 concentration may be a prognostic biomarker of endothelial activation in ali patients . the antigen von willebrand factor ( vwf ) is a macromolecular antigen that is produced predominantly by endothelial cells and in lesser amounts by platelets . vwf is a large multimeric glycoprotein present in blood plasma and produced constitutively in weibel - palade bodies , megakaryocytes ( -granules of platelets ) , and subendothelial connective tissue . some studies have shown that vwf is a marker of poor outcome in patients with ali / ards . paraquat ( 1,1-dimethyl-4 - 4-bipyridium dichloride ; pq ) is used not only as an herbicide but also to produce reactive oxygen species ( ros ) in the oxidative stress model . paraquat mainly accumulates in the lungs , where it is retained even when its concentration in the blood starts to decrease . ros play a crucial role in the pathogenesis of various lung diseases such as asthma , chronic obstructive pulmonary disease , and acute respiratory distress syndrome . paraquat - induced lung injury might be a unique model of dominant ros - induced lung injury . ros can lead to endothelial barrier dysfunction with subsequent increased permeability to fluid , macromolecules , and inflammatory cells . we hypothesized that endothelial cell injury could be involved in early death after paraquat intoxication and that concentrations of ang-1 , ang-2 , and vwf could reflect endothelial cell injury . the aim of our study was to investigate whether ang-2 , vwf , and ang-1 are related to early mortality . therefore , we measured initial plasma levels of these markers in patients who intentionally ingested paraquat . we prospectively collected samples from patients admitted to the soonchunhyang university cheonan hospital for paraquat intoxication from march 2011 through august 2011 . , gastric lavage was performed for all subjects who presented to the emergency room within 2 hours of paraquat ingestion . patients who had ingested paraquat between 2 and 12 hours before presenting to the emergency room were administered 100 g of fuller s earth in 200 ml of 20% mannitol . hemoperfusion was performed if the patient s urinary paraquat test result was positive on arrival at the emergency room . the amount ingested was estimated based on the number of swallows ( 1 mouthful was considered 20 ml ) . high - resolution computed tomography ( hrct ) was performed 7 days after ingestion , and again 1 week later if any abnormality was detected . on hrct , mild lung lesion is < 10% and severe lung lesion is 10% . survivors were defined as those patients who survived more than 3 months after paraquat ingestion and had stable vital signs . the serial sample in 3 patients was obtained at admission ; 6 , 12 , and 24 hours after admission ; and 2 , 3 , 4 , 5 , 6 , and 7 days after admission . paraquat level was measured by high - performance liquid chromatography with a perkin elmer series 200 ( perkin elmer , usa ) . in brief , pq were separated on a reversed - phase column , capcell pak c18 ug120 ( 1504.6 mm i.d . ; particle size , 5 m ; shiseido co. , tokyo , japan ) , by isocratic elution with a mixture of methanol and 200 mm phosphoric acid solution containing both 0.1 m diethyl amine and 12 mm sodium 1-heptane sulfonate ( 1:4 , v commercial enzyme - linked immunosorbent assay ( elisa ) kits were used to measure ang-1 ( quantikine human angiopoietin-1 elisa kit , cat dang10 ; r&d systems , usa ) , ang-2 ( quantikine human angiopoietin-2 elisa kit , cat dang20 ; r&d systems , usa ) , and vwf ( vwf human elisa kit , cat # ka0512 ; abnova , taiwan ) . data are presented as means sd for continuous variables and frequencies ( percentages ) for categorical variables . differences between groups were evaluated with student s t test for continuous variables and test for categorical variables . multivariate logistic regression analysis was used to identify significant determinants of death after paraquat intoxication . the results of logistic regression analyses are reported as odds ratios ( or ) with 95% confidence intervals ( ci ) . statistical analyses were performed using spss for windows ( version 14.0 ; chicago , il , usa ) . data are presented as means sd for continuous variables and frequencies ( percentages ) for categorical variables . differences between groups were evaluated with student s t test for continuous variables and test for categorical variables . multivariate logistic regression analysis was used to identify significant determinants of death after paraquat intoxication . the results of logistic regression analyses are reported as odds ratios ( or ) with 95% confidence intervals ( ci ) . statistical analyses were performed using spss for windows ( version 14.0 ; chicago , il , usa ) . characteristics of the 60 patients with acute paraquat poisoning at the time of their arrival are summarized in table 1 . the mean estimated amount of ingested paraquat dichloride was 83.89 ml ( range , 5500 ml ) . mean ang-1 level was 1.320.75 ng / ml . mean ang-2 level was 4.073.59 pg / ml . comparisons of ang-1 , ang-2 , ang-2 : ang-1 ratio , and vwf between survivors and non - survivors are shown in table 2 . although the 2 groups had no statistically significant difference in ang-1 , the ang-2 and ang-2 : ang-1 ratio were significantly higher in non - survivors than in survivors . hrct was done for 30 patients ( 22 survivors and 8 non - survivors ) . mild lung lesion was found in 21 patients and severe lung lesion in 9 patients . according to hrct , the 2 groups had no statistically significant difference in ang-1 , ang-2 , ang-2 : ang-1 ratio , or vwf ( not shown data ) . serial change of ang-1 , ang-2 , and ang-2 : ang-1 ratio in 3 patients is shown in figure 1 . the ang-2 : ang-1 ratio peaked within 1 day after paraquat ingestion , and then the level of the ratio decreased . the patients were grouped into 3 categories : survivors , early death group ( died within 5 days after ingestion ) , and late death group ( died more than 5 days after ingestion ) . the ang-2 : ang-1 ratio and plasma paraquat level were significantly higher in the early death group than in the others ( table 2 ) . in addition , the pco2 and hco3 ( meq / l ) were lower in this group . univariate analysis showed a statistically significant association between baseline plasma ang-2 level and death ( or , 1.001 ; 95% ci , 1.0001.002 ; p=0.017 ) . moreover , ang-2 : ang-1 ratio was significantly associated with death ( or , 1.928 ; 95% ci , 1.1163.331 ; p=0.019 ) , as was plasma paraquat level ( or , 1.078 ; 95% ci , 1.0521.137 ; p=0.006 ) . ang-1 ( or , 1 ; 95% ci , 0.9991.001 ; p=0.326 ) and vwf ( or , 1.045 ; 95% ci , 0.9931.100 ; p=0.091 ) were not associated with death . in multivariate analysis , ang-2 : ang-1 ratio was significantly associated with early death after adjustment for several combinations of potential confounders : plasma levels of paraquat ( or , 2.644 ; 95% ci , 1.2065.795 ; p=0.015 ) ; plasma levels of paraquat , and age ( or , 2.906 ; 95% ci , 1.2976.512 ; p=0.010 ) ; plasma levels of paraquat , age , and pco2 ( or , 2.644 ; 95% ci , 1.1735.960 ; p=0.019 ) ; and plasma levels of paraquat , age , pco2 , and creatinine ( or , 2.602 ; 95% ci , 1.1066.117 ; p=0.028 ) . the toxicity of paraquat is due to its induction of redox cycling , which leads to oxidative stress - related cell death and inflammation . because of selective accumulation in the lungs , especially in type ii pneumocytes , paraquat causes severe lung injury manifested by edema , hemorrhage , interstitial inflammation , and progressive fibrosis . first , patients who ingest less than 20 mg paraquat cation per kilogram of body weight may be asymptomatic or develop only gastrointestinal tract symptoms ; they recover without sequelae . second , patients who ingest between 20 and 40 mg paraquat cation per kilogram usually die 5 days to several weeks after ingestion . third , patients who ingest more than 40 mg paraquat cation per kilogram usually die within 15 days after ingestion , mainly because of multiorgan failure or shock . we investigated whether initial endothelial cell injury could involve the ros - induced injury model . we suspected that the cause of death in the early death group might be multiorgan failure due to endothelial cell injury . ang-2 : ang-1 ratio and ang-2 were higher in the early death group than in the other groups . these findings suggest that endothelial cell damage may have been involved in the pathogenesis resulting from paraquat intoxication in the early death group . although it has previously been suggested that endothelial cell damage occurs after paraquat poisoning , the underlying mechanism has not been revealed . paraquat could cause an increase in vascular permeability that is attributed to endothelial cell injury . some acute injury markers can predict mortality in cases of ali / ards . among these markers , ang-2 , ang-2 : ang-1 ratio , and vwf can induce endothelial cell injury , which increases vascular permeability . ang-2 , ang-2 : ang-1 ratio , and vwf may be good markers to predict mortality in sepsis models and septic patients [ 2,6,812 ] . increased vascular permeability is the key mechanism in the pathogenesis of sepsis - induced lung injury . paraquat - induced lung injury may be a good model for representing ros - induced lung injury . . the paraquat - induced lung injury model might be different from the lipopolysaccharide model , because ros - induced cell toxicity is a major mechanism in the paraquat model . the ang-2 : ang-1 ratio may reflect endothelial cell injury in the pulmonary microvasculature , which suggests that this mechanism could be involved in early damage to the pulmonary microvasculature and increased vascular permeability to paraquat . vwf did not predict mortality in patients with paraquat poisoning . the use of vwf to predict ald / ards in other situations has been controversial . a study on poisoned patient sample time might be important because of the different dose and time interval after poisoning . sabharwal et al showed that sequential plasma samples obtained from patients with ards had progressively increasing levels of vwf , up to days 4 and 8 . caflee et al showed that vwf did not have differential prognostic value for mortality depending on the presence or absence of infection as a risk factor for ali , but ang-2 is a prognostic marker . indeed , further basic study is needed to understand when and how paraquat injures the pulmonary microvascular system . the serial change of ang-1 , ang-2 , and ang-2 : ang-1 ratio is shown in figure 1 . we thought that paraquat - initiated endothelial injury might be associated with an early mechanism of pathogenesis and sequentially trigger inflammation and fibrosis . according to hrct , early death patients were excluded from this analysis because hrct was performed 7 days after ingestion ; this could be selection bias . ros - directed lung injury may have been the principle pathogenic mechanism in the patients who underwent hrct . second , the origin of ang-2 and ang-1 was unknown . regardless of origin , the ang-2 : ang-1 ratio might reflect the endothelial damage that occurs after paraquat intoxication . in conclusion , endothelial cell damage could play a role in pathogenesis leading to early death after paraquat ingestion .

the prognosis is bleak with the median survival in advanced disease around four months from diagnosis . survival compares unfavourably with other european countries and the usa [ 1 , 2 ] . it has often been felt that the scottish lung cancer population has more comorbidity with poorer performance status thus presenting fewer tolerable therapeutic options . it is difficult to quantify the complex nature of patient frailty to provide a degree of objective assessment of fitness and as a result prediction of survival in patients diagnosed with lung cancer remains problematical . currently , prognosis is based upon a combination of stage of disease and performance status although other factors such as weight loss have been identified in the advanced cancer setting [ 46 ] . however , these host factors ( weight loss and performance status ) included in clinical decisions are recognised to be subjective in nature . recent work shows that the effect of systemic inflammation is detrimental in terms of outcome in cancer in general [ 7 , 8 ] and in lung cancer specifically [ 916 ] . the combination of c - reactive protein and albumin when combined to calculate the modified glasgow prognostic score ( table 1 ) has been previously validated as an independent predictor of survival . the aim of the present study was to examine the clinical utility of this established objective marker of the systemic inflammatory response , the modified glasgow prognostic score ( mgps ) , as the basis of risk stratification in patients with lung cancer . four scottish centres were included in the study : aberdeen , glasgow ( stobhill ) , inverclyde , and west fife . over a defined period from 2005 to 2008 all newly diagnosed lung cancer patients coming through the regional multidisciplinary meetings ( mdts ) were included in the study . in total the details of 882 patients were collected prospectively . at the time of the patient discussion at the mdt annoymised details patient demographics and baseline characteristics ( age , sex , postcode , and smoking history ) , ps ( at time of presentation in addition to that six months prior to attendance as estimated by clinicians on questioning patients about fitness ) , weight loss , laboratory parameters ( c - reactive protein , albumin , and ventilatory function ) , tumour stage and histology , and primary treatment proposed by the mdt were all recorded . if the treatment proposed varied from the 2005 scottish intercollegiate guidelines network ( sign ) guidelines on lung cancer management the reasons why were also recorded ( i.e. , age , poor ps , comorbidity , size of tumour , etc . ) . all four mdts had input from an oncologist and thoracic surgeon and thus we could be confident of accuracy of the clinical management decision . information on date of death was obtained via survival analysis undertaken by the information service division ( isd ) of nhs scotland . death records were complete until 1 june 2011 , which served as the censor date for those alive . information on patients ' individual educational or occupational social class was not available , and postcode of residence was used to identify the 2006 scottish index of multiple deprivation ( simd ) ranking as a proxy indicator of their socioeconomic circumstances . the 2006 simd is a validated area - based index that uses 37 indicators in seven domains to rank 6505 small geographic areas in scotland ( data zones ) from 1 ( most deprived ) to 6505 ( least deprived ) . a venous blood sample was obtained at diagnosis for measurement of crp concentration and albumin . the coefficient of variation for these methods over the range of measurement was less than 5% , as established by routine quality control procedures . the gps was constructed as previously described ( table 1 ) [ 21 , 22 ] . in brief , crp more than 10 mg / l and albumin less than 35 g / dl were each given a score of 1 . the gps was calculated as 0 , 1 , or 2 . since hypoalbuminaemia alone in the absence of an increased crp level did not confer a poorer cancer - specific survival in all patients with cancer [ 8 , 22 ] , the gps was modified to assign a score of 0 in patients with hypoalbuminaemia alone ( table 1 ) . a number of recent studies have supported the use of mgps in predicting outcome both in lung cancer and other tumour types [ 917 ] . as such it was our intention to stratify the group by mgps and then analyse the impact of more conventional staging methods such as tnm stage and performance status . the audit was discussed with the local ethics committee , and since it was classed as a health service clinical practice audit , formal ethical approval was deemed not to be necessary . all statistical testing was conducted at the 5% level so 95% confidence intervals ( ci ) are reported throughout . unless otherwise stated , medians and interquartile range ( iqr ) are used . the survival time defined as the number of months from study entry until death or if alive at follow - up date was calculated . univariate survival analysis was carried out using the kaplan - meier method and the log rank test . survival analysis was carried out using cox 's proportional - hazards model and hazard ratios ( hr ) were calculated . multivariate survival analysis was performed using a stepwise backward procedure to derive a final model of the variables that had a significant independent relationship with survival . to remove a variable from the model , the corresponding p value had to be > 0.10 . statistical analyses were performed using spss v19.0 ( spss inc . , chicago , il ) . in total , 882 patients from a number of different treatment groups were included in the study , comprising 297 from aberdeen , 136 from west fife , 285 from glasgow , and 164 from inverclyde composing ; 59 patients were excluded from the study due to missing survival data ( figure 1 ) . the majority were male , current or ex - smokers , and of good performance status with advanced disease and had treatment with palliative intent . of the patients 24% were diagnosed on the basis of clinical examination and radiological investigations alone and without histological evidence . this compares favourably with the national lung cancer audit data , which had a median rate of 37% of patients who did not have histological confirmation of lung cancer . survival analysis using both gps and mgps was undertaken ( figures 2 and 3 ) . both were highly significantly associated with survival . since the mgps has been most extensively validated and readily extrapolated from previous work using c - reactive protein alone [ 7 , 17 ] , it was used in the remainder of the analysis and to stratify the three groups . there were 213 patients in the mgps score of 0 group , 290 patients in the mgps score of 1 group , and 218 patients in the mgps score of 2 group . the mgps was associated with increasing deprivation ( p < 0.001 ) , pack years smoking ( p < 0.001 ) , poorer performance status ( p < 0.001 ) , more weight loss ( p < 0.001 ) , more advanced disease ( p < 0.001 ) , more radical treatment ( p the relationship between the clinicopathological characteristics and survival in patients with an mgps of 0 is shown in table 4 . on univariate survival analysis , performance status ( p < 0.001 ) , weight loss ( p < 0.01 ) , stage of nsclc ( p < 0.001 ) , radical treatment offered ( p < 0.01 ) , and palliative treatment offered ( p < 0.05 ) were significantly associated with survival . on multivariate analysis , performance status ( hr 1.69 , 95% ci 1.392.06 , and p < 0.001 ) , weight loss ( hr 1.18 , 95% ci 1.041.33 , and p = 0.009 ) , stage of nsclc ( hr 1.06 , 95% ci 1.011.23 , and p = 0.017 ) and palliative treatment offered ( hr 1.30 , 95% ci 1.081.55 , and p = 0.004 ) were independently associated with survival . the relationship between the clinico - pathological characteristics and survival in patients with an mgps of 1 is shown in table 5 . on univariate survival analysis , decreased age ( p < 0.01 ) , performance status ( p < 0.001 ) , weight loss ( p < 0.01 ) , stage of nsclc ( p < 0.001 ) , and radical treatment offered ( p < 0.001 ) were significantly associated with survival . on multivariate analysis , performance status ( hr 1.81 , 95% ci 1.552.13 , and p < 0.001 ) , stage of nsclc ( hr 1.08 , 95% ci 1.031.13 , and p < 0.01 ) and radical treatment offered ( hr 0.70 , 95% ci 0.520.94 , and p < 0.05 ) were independently associated with survival . the relationship between the clinico - pathological characteristics and survival in patients with an mgps of 2 is shown in table 6 . on univariate survival analysis , centre ( p < 0.01 ) , performance status ( p < 0.001 ) , weight loss ( p < 0.001 ) , stage of nsclc ( p < 0.001 ) , and radical treatment offered ( p < 0.01 ) were significantly associated with survival . on multivariate analysis , only performance status ( hr 1.44 , 95% ci 1.211.71 , and p < 0.001 ) and weight loss ( hr 1.13 , 95% ci 1.001.28 , and p < 0.05 ) were independently associated with survival . the relationship between mgps , performance status , and survival at 1 year is shown in table 7 . when used in combination survival at 1 year varied from 72% ( mgps 0 , ps 0 ) to 6% ( mgps 2 , ps 3 ) . the numbers in the ps 4 subgroup were too small to calculate accurately a survival rate . the relationship between mgps , tnm stage ( nsclc patients only ) , and survival at 1 year is shown in table 8 . survival varied from 69% ( mgps 0 , stage i nsclc ) to 2% ( mgps 2 , stage iv nsclc ) . to stratify for stage , the relationship between mgps and ps and survival at 3 months for those patients with advanced nsclc ( st iiib / iv ) is shown in table 9 . survival varied from 100% ( mgps 0 , ps 0 ) to 23% ( mgps 2 , ps 3 ) . the number of patients in the ps 4 group was too small to accurately calculate survival . the relationship between mgps and ps at 3 months for those patients with advanced nsclc ( st iiib / iv ) undergoing palliative chemotherapy is shown in table 10 . survival varied from 92% ( mgps 0 , ps 1 ) to 50% ( mgps 2 , ps 2 ) . the numbers of patients in the ps 0 and 4 groups were too small to accurately calculate survival . the results of the present study show for the first time that , in a large cohort of patients with lung cancer and using the mgps as an objective basis for the prediction of survival , significant factors associated with survival varied significantly . only performance status and to a lesser extent tumour stage furthermore , the combination of the mgps with either performance status or tumour stage effectively stratified the likely outcome in these patients . therefore , this simple scheme based on objective criteria provides a new readily applicable approach to the routine clinical evaluation of patients with lung cancer . in the present study it was of interest that weight loss , a well recognised poor prognostic factor , was inconsistently prognostic when included in the analysis with mgps and performance status . this may suggest that much of the prognostic value of weight loss is attributable to the activation of the systemic inflammatory response and to the progressive loss of lean tissue leading to nutritional and functional decline . indeed , activation of the systemic inflammatory response resulted in a marked reduction in median survival of 13 months ( mgps 0 ) to 2 months ( mgps 2 ) independent of treatment received . this would suggest that the allocation of treatment was suboptimal and it may be that treatment allocated on a more objective scheme as proposed above will result in improved outcomes in all patients . for example , in those patients with mgps of 2 , neither stage nor treatment had independent prognostic value and therefore it would appear that such poor prognosis patients derive little benefit from standard anticancer treatment . in particular a very honest appraisal of both benefits and toxicities of any treatment should be made with the patient irrespective of their tumour stage . however it must be noted that the very small numbers of patients in these groups ( e.g. , only 2 patients underwent surgery and 4 underwent radical radiotherapy ) make it very difficult to interpret and further studies looking only at radically treatable patients are advised . the relationship between poor survival and systemic inflammation ( the mgps ) remains poorly understood , but it is likely to represent an objective marker of the chronic activation of the innate immune response with the consequent up - regulation of proinflammatory cytokines and growth factors and the resultant cancer cachexia [ 2631 ] . it is clear that , in scotland , lung cancer continues to confer a very poor outcome with a median life expectancy of approximately 5 months . even in early disease ( tnm i / ii nsclc ) , with patients undergoing radical treatment with an expectation of cure ( 1015% of total number of patients within scotland ) the 5-year survival is only around 3060% . the advent of more advanced imaging modalities such as pet - ct has improved detection of occult metastasis leading to stage migration and less patients undergoing futile radical local treatment . nevertheless , the present results highlight the importance of also staging the host systemic inflammatory response . the mgps is a simple , cheap , and reproducible prognostic tool that has been shown to be a rational starting point for such work . since the initial work , a decade ago the combination of c - reactive protein and albumin , the glasgow prognostic score ( gps / mgps ) , has been shown to have independent prognostic value in more than 60 studies ( > 30,000 patients with cancer ) . this prognostic value has been demonstrated in a variety of clinical scenarios , in particular primary operable cancer . a more recent study of apporximately 2,500 patients and this present study have demonstrated that the mgps has also clinical utility , together with performance status , in patients with advanced cancer . in conclusion , the results of the present study confirm the independent prognostic value of the mgps . in addition , it demonstrates the clinical utility of the mgps combined with performance status to provide more objective risk stratification in patients diagnosed with lung cancer .

atypical antipsychotics are believed to reduce the risk of extrapyramidal syndrome , including tardive dyskinesia ( td ) . quetiapine is reported to be less likely to induce td side effects and , in fact , has been shown to relieve symptoms of severe td1 ) and reduce td risk.2 ) quetiapine 's low affinity and fast dissociation from postsynaptic dopamine d2 receptors should contribute to low td symptom production.3 ) additionally , quetiapine reduces td side effects from clozapine , a drug already associated with very low td risk.4 ) however , since the first case report of quetiapine - related td side effects was published in 1999,5 ) quetiapine 's safety has been questioned . this case report describes severe , late - onset td side effects after long - term quetiapine use in a patient with psychotic depression . since td onset , the patient has had no significant findings based on comprehensive neurological examinations , brain magnetic resonance imaging , and electroencephalogram . the patient was a female homemaker who has suffered with psychotic depression for 3 years . her symptoms included depressed mood , lack of interest , lack of energy , suicidal ideation , insomnia , feelings of worthlessness , auditory hallucinations ( voices commenting around her ears and commanding her to die ) , and delusions of poverty . diagnosis was based on criteria of the diagnostic and statistical manual of mental disorders iv text revision ( dsm - iv - tr ) and the structured clinical interview for dsm - iv - tr . symptom severity was as follows : brief psychiatric rating scale-18 items ( bprs-18 ) score=31 , especially for hallucinatory behaviors ( 5 points ) , and hamilton rating scales for depression ( hrsd ) score=27 . quetiapine and duloxetine were delivered using gradual titration to relieve symptoms , and the final stable medication doses were quetiapine 600 mg / day and duloxetine 90 mg / day across 2.5 years . the patient 's depressive and psychotic symptoms responded to psychotropic medication and reached partial remission status ( bprs score=9 ; hrsd score=12 ) . she did not have extrapyramidal symptoms during the first two years after initiating the above medications . however , she began to experience severe perioral tremors with involuntary , repetitive , and irregular movements of tongue starting in the third year of quetiapine treatment . the protruding tongue with irregular movements was a significant detriment to her quality of life , as irregular movements occurred all day and were not relieved by anticholinergic medications . quetiapine - related td was suspected , and the quetiapine dose was tapered to 150 mg / day . however , after 3 months , td severity remained the same and seemed unresponsive to interventions , including switching to other atypical antipsychotics such as aripiprazole or ziprasidone . no significant exacerbations of psychotic or mood symptoms were observed after td onset and antipsychotic switch . the patient also received concurrent comprehensive neurological examinations , brain magnetic resonance imaging , and electroencephalograms . no significant alterations in brain anatomy or function were observed , and according to the neurological report , no organic etiology could be found to explain the presence of td . in this case , we observed unique , late - onset , and irreversible td side effects after long - term quetiapine use . late - onset td after 3 years might suggest independent factors . however , comprehensive neurological examinations and imaging studies could not pinpoint organic evidence to suggest that this patient 's td was independent of quetiapine use . late - onset td side effects in this patient appear irreversible even after decreasing quetiapine dose and switching antipsychotics . however , case reports of late - onset td have not been included in previous reports . rather , these reports mention the long - term safety of quetiapine in adolescent6 ) and adult patients.3 ) sacchetti and valsecchi7 ) showed that quetiapine has the lowest risk for td side effects when compared with olanzapine and clozapine in their long - term trial ( 154 weeks ) . quetiapine - related td was noted and described in an early case report about 15 years ago.5 ) however , some reports also mention contrary findings of quetiapine 's ability to relieve td.1,2,8,9,10,11,12,13 ) emsley et al.14 ) found that quetiapine could effectively reduce td severity in patients with established td , but the mechanism of this action is unclear . quetiapine is the atypical antipsychotic most similar to clozapine ( without its hematologic side effects ) based on receptor and pharmacologic profile , which may explain its treatment effects for td.11 ) quetiapine is believed to express lower affinity for d2 receptors in striatal and extrastriatal regions of brain,15 ) which could explain the mechanisms by which it can treat or relieve td side effects . the presence of mood disorders may be a possible reason for quetiapine - related td in this patient . sharma16 ) reported that patients with mood disorders were more prone to treatment - emergent td with quetiapine . recently , reports reveal quetiapine 's td side effects and suggest precautionary attention to td risk characteristics.17 ) a recent dopamine receptor imaging study showed that quetiapine occupies dopamine d2 receptors extensively in striatal regions , which likely contributes to td side effects.18 ) furthermore , low - dose quetiapine also seems to induce early - onset td in the neuroleptic - nave patient.19 ) recent reports provide evidence of quetiapine 's td risk as well as possible mechanisms . previously , it was suggested that quetiapine - related td could be relieved by tapering the dose or switching to another atypical antipsychotic such as aripiprazole.15 ) however , in this case study , td was not abated with quetiapine dose tapering or with a switch to other atypical antipsychotics . therefore , use of quetiapine should be approached with caution , with close attention paid to td risk factors .

india is estimated to have an adult ( 1549 years ) human immunodeficiency virus ( hiv ) prevalence of 0.27% in 2011 . adult hiv prevalence among males and females is estimated at 0.32% and 0.22% , respectively . according to the national aids control organisation ( naco ) the adult hiv prevalence at national level has continued its steady decline from estimated level of 0.41% in 2001 through 0.35% in 20060.27% in 2011 . the adult hiv prevalence rate in maharashtra shows a declining trend from 1.08% in 2002 to 0.67% in 2007 . the total estimated number of hiv positive persons is around 4.62 lakh , which accounts for 20% of the total estimated hiv - infected persons in the country and second highest after andhra pradesh . the clinical course of hiv disease and pattern of opportunistic infections ( ois ) varies from patient to patient and from country to country . tuberculosis is the most common oi and the most common cause of death in hiv / aids patients in india . hiv-1 is a neurotropic virus , central nervous system is among the most frequent and serious target of hiv infection in patients with profound immunosuppression . cryptococcus neoformans being an important cause among etiologies causing fungal meningitis . with the availability of antiretroviral therapy ( art ) at lower cost , the clinical profile of hiv disease in india is now changing to include drug - related toxicities and immune reconstitution syndrome . the clinical presentation and their system - wise profile is not adequately studied as changing the pattern of hiv - positive patients over a period of time . this was retrospective observational study carried out over a period of 1 year ( january 2011december 2011 ) . the study was carried out at tertiary , referral care center in western maharashtra india . all hiv patients diagnosed by naco guidelines were enrolled ( indoor patient ) for detailed analysis from record section . demographic data , clinical features , investigations , and outcome were recorded in a predesigned proforma from the case paper available at record section . all numerical variables were entered in statistical data entry sheet and calculated for mean , standard deviation , and chi - square test . the aim of this study was to study the clinical profile and outcome of hiv / aids patients , which includes demographic and clinical profile of hiv patients and prevalence of different types of oi and nonopportunistic diseases at a tertiary care teaching hospital . the aim of this study was to study the clinical profile and outcome of hiv / aids patients , which includes demographic and clinical profile of hiv patients and prevalence of different types of oi and nonopportunistic diseases at a tertiary care teaching hospital . total 75 ( 67.56% ) were male and 36 ( 32.43% ) were female patients , predominated by male population ( p < 0.05 ) . overall respiratory manifestations were the common presentation in a present cohort of hiv seropositive patients ( relative risk [ rr ] : 0.881 ; odds ratio [ or ] : 0.776 ) . of total 111 patients , 27 ( 24.32% ) patients were presented with neurological manifestation . the patients with cva were outnumbered compared to all other neurological manifestation in seropositive hiv patients ( rr : 0.421 ; or : 0.177 ) table 2 and figure 2 . five patients 15.63% had urinary tract infection table 5 . of total 111 patients , 17 ( 15.31% ) patients were presented with hematological manifestation . mean and standard deviation of various presentations of seropositive patients is shown in table 7 . there was no statistically significant difference in age between succumbed and discharged patients table 8 . respiratory manifestations including pulmonary tb were the most common presentation of patients with hiv positive status compared to the other manifestations ( p < 0.001 ) . clinical presentation and outcome of patients with human immunodeficiency virus patients were presented with the neurological manifestation of which 8 ( 29.62% ) patients had cerebro - vascular accident . the present study reveal cerebro - vascular accident was most common neurological manifestation with p < 0.05 . bilateral apical and mid zone fibro - cavitory lesions of pulmonary tuberculosis in human immunodeficiency virus ( b ) . right apical zone fibro - cavitory lesions with infiltrate of pulmonary tuberculosis ( c ) . bilateral extensive pneumonia in due to pcp ( pneumocystis jirovecii ) neurological manifestations of hiv patients ( a ) . magnetic resonance imaging brain shows diffuse periventricular and subcortical white matter changes of progressive multifocal leukoencephalopathy inhuman immunodeficiency virus patient ( b ) . computed tomography brain right middle cerebral artery + posterior cerebral artery territory and left cerebellar ( posterior inferior cerebellar artery ) infarct ( d ) . computed tomography brain posttuberculous meningitis squeal communicating hydrocephalus gastrointestinal manifestations of hiv patients cardiovascular manifestations of hiv patients miscellaneous manifestations of hiv patients haematological manifestations of hiv patients meansd of age of isolated clinical manifestations in hiv patients mean agesd of male and female patients with hiv ( discharged and death ) clinical presentation and outcome of patients with human immunodeficiency virus the present study highlighted the clinical profile and outcome of hiv patients pertaining to opportunistic and nonopportunistic diseases . reported cryptococcal meningitis was seen in 5 ( 29.4% ) patients , tubercular meningitis in 9 ( 52.9% ) patients , hiv encephalopathy in 2 ( 11.7% ) . similarly , in our study , 27 ( 24.32% ) patients were presented with neurological manifestation of them 5 ( 18.51% ) had cryptococcal meningitis 4 ( 14.81% ) had tubercular meningitis , 4 ( 14.81% ) had aseptic meningitis , 1 ( 3.70% ) had pyogenic meningitis . seizures are common in advanced stages of hiv infection ; all cases of cryptococcal meningitis had seizures and 7% in tuberculous meningitis . in our study , 8 ( 25% ) patients had a general tonic - clonic seizure . studied first 19 indian patients with aids , in which diarrhea was the common presentation and tb was seen in 13 patients and the most common secondary infectious with oropharyngeal candidiasis was found in 11 patients and one patient had cryptococcosis . similarly , in our study , 23 ( 44.23% ) had pulmonary tb , 6 ( 11.53% ) had tubercular effusion , 1 ( 3.22% ) had ileo - cecal tb and 6 ( 19.35% ) had oral candidiasis and 5 ( 18.51% ) had cryptococcal meningitis with overall case fatality rate of 9.90% . the differences in the mortality could be due to early diagnosis and availability of art now - a - days . similarly , misra et al . reported that the tb ( pulmonary and extrapulmonary ) is the major oi affecting 62% of the cases followed by candidiasis seen in 57% of the patients . singh et al . studied 100 aids patients with oral candidiasis ( 59.00% ) was found to be the most common oi , followed by tb ( 56.00% ) and p. jirovecii ( 7.00% ) . in our study , 44.23% patients had pulmonary tb , 5.76% had p. jirovecii pneumonia and 19.35% had oral candidiasis . in contrast to our study , chakraborty et al . reported oral candidiasis in 88% was found to be the most common oi , followed by tb in 57% . studied , 438 hiv positive patients attending the hiv clinic of these 354 were males and 84 were females . the mean age of the study subjects at the time of diagnosis was 32.6 years . the most common oi was tb ( 38.8% ) followed by oropharyngeal candidiasis ( 20.3% ) and diarrhea ( 12.7% ) . sharma et al . studied 135 consecutive , hiv - infected patients ( age 34 10 years ) predominated by male patients tuberculosis was the most common oi ( 71% ) followed by candidiasis ( 39.3% ) , pneumocystis jirovecii pneumonia ( 7.4% ) , cryptococcal meningitis ( 3.7% ) . majority of deaths were due to tb and pcp . similarly in our study 23 ( 44.23% ) had pulmonary tb , 6 ( 11.53% ) had tubercular effusion , 6 ( 19.35% ) had oral candidiasis , and 5 ( 18.51% ) had cryptococcal meningitis with overall case fatality rate of 9.90% . reported that tb was the most common oi with an incidence of 15.4 , oral candidiasis 11.3 and cryptococcal meningitis 1.7 per 100 person - years . studied 61 patients with aids and found that , 31% had chronic diarrhea , tuberculosis in 52% patients was the most common secondary infection , 45 ( 74% ) patients had ois such as oropharyngeal candidiasis 25 ( 41% ) cryptococcal meningitis 4 ( 7% ) , one ( 1.6% ) patient or immunoblastic lymphoma 33 ( 54% ) patients have died . similarly , in our study 38.70% had acute gastroenteritis , 44.23% had pulmonary tb , 11.53% had tubercular effusion , 19.35% had oral candidiasis , and 18.51% had cryptococcal meningitis and 1 ( 5.88% ) had nhl with overall case fatality rate of 9.90% . studied 154 hiv - seropositive subjects with intestinal infections are a significant cause of morbidity and mortality in people living with hiv . similarly , in our study , 75 ( 67.56% ) were male and 36 ( 32.43% ) were female patients with hiv , predominated by male population with male to female ratio of 2.08:1 . similar to our findings , madkar et al . in their 178 hiv positive patients observed that most common opi was tb ( 59% of patients ) followed by oral candidiasis ( 37.6% of patients ) with male to female ratio is 3.3:1 . a maximum number of males ( 79.81% ) as well as females ( 84.77% ) was in the age group of 2039 years . suthar et al . in their retrospective study of 60 hiv patients with pulmonary tb with 35 cases ( 58.33% ) most common total 35% of patients had typical pattern and 65% had atypical pattern of pulmonary tb on chest radiograph . the mean age of these patients were 34.45 8.40 with male to female ratio of 7:1 with tb and candidiasis were common oi . in our study the mean age was 40.8 10.47 years with two - third of male population with hiv . studied 26 hiv patients with mean age of presentation was 40.04 7 years with male : female ratio of 4.2:1 . the most common mode of presentation was tb ( 61% ) , both pulmonary ( 46% ) and extrapulmonary ( 15% ) . similarly , in our study , 67.56% were male and 32.43% were female patients ( p < 0.05 ) with 44.23% had pulmonary tb , 11.53% had tubercular effusion , 22% had ileo - cecal tb and 19.35% had oral candidiasis . overall ops , non - ops , male predominance and mean age of hiv in the present study are comparable with other study . the present report highlights the clinical manifestations related to hiv infection from the maharashtra of india . overall respiratory manifestations were the common presentation in present cohort of hiv seropositive patients and tb was the most common oi . tuberculosis , candidiasis , cryptoccocal meningitis and p. jirovecii pneumonia were the most common ois in the present study . anemia , diarrhea , oropharyngeal candidiasis , ischemic heart disease were the most common hematological , gastrointestinal , and cardiovascular manifestation respectively in hiv patients . clinicians should consider hiv in the differential diagnosis and management of all persons with tb . emphasis needs to be given to the early diagnosis and management of tb in hiv - infected individuals . a wide spectrum of disease , including both ois and noninfectious opportunistic diseases , is seen in hospitalized hiv - infected patients from western maharashtra . tuberculosis remains the most common oi and is the commonest cause of death in these patients . cryptococcal meningitis indicates progression of hiv infection toward aids and is useful as a reference to starting art in settings where facilities for determination of cd4 counts are not available . the study emphasizes that , education ; counseling , early diagnosis of ois , awareness of the disease and early anti - viral drug treatment can help to bring down the number of aids cases . there is need for early screening and increasing awareness in healthcare providers to make a diagnosis of hiv so as to reduce morbidity and mortality .

diarrhea is an important childhood infection globally , accounting for 2.5 million deaths in under - five children every year . passage of blood and mucus in stool associated with high - grade fever , tenesmus , and intestinal cramps are common symptoms . arthritis , conjunctivitis , parotitis , intussusception , rectal prolapse , and hemolytic uremic syndrome ( hus ) may occur as complications , especially in the elderly , children , and immunocompromised patients . resistance to antibiotics such as ampicillin , co - trimoxazole , tetracycline , chloramphenicol , and aminoglycosides have become commonplace over decades . furthermore , resistance to third - generation cephalosporins , azithromycin , and fluoroquinolones are also on the raise . this limits the antibiotic options available for empirical therapy , especially in children who also have a higher incidence of severe infections , complications , and mortality . here , we describe a case of bloody diarrhea in an infant caused by multidrug resistant shigella flexneri , presenting with the clinical features simulating intussusception . although the rapid evaluation with emergency abdominal ultrasonography was imperative to rule out intussusception , treatment with appropriate antibiotic resulted in the resolution of acute bloody diarrhea . a 1-year - old male child was brought by parents to our hospital with several episodes of loose stools lasting 3 days with fever , vomiting , and abdominal pain . icterus , petechial rash , ecchymosis , splenomegaly , and joint tenderness were not detected . it showed edematous loops of terminal ileum with few enlarged mesenteric lymph nodes and minimal free fluid in abdomen suggesting segmental ileitis . hemogram showed hemoglobin 6.2 g / dl , total leukocyte count 15,300/cmm with 46% neutrophils , 50% lymphocytes , 3% monocytes , and 1% eosinophils . the platelet count , serum urea , creatinine , electrolytes , and bilirubin values were within normal range . blood smear revealed normocytic / normochromic red blood cells ( rbcs ) without any acanthocytes , bite cells , and fragmented forms . urine was normal in color and had no pus cells , rbcs , or hemoglobinuria . although the frequency decreased , the child continued to pass loose mucoid greenish stools with streaks of blood . on microscopic examination , the stool was negative for ova , cyst , and trophozoites of intestinal parasites , and it showed 1525 leukocytes and 1015 rbcs per high - power field . a nonmotile , gram - negative bacillus producing nonhemolytic colonies on blood agar and nonlactose fermenting colonies on macconkey 's agar was isolated from the stool . it was identified as s. flexneri based on biochemical reactions ( catalase positive , indole negative , methyl red test positive , voges - proskauer test negative ; citrate not utilized , urea not hydrolyzed ; alkaline slant and acidic butt with no gas and no h2s production in triple sugar iron agar , glucose and mannitol fermented with acid only ; lactose , sucrose and xylose not fermented ; and lysine , arginine , and ornithine not decaboxylated ) . the isolate showed susceptibility to ceftriaxone and gentamicin , but it was resistant to co - trimoxazole , chloramphenicol , tetracycline , ampicillin , furazolidone , nalidixic acid , and ciprofloxacin . hourly ) was continued for 6 days along with intravenous fluids ( for 2 days ) . the baby was discharged on the 7 day with a plan to investigate the cause of anemia on subsequent visits . invasive infections ( i.e. shigella sp . , enterohemorrhagic escherichia coli ( ehec ) , campylobacter jejuni , yersinia enterocolitica , aeromonas hydrophilia , entamoeba histiolytica , and trichuris trichiura infections ) as well as noninfectious diseases such as inflammatory bowel disease ( ibd ) , colitis associated with cow 's milk , necrotizing enterocolitis , intussusception , and intestinal ischemia have been implicated in bloody diarrhea among children . while abdominal ultrasonography and endoscopy are essential for the diagnosis of noninfectious causes , stool microscopy , and culture detect the pathogenic microorganism . among these causes , shigellosis is , especially common in infants in developing countries . in a study from north india , the most prevalent shigella species among hospitalized children with diarrhea between 2001 and 2004 was s. flexneri ( 60% ) , followed by shigella sonnei ( 23.8% ) , shigella dysenteriae ( 9.8% ) , and shigella boydii ( 5.7% ) . s. flexneri , especially serotype 2a , was found to be the most common serotype among children in several other studies from the indian subcontinent . grossly , bloody stools , dehydration , and seizures have been associated more frequently with shigella species when compared with other intestinal bacterial pathogens . unlike s. dysenteriae type 1 and s. flexneri have been less frequently reported to cause severe extraintestinal as well as intestinal manifestations ( i.e. intussusception and rectal prolapse ) . in our case , abdominal cramp , vomiting , pallor , dehydration , and passage of bloody stool with mucus in the infant simulated the clinical picture of intussusception , mandating abdominal ultrasonography to rule it out . surgical intervention for intussusception is indicated in case of bowel perforation and unsuccessful nonoperative reduction . as the baby was anemic , packed cells transfusion was given to prevent the complications of blood loss which might have occurred in the event of surgical reduction of intussusception . since acute bloody diarrhea is unlikely to result in severe anemia , further investigations to rule out ibd , hook - worm infection , and other causes of severe anemia hus is a dreadful complication of shigella and ehec serotype o157:h7 infection , characterized by hemolytic anemia , thrombocytopenia , and acute renal failure . in this patient , normal platelet counts , the absence of microangiopathic hemolytic anemia ( acanthocytes , bite cells , and fragmented rbcs ) , and normal renal parameters were essential to rule out hus . ampicillin , co - trimoxazole , and nalidixic acid were the treatment of choice for shigellosis in developing countries . hence , the third generation cephalosporins , azithromycin , and fluoroquinolones came up as alternatives . re - emergence of multidrug resistant s. dysenteriae type 1 strains during 2002 and s. flexneri type 2a since december 2003 with resultant year - wise increase in quinolone resistance from 15% in 2003 to 25% in 2004 have been reported from west bengal . bhattacharya et al . have studied the trend of antimicrobial resistance in shigella isolates from andaman and nicobar , which showed noticeable increase in resistance against ampicillin ( 72.7100% ) , co - trimoxazole ( 57.680% ) , nalidixic acid ( 27.296% ) , ciprofloxacin ( 0 to 82% ) , and the third generation cephalosporins ( 0 to 12% ) between 2000 and 2009 . both plasmids - coded esbls of shv-11 and ctx - m type and ampc type beta - lactamases have been found in shigella spp . to impart the third generation cephalosporin resistance . in our case , although the s. flexneri isolate was multidrug resistant , it was not an esbl producer . therefore , prompt initiation of ceftriaxone along with good supportive care was effective in shortening the duration of symptoms and preventing complications .

the standard approach of internal jugular vein ( ijv ) for central venous cannulation ( cvc ) using visual and palpable anatomic landmarks is associated with 95% success rate , but it carries the complications from inadvertent puncture of surrounding structures , including the common carotid artery due to significant variation in the degree of overlap of the ijv and carotid artery when patients are placed in a cannulating position . the risk of accidental carotid artery penetration may increase with puncture of the posterior wall of the ijv . carotid artery puncture has an incidence ranging from 3% to 10% independent of the technique chosen or operator experience . although carotid artery puncture is usually a benign event , it can be life - threatening ( inadvertent intra - arterial cannulation , stroke , haemothorax , carotid artery - ijv fistula or airway compromise due to a neck haematoma ) . the true incidence of puncturing the posterior wall of the ijv during cannulation in patients is unknown and underreported . this complication is commonly noted in patients with dehydration , respiratory distress , collapsible ijv and when the ijv diameter is 4 to 5 mm . multiple studies have demonstrated an improved success rate and a decreased complication rate for ultrasound - guided vascular access as compared to the traditional landmark technique . this study compared the incidence of posterior wall puncture with real - time ultrasound during ijv cannulation using the traditional ' blind ' landmark - guided technique versus ultrasound - guided technique . the study was conducted after being passed by due approval from the institutional ethics committee . a written , informed consent was obtained from each patient / relative for central line placements as per hospital policy . both surgical and medical gastroenterology patients in whom central venous lines were indicated as a part of their medical management were included in the study . the exclusion criteria included patient / relatives refusal for central venous line placement , choice of alternate site for central venous cannulation ( besides ijv ) , presence of thrombus within the jugular vein and infection at chosen site of catheter insertion . the patients were randomised using computer - generated random number table , and the allocation was concealed in an opaque - sealed envelope . the envelope was opened just before the central line ( ijv line ) placement . in both groups , multicath 3 ( vygon) triple lumen catheters ( outer diameter 2.5 mm , 7.5 fr , 15 cm length ) were inserted using the seldinger technique . the sonosite m - turbo ( fujifilm , japan ) portable ultrasound machine was used . the linear ( vascular ) high - resolution probe ( 511 mhz ) was selected for visualisation of the needle path in an out of plane ( transverse ) view . in both groups , central venous cannulations were performed by critical care physicians with > 5-year experience in putting internal jugular central venous pressure ( cvp ) lines using landmark - guided technique and more than 1-year experience in real - time ultrasound - guided line placement . in group a , the cvc was placed using the traditional landmark - guided technique . in this , the patient was placed supine with the neck turned to the opposite site , the two heads of the sternocleidomastoid muscle were identified and the apex of the triangle formed by the two heads of the sternocleidomastoid muscles was chosen as the point of entry of needle . the investigator performing the cannulation stood on the head end of the patient while the usg probe was placed transversely ( out of plane ) just below the point of entry of needle by the second investigator who stood by the side of the patient . after feeling for the pulsations of the carotid artery at this point , the needle and syringe assembly was inserted just lateral to it and directed towards the ipsilateral nipple . , the second investigator followed the path of the needle and watched for a posterior wall puncture ( through - and - through puncture ) or an imminent or actual arterial puncture . the person cannulating ( the first investigator ) did not have access to the ultrasound images as the screen of the ultrasound machine was turned away from him / her . in the interest of patient safety , the second investigator warned the cannulating investigator in the event of an imminent arterial puncture . such event was considered equal to an arterial puncture and noted in data collection sheets . in group b , cvc was placed using real - time ultrasound guidance . here , both the investigators , the one cannulating and the other handling the probe were able to follow in real time , the movement and the position of the needle . in this group , the patient was placed supine with the neck turned to the opposite site ; the vascular probe was placed transversely at the apex of the triangle formed by the two heads of the sternocleidomastoid muscle by the second investigator . the vein was identified and centred in the middle of the screen and the needle and syringe assembly was inserted from the midpoint of the probe by the first investigator and the path of the needle tip was followed by both the investigators till successful venous cannulation was confirmed by the visualisation of the needle tip within the lumen of the vein and aspiration of venous blood in the syringe . in both groups , the intraluminal position of the guidewire within the vein was further confirmed sonographically before dilation and insertion of the catheter . after the procedure , the second investigator filled out standardised data collection sheets ( given below ) . they recorded the number of times each physician penetrated the posterior wall of the ijv . if the needle was driven in at an angle and initially penetrated the anterior wall and then the side wall , this was also considered a posterior wall penetration . the following parameters were observed and filled in the data collection sheets : ( a ) the position of ijv relative to carotid artery as seen just before insertion of the needle . here , the second investigator scanned the neck and estimated the degree of overlap between the artery and vein . if there is more than 3/4 ( 75% ) overlap , then the position was deemed anterior , if less than 3/4 was anterolateral , if no overlap then it was considered lateral ; ( b ) number of attempts to locate the vessel ( number of times the needle required reinsertion or was withdrawn and required repositioning ) ; ( c ) posterior wall puncture ( through - and - through puncture of vein ; this was the primary outcome measure for the study ) ( d ) imminent / actual arterial puncture ( e ) opening cvp post - line placement to assess volume status ( f ) final position of needle before insertion of guide wire ( g ) other complications , namely , haematoma formation and pneumothorax . this sample size was determined on the basis of a pilot study previously conducted in this same unit comparing the incidence of posterior wall puncture using a land mark - guided technique and a real - time ultrasound - guided technique . the result of a previously performed pilot study on thirty patients ( 15 per group ) ( 8/15 [ 53% ] in landmark guided technique had posterior wall puncture compared to 3/15 [ 20% ] in the ultrasound guided technique ) suggested that two groups of at least 80 patients would be sufficient for an adequately powered study . one hundred and seventy patients were enrolled in this study . at the time of analysis , group a had eighty patients , and group b had ninety patients . the age , coagulation parameters and opening cvp were also similar in both groups [ table 1 ] . relative position of the ijv with respect to the artery on minimal compression of the neck with ultrasound probe was similar in both groups [ table 2 ] . the most common position in both groups was anterolateral position of vein over the artery , this was seen in 92% of patients in group a vs 86% in group b. the number of attempts at successful cannulation was significantly lesser in group b ( p = 0.00 ) [ table 3 ] . in group a , 37/80 ( 46% ) had inadvertent posterior wall puncture as compared to 19/90 ( 21% ) in group b ( p = 0.00 ) [ table 4 ] . the total incidence of arterial puncture / imminent puncture was 8/80 ( 10% ) in group a as compared to 5/90 ( 5.5% ) in group b. this difference was statistically significant [ table 5 ] . in group a , there were two instances of arterial puncture and six instances of imminent arterial puncture . in group all the arterial punctures in group b ( imminent plus actual ) were associated with a posterior wall puncture . the incidence of other complications ( namely haematoma formation and pneumothorax ) was significantly lesser in group b as compared to group a. 11/80 ( 14% ) patients in group a had hematoma formation vs 1/90 ( 1.1% ) in group b ( p = 0.003 ) . 1 patient had a pneumothorax formation in group a and no patient had pneumothorax in group b. [ table 6 ] age , coagulation profile , number of attempts and opening central venous pressure relative position of internal jugular vein with respect to carotid artery number of attempts for successful cannulation incidence of posterior wall puncture of internal jugular vein incidence of inadvertent arterial puncture incidence of other complications the true incidence of posterior wall puncture ( through - and - through puncture ) of the ijv during cannulation is unknown and underreported in literature . it is usually considered to be a benign event , and at times , a universal occurrence in patients who are dehydrated wherein the blood may be aspirated on withdrawing the needle rather than on the first pass . in the classical cannulation position ( neck turned to contralateral side ) , the jugular vein overlaps the carotid artery in 70%90% of the time and more so with increasing age and with increasing degree of neck rotation . in our study the true lateral position of vein to artery with no overlap was seen only in 3.3% of cases , similar to another study findings by maecken et al . the use of ultrasound for real - time jugular venous cannulation has improved the success rate and reduced the complications associated with blind ' landmark ' guided cannulation . even with the use of ultrasound , complications using ultrasound guidance for needle placement have been described , and lack of visualisation of the needle tip during needle advancement may contribute to inadvertent puncture of vital anatomic structures and failed vascular access attempts . besides even with ultrasound guidance , the true incidence of posterior wall puncture is not known . the most common approach to ultrasound guidance is a short - axis or transverse visualisation of the central vein . short - axis technique appears to be favoured by novice sonologists and is described as being easier to perform and taking less time than the alternative longitudinal guidance approach . a study found that residents with some prior experience in ultrasound - guided vascular access , regularly had a through - and - through puncture of the ijv in a life - sized torso vascular access mannequin . the residents not only lost track of the needle and regularly penetrated the posterior vessel wall but also cannulated the carotid artery on several instances . ijv cannulation by emergency physicians in a simulated model ( by evaluating the path of needles that had been previously placed into vascular access tissue phantoms ) , the incidence of posterior vein wall penetration was found to be 34% . present study was performed on humans ( previous published studies have been only on mannequins or vascular tissue phantoms ) . results of present study suggests posterior venous wall penetration ( through - and - through puncture ) of the ijv is a common occurrence during traditional landmark alone guided cvc placement , the use of real - time ultrasound reduces but does not totally eliminate the occurrence of this event . indeed , the reported incidence in this study may be higher than the previous studies despite the presence of experienced personnel . this may be because , this study was performed on live individuals where the respiratory variation in vessel size and smaller vessel size in hypovolaemia could have played a role . accidental penetration of the posterior wall of the vessel may also be attributed to other factors which could include the angle and speed of insertion of needle , the distance from needle entry point and the transducer margin and loss of needle track during insertion . the clinical implications of such through - and - through puncture however still remain unclear . posterior wall punctures did predispose to arterial punctures in both groups but not all patients who had posterior wall puncture had subsequent arterial puncture . in addition , special precautions can be taken in patients whose veins are collapsed or show significant variation with respiration such as reducing the angle of insertion , reducing the speed of insertion and close watch over the track of the needle tip . with respect to loss of needle track , an in - plane approach may be beneficial as it allows better visualisation of the entire needle path and may help in reducing the incidence of posterior wall puncture . further studies on hypovolaemic subset of patients may be required to further clarify the implications and precautions that can be taken . in addition , a larger study group with higher numbers of posterior wall punctures would have to be studied to determine the actual incidence of arterial punctures following a posterior wall puncture . real - time ultrasound - guided ijv cannulation significantly reduces but does not wholly eliminate the incidence of posterior venous wall ( through and through ) penetrations . real - time ultrasound guidance does reduce the number of attempts for successful cannulation and significantly reduces the risk of complications associated with blind cannulations and at the same time allowed prompt diagnoses complications including arterial punctures , haematoma formation and pneumothorax and hence contributes to increased patient safety profile .

pioglitazone is an established insulin sensitizer hugely successful in therapeutic management of type 2 diabetes mellitus . the beneficial effects extend beyond glycemic control and have positive effects on lipid metabolism , blood pressure , endothelial function , adiponectin , and c - reactive protein levels . pioglitazone was a safer option for patients till it got involved in a controversy because of the side effects . due to the concerns over bladder cancer as an adverse effect , the manufacture , sale , and distribution of the drug was temporarily banned in india . it was subsequently revoked , but with the boxed warning relating to bladder cancer . the clinical use is further limited by the spectrum of side effects that include weight gain , decrease in hematocrit values , edema , heart failure , fractures , and worsening of diabetic macular edema . heart failure is well documented in patients with known left ventricular dysfunction . however , in patients with normal left ventricular systolic and diastolic function , it has not been widely reported . we report a patient who developed congestive heart failure and pulmonary edema with normal left ventricular function within 1 year of starting pioglitazone therapy . a 65-year - old , non - obese male , a diabetic for the last 10 years on oral hypoglycemic agents ( oha ) , was admitted in the emergency department with the chief complaint of progressive breathlessness for last 15 days . there was no history of chest pain , palpitations , syncope , fever , cough , wheeze , and abdominal distension . , patient was taking combination of pioglitazone 30 mg , metformin 1 g , and glimepride 2 mg for the last 1 year . at the time of admission , pulse was 100/minute and was regular . vesicular breath sounds were heard with extensive fine end - inspiratory crackles up to the interscapular area . further examination was normal . on investigations , hemoglobin was 11.6 g% and total leukocyte count was 11,200/mm . biochemistry revealed the following values : random blood glucose- 276 mg% , blood urea nitrogen- 20 mg% , serum creatinine- 0.9 mg% , serum aspartate aminotransferase- 58 u / l , alkaline phosphatase- 215 u / l , and serum albumin 3.6 g% . chest x - ray revealed picture of pulmonary edema with evidence of bilateral pleural effusion [ figure 1a ] . a contrast - enhanced computed tomography ( cect ) of chest done in a secondary care hospital was brought by the patient , which revealed ground glass opacities , particularly in the perihilar area , thickened interlobular septa , and bilateral pleural effusion [ figure 1b ] . with this clinical and investigational profile , possibility of ( b ) computed tomography chest showing ground glass opacities , interlobular septal thickening , and bilateral effusion patient was started on heart failure treatment protocol . oha were stopped and patient was started on premixed insulin ( 30:70 ) for control of blood glucose . on the 2 day of admission , patient 's symptoms improved and 2d echocardiogram was done which documented normal systolic and diastolic functions , normal cardiac chambers , and no regional wall motion abnormality . the measurements on m - mode were : ivsed- 10 , ivses- 13 , lved- 56 , lves- 35 , pw ( lv ) ed- 10 , and pw ( lv ) es- 13 . over the next few days , his symptoms improved gradually . on chest examination , the crackles disappeared , and pedal edema and raised jugular venous pressure resolved . serial chest x - rays and repeat cect chest showed radiological clearance [ figure 2a and b ] . as no other cause for biventricular failure could be made and patient was on pioglitazone for the last 1 year , possibility of pioglitazone - induced pulmonary edema was suspected . the adverse drug reaction ( adr ) was reported to the hospital adr monitoring center . ( a ) chest radiograph and ( b ) computed tomography chest showing resolution of ground glass opacities , interlobular septal thickening , and bilateral effusion numerous drugs , either in monotherapy or in combination therapy , are used in the treatment of type 2 diabetes to achieve glycemic goals and subsequently reduce complications . we diagnosed a diabetic patient with congestive heart failure and pulmonary edema , who had preserved left ventricular ejection function on echocardiogram within 1 year of starting pioglitazone therapy . pioglitazone is the only thiazolidinedione approved currently for use in type 2 diabetes mellitus after it faced uncertainty due to safety issues . in addition to lowering blood glucose , pioglitazone has a beneficial effect on lipid profile , blood pressure , inflammatory biomarkers , endothelial function , and fibrinolytic status . however , weight gain , decrease in hematocrit values , edema , cardiac failure , fractures , and possible worsening of diabetic macular edema are well - recognized complications of pioglitazone . it is due to reduction in renal excretion of sodium and an increase in sodium and free water retention . increased sympathetic nervous system activity , altered interstitial ion transport , alterations in endothelial permeability , and peroxisome proliferator activated receptor mediated expression of vascular permeability growth factor represent other possible mechanisms for edema . fluid retention exacerbates pre - existing heart failure or precipitates heart failure in a patient with underlying left ventricular dysfunction . however , pathogenesis of heart failure in a patient with normal left ventricular function is not known . it is also known that thiazolidinedione therapy does not have significant effects on left ventricular function based on echocardiographic studies . studies of rosiglitazone indicate that a dose - related effect on pulmonary endothelial permeability , rather than alterations in left ventricular mass or ejection fraction , is probably responsible for the development of pulmonary edema and , in susceptible patients , cardiac failure . the prospective pioglitazone clinical trial in macrovascular events ( proactive ) study examined the incidence of heart failure in patients taking pioglitazone compared with placebo . heart failure leading to hospital admission occurred significantly more often in patients taking pioglitazone compared with placebo ( 5.7% vs 4.1% ) . however , the mortality rate due to heart failure was proportionately lower with pioglitazone ( 26.8% vs 34.3% ) . the reason for the reduced all - cause mortality despite increased risk of heart failure is better glycemic control and lesser myocardial infarction and stroke . the risk of heart failure associated with the use of non - insulin blood glucose lowering drugs has been reviewed . information on the risk of heart failure in glitazone users compared with sulfonylureas users is very scarce , and the risk of heart failure in sulfonylurea users is higher compared with metformin users . in view of this , the probability of occurrence of heart failure due to second generation of sulfonylureas can not be ruled out . however the findings of the same meta - analysis show that the likelihood of occurrence of heart failure due to pioglitazone is higher as compared to sulfonylureas . to summarize , evaluate every patient on pioglitazone , particularly with multiple medications , old age , and chronic renal disease , for all possible side effects by careful monitoring and follow - up .

chronic helicobacter pylori infection is causally associated with gastritis , gastroduodenal ulcer disease , gastric adenocarcinoma and mucosa - associated lymphoid tissue lymphoma . a genome - wide association study ( gwas ) of anti - h . pylori serologic status among europeans identified inverse associations with single nucleotide polymorphism ( snps ) in the toll - like receptor ( tlr ) locus at 4p14 and the fc receptor 2a ( fcgr2a ) locus at 1q23.3 . pylori immunoglobulin g ( igg ) antibody levels in the highest quartile vs. lower levels , the 4p14 associations were strongly significant ( top - ranked snp , rs10004195 ; p=1.4e-18 ) and the 1q23.3 associations were borderline ( top - ranked snp , rs368433 ; p=2.1e-8 ) . in contrast , there were no genome - wide significant associations with anti - h . pylori antibody levels in a gwas among mexican - americans . to extend the previous findings among caucasians pylori igg with 4p14 and 1q23.3 loci in the alpha - tocopherol , beta - carotene cancer prevention study ( atbc ) . the per - allele odds ratio ( or ) was 0.61 ( 95% confidence interval ( ci)=0.470.79 ; p=2.2e-4 ) , consistent with the previous report . in contrast , seropositivity ( 73% of participants ) was not associated with rs10004195 ( or=1.00 ; 95% ci=0.791.27 ; p=9.9e-1 ) . indeed , the minor allele frequency ( maf ) of rs10004195 among seronegative individuals ( maf=0.15 ) was intermediate between the subjects with the highest 25% of antibodies ( maf=0.11 ) and all other seropositives ( maf=0.17 ) ( supplementary table 1 ) . notably , the statistical significance as well as magnitude of effects was accentuated when we restricted our analysis to seropositive individuals ( figure 1 ) . for example , the per - allele beta coefficients of rs10004195-a were 0.15 ( standard error ( se)=0.05 ; p=3.4e-3 ; pfdr = 4.0e-2 ) and 0.20 ( se=0.04 ; p=3.8e-7 ; pfdr = 4.8e-6 ) among all and seropositive participants , respectively . the strongest signal was observed at rs6835514 ( maf=0.17 ; beta=0.23 ; se=0.04 ; p=1.6e-9 ; pfdr = 1.9e-6 ) , which was in moderate linkage disequilibrium ( ld ) with rs10004195 ( r=0.62 ) . fifty - one nearby snps within moderate to high ld ( r > 0.6 ) of rs6835514 had p - values ranging from 4.7e-7 ( pfdr = 5.6e-6 ) to 3.0e-9 ( pfdr = 1.9e-6 ) ( supplementary table 2 ) . in analyses controlling for rs6835514 , the effect of rs10004195 did not remain significant ( beta=0.03 ; se=0.06 ; p=5.5e-1 ) , indicating that the two snp associations with igg levels are not independent . furthermore , we explored putative functional effects of these 52 4p14 snps based on publically available data . except for rs4833095 ( asn248ser ) and synonymous rs5743614 in tlr1 , all other variants were located in intronic or non - coding regions . however , many of these remaining snps fall within proximal or distal regulatory elements ( supplementary table 3 ) . using the roadmap chromhmm track , we found 7 promoter snps and 8 enhancer snps in cd19-positive primary blood cells . in encode cell lines , 17 snps were mapped to dnasei hypersensitive regions and 42 snps altered binding motifs of at least one transcription factor . gtex expression quantitative trait loci ( eqtl ) data on whole - blood samples identified multiple snps significantly correlated ( p<0.01 ) with mrna transcript levels of tlr6/10 or fam114a1 but not with tlr1 ( supplementary table 3 ) . the low igg allele ( g ) of rs6835514 was inversely associated with mrna levels of tlr6 ( beta=0.11 ; p=3.2e-3 ) and positively associated with fam114a1 expression ( beta=0.31 ; p=3.7e-5 ) ( figure 2 ) . of particular interest , rs10034903 , which was mapped to an active promoter of tlr10 as well as a transcription factor binding site , appeared to be a significant eqtl for both tlr10 and fam114a1 ( figure 2 ) . similar to rs6835514 , the low igg allele ( g ) of rs10034903 was associated with decreased mrna expression of tlr10 ( beta=0.13 ; p=6.5e-3 ) and increased mrna expression of fam114a1 ( beta=0.27 ; p=8.0e-4 ) . although roles in pathogen recognition and innate immunity have been well established , little is known about tlr1/6/10 with respect to h. pylori response . in a recent report , heterodimeric tlr2/tlr10 was suggested to mediate h. pylori lipopolysaccharide recognition in activating the nf - kb signaling pathway . additionally , a growing number of studies suggest genetic polymorphisms in tlr genes are associated with infectious disease susceptibility . for example , rs5743618 ( ser602ile of tlr1 ; r=0.80 among ceu ) in high ld with rs6835514 ( r=0.80 among ceu ) has been associated with susceptibility to tuberculosis , chlamydia trachomtis infection and leprosy ; while rs4833095 ( asn248ser of tlr1 ; r=0.85 with rs6835514 among ceu ) has been associated with atopobium vaginae infection and placental malaria . intriguingly , 4p14 has also been implicated as a susceptibility locus for ige - mediated allergic sensitization and for hay fever - related asthma ; minor alleles were associated with decreased levels of ige and decreased risk of asthma in caucasians , in parallel with our finding of an inverse association with anti - h . pylori antibody levels . however , three tlr snps included in the current report were inconsistently associated with active h. pylori infection determined by c - urea breath test in a chinese population . further studies are warranted of these genetic polymorphisms in relation to target gene regulation and disease consequences . fine mapping studies are also needed to pinpoint the functionally relevant causal variants . as for the borderline association reported for 1q23.3 , we did not find qualitative or quantitative associations with anti - h . pylori antibodies . in particular , rs368433 was not associated with the highest quartile of antibody levels ( or=1.05 ; 95% ci=0.721.52 ; p=8.1e-1 ) nor with seropositivity overall ( or=0.84 ; 95% ci=0.581.23 ; p=3.8e-1 ) ( supplementary table 1 ) . moreover , there were no significant associations with continuous igg levels among either all participants or seropositives only ( supplementary figure 1 ) . based on our observed 0.06 maf of rs368433 , estimated power to detect the previously reported 0.73 or at a 5% significance level was 71% among all individuals and 80% among seropositives . in conclusion , we confirmed the association of the 4p14 locus with anti - h . our findings suggest that the 4p14 locus may modulate intensity of host immune response rather than acquisition of h. pylori infection per se . the clinical significance of higher levels of antibody to h. pylori remains to be determined ; conflicting associations with either increased or decreased gastric cancer risk have been reported . these findings await extension to other ethnic / racial groups with differences in exposure patterns , bacterial strain pathogenicity , host genetic characteristics and population burdens of h. pylori - associated diseases . our study included participants from the atbc , a randomized , double - blind , placebo - controlled trial conducted 19851993 in 29,133 finnish male smokers aged 50 to 69 years . participants completed questionnaires at enrollment and serum samples were collected and stored at 70c for future analyses . the current study included 1,402 participants who had both genotyping and h. pylori serology data available . antibodies to h. pylori were measured by enzyme - linked immunosorbent and multiplex bead - based assays , as described previously . in order to combine measurements based on different technologies , we standardized levels using laboratory - specific means and standard deviations . imputation was performed using the hidden - markov model algorithm implemented in mach , based on hapmap ceu reference panel build 36 , r22 . we defined candidate regions as 200 kb from the previously reported top snps , rs10004195 at 4p14 ( chr4:3858472438984724 , hg19 ) and rs368433 at 1q23.3 ( chr1:161284210161684210 , hg19 ) . we additionally included snps located in flanking regions of rs6835514 ( chr4:3869438039094380 , hg19 ) , the most significant 4p14 snp in our linear regression analysis . after quality control , 1,380 snps at 4p14 and 1,127 snps at 1q23.3 were available from genotyping or imputed data . average call rate for the genotyped snps was 1 and average quality score for imputed snps ( rsq ) was 0.92 . to refine the phenotype affected by gene polymorphisms , we tested several definitions of h. pylori serologic status . first , we used the same definition as the previous report in caucasians , which compared individuals with igg levels in the highest 25% to individuals in the other 75% . second , to determine whether the loci are associated with h. pylori acquisition , we compared seropositives to seronegatives . we assumed an additive genetic model with number of minor alleles as a predictor , using 10-year age groups and genotying principal components as covariables . we used logistic regression for dichotomous outcome variables and linear regression for the continuous outcome variable . adjustment for multiple comparisons at 4p14 was performed by the false discovery rate ( fdr ) based on 1,380 snps , ignoring the high correlation among the tested snps . analyses were conducted using sas v9.3 ( sas institute inc . , cary , nc ) and r v3.1.3 . we focused functional annotation on the 52 snps located in moderate to high ( r>0.6 ) ld with rs6835514 . the ucsc genome browser ( http://genome.ucsc.edu ) was used to confirm genomic regions and to screen nih epigenomics roadmap ( http://www.roadmapepigenomics.org/ ) and encode ( http://genome.ucsc.edu/encode/ ) tracks . haploreg ( http://www.broadinstitute.org/mammals/haploreg/haploreg.php ) and regulomedb ( http://regulomedb.org/ ) were also used to confirm snp functions and to compile summary results . to map promoter , enhancer , polycomb - repressed or heterochromatin regions , we used the chromatin state segmentation by hidden markov model ( chromhmm ) track from roadmap reported for cd19 primary cells ( presumably , circulating b - lymphocytes ) . dnase cluster assigned by dnase i hypersensitive assay results from 125 cell types , transcription factor binding sites defined by chromatin immunoprecipitation sequencing for 161 factors , and transcription levels determined by rna - seq in gm12878 were tracked using encode . to identify putative target genes regulated by snps , we compiled eqtl results assessed in whole blood samples ( n=168 ) from genotype - tissue expression ( http://www.gtexportal.org/home/ ) . linear regression was conducted for the 52 snps on log and quantile normalized rna - seq levels of four genes to which any of these snps were mapped , including tlr1 ( ensg00000174125.3 ) , trl6 ( ensg00000174130.8 ) , tlr10 ( ensg00000174123.6 ) , and fam114a1 ( ensg00000197712.7 ) . based on the number of genes tested , our bonferroni - corrected significance threshold was p=0.012 ( 0.05/4 genes ) . the study was approved by irbs of the national public health institute of finland and the us national cancer institute . supplementary figure 1 : 1q23.3 locus ( log10 p ) associations with anti - h . pylori antibodies estimated among ( a ) all and ( b ) seropositive atbc participants . genomic region was defined as 200 kb surrounding the index snp ( rs368433 , purple ) . figure was generated with locuszoom version 1.1 ( http://csg.sph.umich.edu/locuszoom/ ) using hg18/hapmap phase ii ceu as genome build / ld population . supplementary table 1 : associations of previously reported top snps with h. pylori serologic status in atbc participants ( n=1,402 ) associations of anti - h . pylori igg antibody levels with 4p14 snps among all or seropositive atbc participants functional annotation of snps within moderate to high ld ( r>0.6 ) of rs6835514

malignant ascites ( ma ) , defined as the abnormal accumulation of cancer - containing fluid in the peritoneal cavity , is a sign of peritoneal carcinomatosis.1 it is caused by a variety of primary abdominal and extra - abdominal cancers . the former category mainly includes gastric cancer ( gc ) , colorectal cancer , pancreatic cancer , ovarian cancer , and primary peritoneal carcinomatosis . the category of extra - abdominal cancers includes breast cancer , lung cancer , lymphoma , and some unknown primary cancers.1,2 ma presents a difficult clinical problem that causes distressful symptoms , including abdominal pain and distension , nausea , vomiting , dyspnea , and anorexia . ma is a grave prognostic sign with a median survival time of less than 6 months.14 there are no curative therapies for ma . the current treatments include sodium - restricted diets , paracentesis , diuretic therapy , peritoneovenous shunting , and chemotherapy . these are palliative procedures mainly to relieve symptoms , and they have limited efficacy and some degree of risk.1,2 ma is distinctive exudate containing cancer cells , macrophages , mesothelium cells , lymphocytes , and other cell components . macrophages and lymphocytes are immunocytes ; both have been confirmed to play important roles in cancer progression.59 at present , studies on ma at the cellular level are limited . in clinical practice , ma is mainly used to prepare cell smears to observe whether there are cancer cells . the staining methods mainly include giemsa staining , haematoxylin and eosin ( he ) staining , and immunohistochemistry ( ihc ) . giemsa staining and he staining are single - color staining techniques that focus on cancer cells and are not able to reveal different cell types simultaneously . although ihc can be visible with multiplexed imaging , ihc multiplexed imaging techniques based on organic dyes and fluorescent proteins such as fluorescein isothiocyanate and rhodamine have several shortcomings in common.79 as a result , these organic dye - based or fluorescent protein - based ihc multiplexed imaging technologies are usually limited to research purposes and have few clinical applications.1012 quantum dots ( qds ) are inorganic nanoparticles with unique optical and electronic properties . they have promising potential applications in many fields , such as medicine and energy.10,11 compared with organic dyes and fluorescent proteins , qds have higher fluorescence efficiency , intensity , stability , and constant excitation wavelength , and they have narrower emission spectra ; these properties allow simultaneous excitation of multiple qds in the same area with one light source.9,12 in this work , we developed a qd - based multiplexed imaging technique for ma , and a new model for ma classification and prognosis . before using qd - based ihc staining with ma , we stained with gc tissue to optimize the staining conditions and explore the gc microenvironment . then we developed the qd - based multiplexed imaging of ma with the optimized staining conditions . we obtained ten gc tissues and 20 ma samples from the department of oncology , zhongnan hospital of wuhan university ( wuhan , people s republic of china ) , and the study protocol was approved by ethics committee of hospital . the tumor tissues were prepared with conventional histopathology procedures , and tissue slides ( 3 m thickness ) were used for qd - based multiplexed imaging as detailed in the next section . all gc tissue slides were preheated at 65c for 1 hour and then deparaffinated in xylene three times , each for 6 minutes . then , tissue and cell hydration was carried out by successively immersing the samples in 100% , 95% , 95% and 85% ethanol for 6 minutes , 3 minutes , 3 minutes , and 3 minutes , respectively , and was followed by rinsing the samples in water for 5 minutes . then , for antigen retrieval , the slides were pretreated in 0.01 m citrate buffer ( ph 6.0 ) and heated at 98c for 15 minutes . after cooling in the citrate buffer at room temperature , the slides were washed in water and stored in a solution of tris - buffered saline ( tbs ) and buffer containing 0.05% tween 20 until they needed to be used . for conventional ihc , gc tissue slides were immersed in 3% h2o2 for 10 minutes at 37c to quench endogenous peroxidase activity . then , slides were blocked with 2% bovine serum albumin ( bsa ) for 10 minutes at 37c . next , the slides were incubated overnight at 4c in these combinations : 1 ) mouse anti - human monoclonal antibody against carcinoembryonic antigen ( cea ) ( ab-4451 , dilution 1:300 ) ( abcam , cambridge , uk ) ; 2 ) mouse anti - human monoclonal antibody against ca125 ( ab-693 , dilution 1:300 ) ( abcam ) ; 3 ) mouse anti - human monoclonal antibody against ca19 - 9 ( ab-15146 , dilution 1:300 , for labeling cancer cells ) ( abcam ) ; 4 ) rabbit anti - human polyclonal antibody against cd68 ( ab-125157 , dilution 1:100 , for labeling macrophages ) ( abcam ) ; 5 ) goat anti - human polyclonal antibody against cd3 ( sc-1127 , dilution 1:300 ) ( santa cruz , usa ) ; and 6 ) goat anti - human polyclonal antibody against cd20 ( sc-7733 , dilution 1:300 , for labeling lymphocytes ) ( santa cruz , usa ) . slides were then incubated for 30 minutes at 37c with corresponding secondary antibodies : 1 ) horseradish peroxide ( hrp)-labeled goat anti - mouse igg ( h+l ) antibody ( 074 - 1806 , dilution 1:200 ) ( kpl , gaithersburg , md , usa ) ; 2 ) hrp - labeled goat anti - rabbit igg ( h+l ) antibody ( 074 - 1506 , dilution 1:200 ) kpl ) ; and 3 ) hrp - labeled rabbit anti - goat igg ( h+l ) antibody ( 14 - 13 - 06 , dilution 1:200 ) ( kpl ) . finally , the reaction products were visualized with diaminobenzidine ( dako , glostrup , denmark ) . as a negative control , the primary antibody was replaced by tbs . for simple qd - based ihc staining , after being blocked with 2% bsa for 30 minutes at 37c , the slides were incubated with the primary antibodies overnight at 4c . the slides were then incubated for 2 hours with the corresponding qd probes that were secondary antibodies conjugated with qd on the f(ab)2 fragments : 1 ) qd-525 goat f(ab)2 anti - mouse igg conjugate ( qd-525 ) , which emits green light and whose dilution was 1:200 ( thermo fisher scientific , waltham , ma , usa ) ; 2 ) qd-585 goat f(ab)2 antirabbit igg conjugate ( qd-585 ) , which emits yellow light and whose dilution was 1:200 ( thermo fisher scientific ) ; and 3 ) qd-655 rabbit f(ab)2 anti - goat igg conjugate ( qd-655 ) , which emits red light and whose dilution was 1:500 ( thermo fisher scientific ) . for multiple qd - based ihc staining , in order to improve the staining sensitivity , cancer cells were simultaneously marked with anti - cea , anti - ca125 , and anti - ca19 - 9 antibodies , and lymphocytes were simultaneously marked with anti - cd3 and anti - cd20 antibodies . cells were recognized as cancer cells if only green was visible and as lymphocytes if only red was visible . the primary antibodies of three different species ( mouse , rabbit , and goat ) were mixed and simultaneously incubated with the slides . correspondingly , the three qd probes were mixed to incubate and to stain the corresponding antibodies . part of the cell mixture ( 10 l ) was transferred to a slide , and cell smears were made . they were dried at room temperature for 2 hours , then fixed in 4% paraformaldehyde for 20 minutes . the smears were washed in tbs three times each for 3 minutes , and then dried at room temperature for 1 hour . the prepared spears were either subjected to giemsa staining or sealed in tin foil and stored in dark at 20c until they needed to be used . the rest of the cell mixture was centrifuged at 3,000 rotations per minute at room temperature for 5 minutes again . paraffin was embedded in accordance with a standard procedure for serial slides ( 3 m thickness ) and subjected to either conventional he staining or qd - based ihc staining as detailed in the next section . for qd - based ihc staining , before being labeled with a primary antibody , ma cell slides were prepared as gc slides by using the procedure previously mentioned . ma cell spears were first treated with 0.01% triton - x 100 as surfactant for 10 minutes to improve the permeability of cell membranes and expose antigenic determinants . all ma cell spears and slides were incubated with mixed primary antibodies from different specimens overnight at 4c . then , they were incubated with corresponding mixed qd probes for 2 hours after being blocked with 2% bsa for 30 minutes at 37c . all slides and smears were examined under an olympus bx51 fluorescence microscope ( olympus corporation , tokyo , japan ) equipped with an olympus dp72 camera ( olympus corporation ) and cri nuance multiplexed imaging systems ( cambridge research and instrumentation , inc . , hopkinton , ma , usa ) . qd - based ihc staining sections were excited by ultraviolet light ( 330385 nm ) . he , conventional ihc , and qd - based ihc staining images were captured by the dp72 camera under the same conditions . qd - based ihc staining cubes that contained complete spectral information at 10-nm wavelength intervals from 420720 nm were captured by cri nuance systems under the same conditions . for the spectral unmixing of the cubes , the analysis software package that came with the nuance system was used . for each ma slide , cell images were captured at magnifications of 100 times , 200 times , and 400 times . chicago , il , usa ) . a t - test was used to compare the ratio of cancer cells and immunocytes between groups . we obtained ten gc tissues and 20 ma samples from the department of oncology , zhongnan hospital of wuhan university ( wuhan , people s republic of china ) , and the study protocol was approved by ethics committee of hospital . the tumor tissues were prepared with conventional histopathology procedures , and tissue slides ( 3 m thickness ) were used for qd - based multiplexed imaging as detailed in the next section . all gc tissue slides were preheated at 65c for 1 hour and then deparaffinated in xylene three times , each for 6 minutes . then , tissue and cell hydration was carried out by successively immersing the samples in 100% , 95% , 95% and 85% ethanol for 6 minutes , 3 minutes , 3 minutes , and 3 minutes , respectively , and was followed by rinsing the samples in water for 5 minutes . then , for antigen retrieval , the slides were pretreated in 0.01 m citrate buffer ( ph 6.0 ) and heated at 98c for 15 minutes . after cooling in the citrate buffer at room temperature , the slides were washed in water and stored in a solution of tris - buffered saline ( tbs ) and buffer containing 0.05% tween 20 until they needed to be used . for conventional ihc , gc tissue slides were immersed in 3% h2o2 for 10 minutes at 37c to quench endogenous peroxidase activity . then , slides were blocked with 2% bovine serum albumin ( bsa ) for 10 minutes at 37c . next , the slides were incubated overnight at 4c in these combinations : 1 ) mouse anti - human monoclonal antibody against carcinoembryonic antigen ( cea ) ( ab-4451 , dilution 1:300 ) ( abcam , cambridge , uk ) ; 2 ) mouse anti - human monoclonal antibody against ca125 ( ab-693 , dilution 1:300 ) ( abcam ) ; 3 ) mouse anti - human monoclonal antibody against ca19 - 9 ( ab-15146 , dilution 1:300 , for labeling cancer cells ) ( abcam ) ; 4 ) rabbit anti - human polyclonal antibody against cd68 ( ab-125157 , dilution 1:100 , for labeling macrophages ) ( abcam ) ; 5 ) goat anti - human polyclonal antibody against cd3 ( sc-1127 , dilution 1:300 ) ( santa cruz , usa ) ; and 6 ) goat anti - human polyclonal antibody against cd20 ( sc-7733 , dilution 1:300 , for labeling lymphocytes ) ( santa cruz , usa ) . slides were then incubated for 30 minutes at 37c with corresponding secondary antibodies : 1 ) horseradish peroxide ( hrp)-labeled goat anti - mouse igg ( h+l ) antibody ( 074 - 1806 , dilution 1:200 ) ( kpl , gaithersburg , md , usa ) ; 2 ) hrp - labeled goat anti - rabbit igg ( h+l ) antibody ( 074 - 1506 , dilution 1:200 ) kpl ) ; and 3 ) hrp - labeled rabbit anti - goat igg ( h+l ) antibody ( 14 - 13 - 06 , dilution 1:200 ) ( kpl ) . finally , the reaction products were visualized with diaminobenzidine ( dako , glostrup , denmark ) . as a negative control , the primary antibody was replaced by tbs . for simple qd - based ihc staining , after being blocked with 2% bsa for 30 minutes at 37c , the slides were incubated with the primary antibodies overnight at 4c . the slides were then incubated for 2 hours with the corresponding qd probes that were secondary antibodies conjugated with qd on the f(ab)2 fragments : 1 ) qd-525 goat f(ab)2 anti - mouse igg conjugate ( qd-525 ) , which emits green light and whose dilution was 1:200 ( thermo fisher scientific , waltham , ma , usa ) ; 2 ) qd-585 goat f(ab)2 antirabbit igg conjugate ( qd-585 ) , which emits yellow light and whose dilution was 1:200 ( thermo fisher scientific ) ; and 3 ) qd-655 rabbit f(ab)2 anti - goat igg conjugate ( qd-655 ) , which emits red light and whose dilution was 1:500 ( thermo fisher scientific ) . for multiple qd - based ihc staining , in order to improve the staining sensitivity , cancer cells were simultaneously marked with anti - cea , anti - ca125 , and anti - ca19 - 9 antibodies , and lymphocytes were simultaneously marked with anti - cd3 and anti - cd20 antibodies . cells were recognized as cancer cells if only green was visible and as lymphocytes if only red was visible . the primary antibodies of three different species ( mouse , rabbit , and goat ) were mixed and simultaneously incubated with the slides . correspondingly , the three qd probes were mixed to incubate and to stain the corresponding antibodies . part of the cell mixture ( 10 l ) was transferred to a slide , and cell smears were made . they were dried at room temperature for 2 hours , then fixed in 4% paraformaldehyde for 20 minutes . the smears were washed in tbs three times each for 3 minutes , and then dried at room temperature for 1 hour . the prepared spears were either subjected to giemsa staining or sealed in tin foil and stored in dark at 20c until they needed to be used . the rest of the cell mixture was centrifuged at 3,000 rotations per minute at room temperature for 5 minutes again . then paraffin was embedded in accordance with a standard procedure for serial slides ( 3 m thickness ) and subjected to either conventional he staining or qd - based ihc staining as detailed in the next section . for qd - based ihc staining , before being labeled with a primary antibody , ma cell slides were prepared as gc slides by using the procedure previously mentioned . ma cell spears were first treated with 0.01% triton - x 100 as surfactant for 10 minutes to improve the permeability of cell membranes and expose antigenic determinants . all ma cell spears and slides were incubated with mixed primary antibodies from different specimens overnight at 4c . then , they were incubated with corresponding mixed qd probes for 2 hours after being blocked with 2% bsa for 30 minutes at 37c . all slides and smears were examined under an olympus bx51 fluorescence microscope ( olympus corporation , tokyo , japan ) equipped with an olympus dp72 camera ( olympus corporation ) and cri nuance multiplexed imaging systems ( cambridge research and instrumentation , inc . , hopkinton , ma , usa ) . qd - based ihc staining sections were excited by ultraviolet light ( 330385 nm ) . he , conventional ihc , and qd - based ihc staining images were captured by the dp72 camera under the same conditions . qd - based ihc staining cubes that contained complete spectral information at 10-nm wavelength intervals from 420720 nm were captured by cri nuance systems under the same conditions . for the spectral unmixing of the cubes , the analysis software package that came with the nuance system was used . for each ma slide , cell images were captured at magnifications of 100 times , 200 times , and 400 times . chicago , il , usa ) . a t - test was used to compare the ratio of cancer cells and immunocytes between groups . gc tissue samples were successfully stained with all antibodies , including anti - cea , anti - ca125 , anti - ca19 - 9 , anti - cd68 , anti - cd3 , and anti - cd20 antibodies ( figures 1a1f , respectively ) , with good sensitivity and specificity for both conventional ihc staining ( figures 1a11c1 and 1d11f1 ) and qd - based ihc staining ( figures 1a21c2 and 1d21f2 ) . from these simple staining samples , furthermore , they presented lineal distribution on one side of the tumor nests and cancer cells , and there were some macrophages in tumor nests as well ( figures 1d1 and 1f1 , black arrow ) . moreover , more macrophages and lymphocytes presented at the cancer invasion front . then , a qd - based multiplexed imaging technique was developed to simultaneously reveal the presence of gc cells , macrophages , and lymphocytes in the tumor microenvironment ( figure 2 ) . it was found that macrophages and lymphocytes tended to present lineal distribution synchronously . by using this qd - based multiplexed imaging technology of gc tissue as a basis , we have established an optimized protocol for qd - based multiplexed imaging of cancer cells and immunocytes in ma . this technology revealed four types of interactions between cancer cells and immunocytes in the ma ( figure 3 ) : 1 ) a few cancer cells were surrounded by numerous lymphocytes ( figures 3a1 and 3a2 ) ; this finding suggests enhanced host immune reactions against cancer cells in ma ; 2 ) cancer cells were approached but could not be surrounded by macrophages ( figures 3b1 and 3b2 ) ; this finding suggests the reduced ability of the host 's immune system to limit cancer cells ; 3 ) numerous cancer cells surrounded the macrophages ( figures 3c1 and 3c2 ) ; this finding indicates significantly compromised host immune reactions against cancer cells ; and 4 ) cancer cells and immunocytes were unrelated ( figures 3d1 and 3d2 ) ; this finding suggests possible host immune tolerance for cancer cells . to further investigate the relationship between cancer cells and immunocytes in ma , we calculated the ratio of cancer cells to immunocytes , and such quantitative analysis revealed four types of quantitative relations between cancer cells and immunocytes ( figure 4 ) : 1 ) numerous cancer cells and immunocytes coexisted in the same slide ( figures 4a14a5 ) ; this finding suggests that there were strong immune reactions to highly proliferating cancer cells in ma ; 2 ) numerous cancer cells and almost no immunocytes existed in the slide ( figures 4b14b5 ) ; this finding suggests that immune reactions against cancer cells and highly proliferating cancer cells in ma were destroyed ; 3 ) a few cancer cells were immersed in numerous immunocytes ( figure 4c14c5 ) ; this finding suggests that the effect of boosting host immunity against cancer cells in ma and the cancer cells proliferation was limited ; and 4 ) a few cancer cells and immunocytes coexisted in the slide ( figure 4d14d5 ) ; this finding indicates fewer immune reactions against less active cancer cells in ma . on the basis of the interactions and quantitative relations of cancer cells and immunocytes and on the basis of the different immune reactive states , ma can be divided into four categories : 1 ) immunocyte - dominant type ; 2 ) immunocyte - reactive type ; 3 ) cancer cell - dominant type ; and 4 ) cell - deletion type ( table 2 ) . to quantitatively investigate the relationships between cancer cells and immune cells , we calculated the ratio of cancer cells to immunocytes for each ma ( table 3 , figure 5 ) . the mean ratios for the four categories of ma were 0.50 , 1.43 , 6.67 , and 0.74 ( p<0.01 ) , respectively , for immunocytes - dominant type , immunocytes - reactive type , cancer cells - dominant type , and cell - deletion type ; this finding indicates the different immunological statuses of the patients and cancer cell activity ( table 3 , figure 5a ) . the ratios of cancer cells to immunocytes in ma from different primary tumors were also different . the mean ratios of ma from gc , colorectal cancer and ovarian / endometrial cancer were 1.70 , 0.67 and 2.36 , respectively ( table 3 , figure 5b ) . although such differences were not statistically significant ( p=0.530 ) , they do reflect the clinical reality that ma from colorectal cancer has a relatively slower progression than ma from either gc or gynecological malignancies . gc tissue samples were successfully stained with all antibodies , including anti - cea , anti - ca125 , anti - ca19 - 9 , anti - cd68 , anti - cd3 , and anti - cd20 antibodies ( figures 1a1f , respectively ) , with good sensitivity and specificity for both conventional ihc staining ( figures 1a11c1 and 1d11f1 ) and qd - based ihc staining ( figures 1a21c2 and 1d21f2 ) . from these simple staining samples , furthermore , they presented lineal distribution on one side of the tumor nests and cancer cells , and there were some macrophages in tumor nests as well ( figures 1d1 and 1f1 , black arrow ) . moreover , more macrophages and lymphocytes presented at the cancer invasion front . then , a qd - based multiplexed imaging technique was developed to simultaneously reveal the presence of gc cells , macrophages , and lymphocytes in the tumor microenvironment ( figure 2 ) . by using this qd - based multiplexed imaging technology of gc tissue as a basis , we have established an optimized protocol for qd - based multiplexed imaging of cancer cells and immunocytes in ma . this technology revealed four types of interactions between cancer cells and immunocytes in the ma ( figure 3 ) : 1 ) a few cancer cells were surrounded by numerous lymphocytes ( figures 3a1 and 3a2 ) ; this finding suggests enhanced host immune reactions against cancer cells in ma ; 2 ) cancer cells were approached but could not be surrounded by macrophages ( figures 3b1 and 3b2 ) ; this finding suggests the reduced ability of the host 's immune system to limit cancer cells ; 3 ) numerous cancer cells surrounded the macrophages ( figures 3c1 and 3c2 ) ; this finding indicates significantly compromised host immune reactions against cancer cells ; and 4 ) cancer cells and immunocytes were unrelated ( figures 3d1 and 3d2 ) ; this finding suggests possible host immune tolerance for cancer cells . to further investigate the relationship between cancer cells and immunocytes in ma , we calculated the ratio of cancer cells to immunocytes , and such quantitative analysis revealed four types of quantitative relations between cancer cells and immunocytes ( figure 4 ) : 1 ) numerous cancer cells and immunocytes coexisted in the same slide ( figures 4a14a5 ) ; this finding suggests that there were strong immune reactions to highly proliferating cancer cells in ma ; 2 ) numerous cancer cells and almost no immunocytes existed in the slide ( figures 4b14b5 ) ; this finding suggests that immune reactions against cancer cells and highly proliferating cancer cells in ma were destroyed ; 3 ) a few cancer cells were immersed in numerous immunocytes ( figure 4c14c5 ) ; this finding suggests that the effect of boosting host immunity against cancer cells in ma and the cancer cells proliferation was limited ; and 4 ) a few cancer cells and immunocytes coexisted in the slide ( figure 4d14d5 ) ; this finding indicates fewer immune reactions against less active cancer cells in ma . on the basis of the interactions and quantitative relations of cancer cells and immunocytes and on the basis of the different immune reactive states , ma can be divided into four categories : 1 ) immunocyte - dominant type ; 2 ) immunocyte - reactive type ; 3 ) cancer cell - dominant type ; and 4 ) cell - deletion type ( table 2 ) . to quantitatively investigate the relationships between cancer cells and immune cells , we calculated the ratio of cancer cells to immunocytes for each ma ( table 3 , figure 5 ) . the mean ratios for the four categories of ma were 0.50 , 1.43 , 6.67 , and 0.74 ( p<0.01 ) , respectively , for immunocytes - dominant type , immunocytes - reactive type , cancer cells - dominant type , and cell - deletion type ; this finding indicates the different immunological statuses of the patients and cancer cell activity ( table 3 , figure 5a ) . the ratios of cancer cells to immunocytes in ma from different primary tumors were also different . the mean ratios of ma from gc , colorectal cancer and ovarian / endometrial cancer were 1.70 , 0.67 and 2.36 , respectively ( table 3 , figure 5b ) . although such differences were not statistically significant ( p=0.530 ) , they do reflect the clinical reality that ma from colorectal cancer has a relatively slower progression than ma from either gc or gynecological malignancies . the major components of the tumor microenvironment are stromal cells , collagens , matrix metalloproteinases , infiltrating inflammatory cells , and tumor neovessels . the tumor microenvironment is becoming more widely recognized as playing an important role in tumor growth , progression , and metastasis.7,8,12 tumor - infiltrating immunocytes have strong influence on tumor growth , progression , and metastasis , and they are independent of the tnm stage.13,14 tumor - infiltrating macrophages are also involved in tumor development and are associated with a poor clinical prognosis.1517 tumor - infiltrating lymphocytes are associated with effective therapeutic responses and favorable clinical outcomes.18,19 however , current research about tumor microenvironments has been mainly focused on primary cancers , and to our knowledge , seldom has attention been paid to the microenvironment of malignant ascites,20,21 particularly the complex interactions between cancer cells and immunocytes . until now , the pathological characteristics of ma microenvironment have been unclear . in this work , in order to gain direct insight into the ma microenvironment , we developed a new qd - based multiplexed imaging method for studying the interaction and the quantitative relations of cells in ma . to improve diagnostic accuracy , researchers explored many methods in addition to cytology . from previous studies , methods such as relative lymphocyte count , ma tumor markers , and telomerase activity were examined.2226 all of these new methods have their own advantages ; however , cytology is still the recognized gold standard in clinical practice . although cytology has long been used to detect malignant cells , it has rarely been used in studying the tumor cell microenvironment , except for studying mesothelial cells.27,28 in the study by matte et al28 mesothelial cells and cancer cells had important interactions , and mesothelial cell proliferation was stimulated by cancer cells . however , this study did not directly observe the cancer cells and mesothelial cells in ma . in this work , we combined the synchronous multiplexed staining properties of qds with ma cytology to develop the qd - based multiplexed imaging technique , which provided a new method to synchronously observe cancer cells , macrophages , and lymphocytes in ma . with this method , the complex components of the tumor microenvironment could be observed directly , an ability that will be helpful in the studies focusing on the relationship between cancer cells and tumor microenvironments . moreover , we synchronously labeled cancer cells with anti - cea , anti - ca125 , and anti - ca19 - 9 antibodies to improve the diagnostic accuracy . regard , previous studies were mostly focused on searching for the optimal chemotherapy regimens against ma , including the paclitaxel with tegafur regimen , the combined intraperitoneal recombinant human endostatin and chemotherapy regimen , the catumaxomab regimen , the cediranib regimen , immunotherapy , and others.2936 however , the rate of complete remission with these regimens remains less than 30% . fundamentally , all of these studies have been focused on finding the best regimen to cure all ma . the fact that tumors are highly heterogeneous can not be ignored,12,3740 and one regimen to treat all ma is impossible . it is imperative to gain further insight into the biological features of ma , to understand the microenvironment of ma , and to classify ma . in our study , by using qd - based multiplexed imaging , we could observe different interactions and quantitative relations among the cancer cells and immunocytes . on the basis of such information , ma can be divided into four categories : 1 ) immunocyte - dominating type ; 2 ) immunocyte - reactive type ; ( 3 ) cancer cell - dominating type ; and ( 4 ) cell - deletion type . on the basis of the different immune reactive states of ma , therapies should be individualized . for ma in hosts with a better immune reactive state , the therapy regimen should focus on killing cancer cells , and for ma in hosts with a worse immune reactive state , the therapy regimen should focus on boosting immunotherapy . thus , therapeutic efficacy of ma may be improved on the basis of this new classification . due to the insufficient number of ma samples , the specific ratios of different categories of ma were not defined . in our future work , we will increase the number of ma samples and achieve longer follow - up . on the basis of the overall survival of these patients with ma and on the basis of the ratios of cancer cells to immunocytes in this work , we developed a new qd - based multiplexed imaging method to directly reveal cancer cells , lymphocytes , and macrophages in the ma microenvironment . on the basis of this new method , we were able to classify ma by different interactions and quantitative relations of cancer cells and immunocytes , and we were able to further predict prognosis and improve therapeutic efficacy .

blood is a complex tissue consisting of a very specialized network of circulating immune cells and soluble factors that are the morphological substrate of the human immune response . among immune cells , the monocyte , neutrophil , and natural killer ( nk ) compartments are essential for first - line , innate immune responses , while t cells , b cells , and the latter s cognate immunoglobulin ( [ ig ] antibody ) repertoire are essential for effective adaptive immune response to a wide variety of pathogens . dysregulated immune cell or ig numbers and/or functions can lead to an increased susceptibility to infections or to immune - mediated inflammatory disorders such as autoimmune diseases or allergy ( cho and feldman , 2015 , tangye et al . , both genetic and non - genetic factors may contribute to variations in the number and function of human immune cells , as well as the concentration of soluble mediators , resulting in considerable heterogeneity in individual immune responses . recent cohort - based studies have highlighted the effect of both genetic ( brodin et al . 2015 ) and non - genetic factors , including cohabitation , chronic infection , aging , and microbiome ( carr et al . , 2016 , , 2015 , shaw et al . , 2013 ) on the variation of human immune cell levels . however , a comprehensive analysis characterizing the interrelationship between different immune cell types ( innate and adaptive ) and ig levels in freshly drawn ( non - frozen ) human blood as well as the effect of genetic and non - genetic factors on the variation in these immune traits has been lacking . the human functional genomics project ( hfgp ) is an initiative comprising several cohorts of healthy individuals and patients that aims to identify the factors responsible for the variability of immune responses in health and disease ( http://www.humanfunctionalgenomics.org ) . while three other studies accompanying this present study describe environmental ( ter horst et al . , 2016 ) , genetic ( li et al . , 2016 ) , and host microbiome ( schirmer et al . , 2016 ) factors that affect pathogen - induced peripheral blood cytokine responses , this study is a comprehensive assessment of the impact of environmental and genetic host factors on circulating cell populations , focusing on both t cells and b cells and including associations of b cells with ig concentrations . our results provide a full picture of humoral immunity , as seen in serum igs , and its interrelationship with immune cell levels . we analyzed the determinants of variation in t and b cell counts and ig levels by testing the association between immune traits and non - heritable factors such as age , gender , and season . we estimated the genetic heritability of different immune cells and show that the variation in t cell counts is predominantly ( 37% ) explained by genetic factors , which is in contrast to b cell counts , which are more strongly influenced by the environment . we also tested the effect of genome - wide genetic variation on cell - level variation by using cell - count quantitative trait loci ( ccqtl ) mapping and identified eight independent genomic loci associated with lymphocyte counts , four of which have not been described before , and with four cell subsets that have not been characterized in previous studies . we also performed an integrative genomics analysis by using rna - sequencing ( rna - seq ) data from blood samples of 628 healthy individuals to identify putative causal genes , including long non - coding rnas , at ccqtls that may regulate cell counts . lastly , we show that the genetics behind ccqtls partially overlap with the previously described genetics of immune - mediated / related disease . both the cellular and humoral arms of our immune system are crucial for an effective immune response . however , information on the interrelationship between the cellular and humoral components is scarce . to analyze the underlying patterns of the variation within these immune components at the population level , we performed unsupervised hierarchical clustering within our measured immune cell populations and within ig levels , after correcting for age , sex , and season effects . for immune cells , we identified four clusters of biological relevance ( figure 1a ) in which subpopulations of b cells , t cells , and myeloid immune cells clustered into clusters 1 , 2 , and 3 , respectively . cluster 4 contains plasma cells and their precursors , as well as plasmablasts , with both groups clustering separately from the b cell cluster ( cluster 1 ) . these observations suggest that plasma cells and cd4+cd45ra+cd27 terminally differentiated effector t cells are co - regulated by similar factors . moreover , using a nonmetric multi - dimensional scaling approach , we revealed , in a data - driven way , a separation between b cells and the other immune subpopulations at the second dimension ( figure 1b ) . this suggests that b cells might also be co - regulated independently of the other immune subsets . the clustering patterns of ig ( sub)classes formed two major clusters , one containing igm and igg3 and the other containing igg , igg1 , igg4 , and iga ( figure 1c ) . for the igm and igg3 cluster , they are known to have the strongest complement binding capacity , a function that is required for optimal protection against ( intracellular ) pathogens ( schroeder and cavacini , 2010 ) . interestingly , the regulation of both igm and igg3 appears to be controlled by the cytokines interleukin ( il)-4 and transforming growth factor ( tgf- ) , indicating functional homogeneity under similar regulatory control ( brggemann et al . , 1987 , coffman et al . , 1989 , mcintyre et al . , 1993 , having established the hierarchical clustering of immune cell populations and ig levels , we analyzed the association between immune cell counts and ig levels by using spearman correlation ( figure 1 ) . out of 511 possible relations , nine significant correlations ( false discovery rate [ fdr ] 0.05 ) were identified between ig subclass and immune cell populations ( figure 1c ) . cd4 + effector t cells ( cd45ra+ cd27 ) , which cluster with the plasma cells and plasmablasts ( cluster 4 ) , show a significant correlation with igg levels ( r = 0.2 , p = 8.5e6 ) ( figures 1d and 1e ) . this correlation may partly reflect the connection between these cell types in humans , where effective recall of antibody responses is dependent on t - cell - dependent memory b cell generation ( kurosaki et al . , 2015 ) . a significant correlation was also observed between igm - only b cell levels and igm serum levels ( r = 0.24 , p = 6.3e8 ) , and a negative was correlation observed between igm serum levels and igd+igm b cells ( r = 0.2 , p = 1.0e8 ) ( figures 1c and 1d ; table s1 ) . this correlation between igm - only b cells in peripheral blood and igm in serum suggests that high levels of igm - only b cells predict higher levels of plasma cells in tissue . these results stress the importance of identifying the key factors driving the underlying inter - individual variation in the immune system . we investigated the distribution of immune cell counts and subset frequencies among 500 individuals in our cohort ( 500 functional genomics project cohort [ 500fg ] from the hgfp ) . we observed substantial variation in total white blood cell ( wbc ) counts ( figure 2a ) and the levels of the lymphoid and myeloid cell populations ( figures 2b and 2e ) between individuals . we then systematically tested the association of this variation with age , gender , and season . aging plays a major role in shaping the immune profile ( lemaoult et al . , 1997 , shaw et al . , 2013 , solana et al . , 2006 ) . using spearman correlation , we observed consistent correlation with age ( 64% of the cell subpopulations studied are significantly correlated ) , both negative and positive . aging was significantly associated ( fdr 0.05 , corrected for 73 tests ) with a decrease in lymphoid immune cell levels ( naive t cells , b cell subsets ) and with a concomitant increase in myeloid immune cell levels of granulocytes , pro - inflammatory non - conventional monocytes ( cd14++cd16 + ) , and intermediate monocytes ( cd14+cd16 + ) and levels of proliferating cd4 + regulatory t cells ( tregs ) ( figure 3a ; table s2 ) . to show the robustness of age effect on immune traits we randomly selected 90% of all the samples and tested for age effect on immune traits . we iterated this 100 times and observed that 91% of traits showed consistent results when compared with the original full dataset in more than 70% of the sampling iterations ( figure s1 ) . we also compared the variation within cell counts in younger subjects ( lower quartile of age distribution in the 500fg cohort ; median age = 19 years ) versus older subjects ( upper quartile ; median age = 65 years ) . we observed significant differences ( p 0.05 ) in the variations of cd4 + ( cd45racd27 + ) effector t cell , nk cell ( cd56+cd16 ) , and cd3+cd56 + t cell subpopulations ( figure s2a ) . upon testing of associations between age and ig levels , only igg2 and iga levels showed a significant positive correlation age ( fdr 0.05 , corrected for seven tests ) . these observations support the hypothesis that immune response shifts class in elderly individuals with de novo infections , with a restricted adaptive response being replaced by an innate type of immunity ( le garff - tavernier et al . , 2010 , hazeldine et al . , 2012 , lemaoult et al . , 1997 , solana et al . , 2006 ) . we observed a significant increase ( fdr 0.05 ) in mature b cell subsets , igm - only b cells , plasmablast b cells , proliferating and memory ( cd45ra ) treg cells , nk cell subsets , and igm serum levels in women as compared to men ( figure 3b ; table s2 ) . the significant association between higher levels of igm - only b cells ( p = 0.0005 ) and increased serum igm levels ( p = 0.0002 ) in women highlights the functional link between the cell type and its product ( amadori et al . , 1995 ) . by using the resampling approach , we observe that 87% of traits show consistent results when compared with the original full dataset in more than 70% of the iterations , we observed an increased level of effector and effector memory t cells ( figure s2c ) and a reduced level of igg4 and iga with nominal p values < 0.01 ( see also ter horst et al . ) because we observed a significant effect of gender on different b cell and ig levels , we investigated whether this effect was due to a difference in gender - associated hormone levels . we first tested whether the immune cell counts correlated with hormone levels in the 500fg cohort , but found no statistically significant correlation ( figure s2b ) . as expected , we observed lower testosterone concentrations in women than in men ( figure s2c ) . although testosterone has been shown to inhibit ig levels of human peripheral - blood mononuclear cells in vitro ( kanda et al . , 1996 ) , our analysis indicates that higher testosterone levels in women are significantly associated with increased igg levels . moreover , we observed a significant association of hydroxyprogesterone with igg levels in women ( figure s2c ) . hydroxyprogesterone levels vary with menstrual cycle , being highest in the luteal phase and lowest prior to ovulation . in men we found a consistent seasonal effect on immune cell subpopulations , with 67% of the measured cell types showing a significant association with season ( fdr 0.05 ) . b cell subsets were the most consistently affected , with all b cell subpopulations showing significantly higher levels in winter . treg , nk(t ) , and classical monocytes ( cd14++cd16 ) were also significantly higher in winter , while granulocytes , proliferating cd8 + t cells and cd4 + effector memory cells showed a higher peak during the summer months ( figure 3c ; table s2 ) . igg , igg1 , and igg4 levels were also higher in winter , with nominal p values < 0.01 ( see also ter horst et al . , 2016 ) . by using the resampling approach , we observed that 94% of traits show consistent results when compared with the original full dataset in more than 70% of the iterations ( figure s1 ) . altogether , these results point to an important role for environmental factors that vary with season ( e.g. , allergies and viral infections ) in the regulation of the magnitude of both the cellular and the humoral immune response ( dopico et al . , 2015 ) . we observed that cell counts show high variability across individuals and that this variation could be partially ascribed to age- , gender- , or season - related factors . to further explore this inter - individual variation , we estimated the proportion of variance explained by genome - wide snps for each of 73 independent cell types after controlling for age , gender , and seasonal variation . as shown in figure 4a and figure s3 , the majority of immune cell population variation is explained by non - heritable rather than heritable influences . the proportion of immune cell variation that was explained by genetics varies for each cell subpopulation . it was significantly higher for the 29 t cell immune traits as compared to the 27 b cell immune traits ( median of 30% versus 18% , respectively ; student s t test , p 0.05 ) . effector memory and effector cd4 + and cd8 + and cd4 + tregs were also strongly influenced by genetic factors ( figure s3 ) . the seemingly interdependent igd+igm+ and igd+igm b cell populations showed completely opposing heritability estimates ( figure s3 ) , likely reflecting the heterogeneity of the igd+igm+ population , which consists of both t - cell - dependent naive cd27 b cells and presumed t - cell - independent cd27 + memory b cells ( weller et al . , more than 50% of the variance in transitional monocytes ( cd14 + cd16 + ) , nk cells ( cd3cd56 + ) , and nk - bright cells ( cd56++cd16 ) was explained by genetic variation . notably , 50% ( 20% ) of the variance in igm can be explained using genotype information . for the remaining igs , we did not identify any contribution of genetics to the variance ( figure s3 ) . to identify the genetic variants determining cell counts and ig levels , we mapped ccqtl and ig level qtls ( igqtls ) using genome - wide snp genotype data . after controlling for the effect of age , gender , and season , we identified eight independent genome - wide significant ccqtls specific for three cell types : t cells ( five ccqtls ) , b cells ( two ccqtls ) , and nk cells ( one ccqtl ) ( figures 4b and 4c ; table 1 and table s3 ) . four of these ccqtls have been reported before ( table 1 , figures 4a4d ) , providing validation for our analytical approach ( orr et al . , 2013 , one of these b cell ccqtl snps was also associated to ig levels , although not at genome - wide significance ( rs62433089 , p < 5e8 ) ( figure s4f ) . the higher numbers of t cell ccqtls compared to b cell ccqtls , when combined with our finding that a greater proportion of the variance in t cells ( but not b cells ) can be explained by genetics , would suggest a stronger genetic component for t cell immunity when compared to b cells . furthermore , we also found that the igg1 level is suggestively associated with a b - cell - specific ccqtl ( rs10277809 , p 0.001 ) , implying a shared regulation of b cell and certain ig levels in blood . we found a b - cell - specific ccqtl ( rs10277809 , chromosome 7 ) ( figures 4b and 4c ; table 1 ) that showed a genome - wide significant association with three b cell subpopulations ( cd24dim cd38dim , igm+-only , and igm - only memory igd igm+ cd27 + b cells ) ( figures 5a and 5b ) . to explore the biological role of the myo1b locus , we mapped expression qtls ( eqtls ) by using rna - seq data from peripheral - blood cells of 629 healthy individuals from the lifelines deep ( lldeep ) cohort ( tigchelaar et al . , 2015 ) . we observed that snp rs10277809 affects the expression levels of both lncrna rp4 - 647j21 and the myo1 g protein - coding gene ( figure 5c ) . this further supports our finding that this ccqtl is associated with the abundance of peripheral b cell subsets in human peripheral blood . co - expression analysis and pathway predictions using over 10,000 rna - seq samples collected from public databases ( fehrmann et al . , 2015 ) show a significant enrichment of b - cell - related functions for both myo1 g and rp4 - 647j21 ( figure 5d ) . we found a t - cell - specific ccqtl , rs280499 on chromosome 19 , that ( figures 4b and 4c ; table 1 ) particularly associated with cd8 + cm cd45ro+ cd27 + cells ( figures 5e and 5f ) . we then mapped cis - eqtls for snp rs280499 and found its effect on expression levels of pde4a ( figure 5 g ) . pde4a encodes the protein phosphodiesterase 4a and has been implicated in t cell differentiation ( peter et al . pde4a hydrolyses cyclic amp , which modulates a variety of cellular responses to extracellular stimuli , including regulating lymphocyte proliferation and the biosynthesis of il-2 . because pde4a plays a role in inflammatory processes , it is therapeutically targeted in the treatment of a number of immune - mediated diseases ( mazur et al . , 2015 ) . three out of our eight ccqtls have been previously associated with immune - mediated diseases ( table 1 ) . in particular , rs1801274 , which is a ccqtl for multiple cell types , is associated with several auto - immune diseases ( table 1 ; figure s4a ) , including ulcerative colitis and kawasaki disease , and has also been replicated in previous studies ( orr et al . , 2013 , roederer et al . , 2015 ) . on chromosome 19 , the snp rs2164983 associated to nk cells ( table 1 ) has been previously reported to be a risk factor for atopic dermatitis ( paternoster et al . , 2011 ) . furthermore , ccqtl rs280499 overlaps with immunobase regions associated with immune - mediated diseases such as multiple sclerosis and rheumatoid arthritis ( https://immunobase.org/studies/ ) . in addition , we make use of ccqtls and igqtls at a suggestive significance threshold ( p < 1e5 ) and genome - wide association study ( gwas ) catalog snps known to influence susceptibility to various diseases ( figure 6 ) . interestingly , snps that affect t cells levels are also enriched for snps associated to auto - inmmune and inflammatory diseases . in contrast , ccqtls that affect b cells are enriched for snps associated with allergy - related diseases ( figure 6 ) . the hfgp project was initiated to better understand the variation of the immune landscape of human beings and to identify targets for personalized treatment interventions . to explore the determinants of variation in t and b lymphocytes and ig levels , we tested the association between these immune traits and both heritable factors and non - heritable factors , such as age , gender , and seasonality , in the hfgp 500fg cohort of healthy volunteers . the abundance of circulating t cells appears to be influenced more by genetics than the numbers of circulating b cells . this hypothesis is based on our observation that a higher percentage of variation is explained by genetics for t cells ( 30% ) than for b cells ( < 18% ) and on our identification of five t cell ccqtls versus only two b cell ccqtls . most b cell subsets ( and ig levels ) consistently showed seasonality effects , peaking during winter , suggesting that environmental factors might be more important in driving b cell count variation . this hypothesis is supported by results of multi - dimensional scaling analysis , revealing a separation between b cells and other immune cell subpopulations . despite the impact of environmental cues on b cell counts , b cell function is still affected by genetics . moreover , only one type of ig showed a significant genetic component to its variation : 50% of the proportion of variance in igm levels was explained by genetics , while none of the other igs we measured showed any genetic component . we also identified an igm - specific qtl but did nt find qtls for any of the other igs that we investigated . both the igm qtl and the ccqtl associated to igm - only b cells , and this may be representative for that part of the b cell response that has innate - like features , such as the production of natural antibodies by dedicated b cell types . in contrast , the adaptive b cell response , featuring receptor editing and affinity maturation , might be under more stringent environmental control , as previously reported in a study of the seasonal pathogen influenza ( baumgarth et al . , 1999 ) . non - genetic factors such as age and gender have extensively been associated with changes in immune profiles . fluctuating gender - associated hormone levels and the accumulation of environmental factors , such as an increasing infection burden with age , both leave a strong imprint on the nature and dynamics of the immune response ( lemaoult et al . , 1997 , notably , our results appear to support the hypothesis that aging is associated with an overall decrease in lymphoid immune cell levels and an increase in myeloid cell types , as well as increased treg activity . this suggests that immune response type and regulation is altered toward a more innate - type of immunity with age , as previously reported ( le garff - tavernier et al . , 2010 , , we replicate a number of previously reported age - related changes in the human immune system , such as depletion of naive b cells and t cells and a concomitant increase of memory b and t cells ( lemaoult et al . we also identify age - related changes in specific cell subsets , such as monocyte subclasses , granulocytes , and proliferating t cell populations , that were not reported before . with regard to gender , we see overall higher immune cell counts and ig levels for women , with the notable exception of effector / memory t cells , which are more abundant in men . the significant correlation we observed between the higher levels of igm - only b cells and increased serum levels of igm in women could be explained by the functional link between these cell types and overall serum ig levels in humans ( amadori et al . , 1995 ) . the enhanced antibody responses found in women upon vaccination fits this profile ( butterworth et al . , 1967 , rowley and mackay , 1969 ) , as does the previously established positive correlation between estrogens and igm and igg levels ( kanda and tamaki , 1999 ) . the generation of heterogeneous human memory t cell subsets , and how they develop upon activation of naive t cells , is a subject of intense research ( farber et al . , 2014 ) . either ( 1 ) memory t cells arise directly from effector cells or ( 2 ) naive cells develop directly into memory cells without effector stage transition ( restifo and gattinoni , 2013 ) . in our unsupervised approach to study the inter - relationship between cell types , we observed that naive and central memory t cells co - cluster within the t cell cluster , while effector and effector memory t cells co - cluster with innate effector cells . although we were nt able to decipher the developmental route of these t cell maturation stages , this differential clustering of more quiescent naive and central memory t cells versus innate effector - like effector and effector memory t cells suggests clustering based on function . meanwhile , the cluster composed of plasmablast b cells also grouped the t helper cytokine ( th2 ) subpopulation , and these two subpopulations of immune cells have previously been functionally linked given that they are increased in patients with igg4-related disease ( akiyama et al . , 2015 ) . unfortunately , we were nt able to find any significant association between igg4 levels and plasmablast or th2 t cells within the general population . the generation and isotype switching of ig - producing plasma cells can be mediated in either a t - cell - dependent or a t - cell - independent fashion . we found that cd4 + effector t cells ( cd27 cd45ra+ ) show a strong association with igg levels , implying a functional link between these cell types in humans , which is in line with the finding that effective recall of antibody responses requires the generation of memory b cells controlled by t cell subsets ( kurosaki et al . , 2015 ) . we found a significant positive correlation between igm - only b cell counts and igm serum levels and a negative correlation between igm serum levels and igd+igm b cells . igm - only peripheral - blood lymphocytes are non - activated resting b cells that resemble classical , class - switched memory b cells and express higher levels of mrna than naive b cells ( klein et al . , 1997 ) . a similar positive impact of genetics on nk cells was described previously ( roederer et al . , 2015 ) . with respect to the effector memory and effector t cells , high levels of variance explained by genetics are in agreement with recent findings in twins ( brodin et al . , 2015 ) . we also observed a genetic contribution to treg counts , which is in contrast to the study by brodin et al . for the majority of b cell subsets , with the exception of igd+ igm and transitional b cells , the variance in cell counts explained by genetics was low ( median < 18% ) . this result could suggest that b cell immunity is more susceptible to environmental cues , which is further exemplified by a prominent seasonal effect on both b cell counts and ig levels . additionally , in a recent vaccination cohort study , it was discovered that the inter- and intra - individual variations in immune response before and after vaccination can be influenced by age and gender , which also corroborates our findings ( frasca et al . , 2012 , tsang et al . , 2014 ) . identifying ccqtls associated with genomic regions of relevance to disease provides insight into disease etiology . we identified eight ccqtls , four of which were not reported before . those specific b cell subpopulations ( which had a ccqtl effect ) a multi - omics approach combining cell count data , genomics , and transcriptomics was applied to identify the functional and clinical relevance of the ccqtls . given the comprehensive analysis of b cell subpopulations , we identified eqtl - effects on myo1 g expression and on the expression of a neighboring lncrna . myo1 g has previously been implicated in b cell biology and blood cell numbers in a mouse model ( maravillas - montero et al . , these results suggest the involvement of myo1 g in the active regulation of b cell levels in humans . the lncrna might or may not be involved in regulation of myo1 g expression ( quinn and chang , 2016 ) . furthermore , we identified a t - cell - specific ccqtl in the pde4a locus that modulates its expression . the fact that pde4a is a common therapeutic target for immune - mediated diseases ( mazur et al . , 2015 ) further supports an immune - associated role for our ccqtls . there are some drawbacks to the approach that we have used for our current study . , 2015 , carr et al . , 2016 , orr et al . , 2013 , roederer et al . , 2015 ) is that circulating immune cells are used and do not represent the full landscape of human immunity . strong differences in immune cell composition have been reported between human peripheral blood , bone marrow , spleen , and lymph nodes ( peters et al . , 2013 ) . however , obtaining samples of lymphoid organs in a cohort of healthy individuals is not feasible for ethical and practical reasons . moreover , given the sample size of our study , the standard error on the calculation of percentage of explained variance by genetics per trait can be substantial ( yang et al . , 2010 ) . finally , in this study we were unable to set a discovery - replication scheme for the immune trait qtl mapping due to the limited sample size . despite these drawbacks , we were able to identify a differential contribution of genetic versus environmental factors on lymphocyte subpopulations , we confirmed previously reported ccqtls , and we identified ccqtls for b cell and t cell subpopulations . in conclusion , we assessed the influence of genetics , age , gender , and seasonality on cell count variation , ig levels , and their interrelationship in healthy volunteers participating in the hfgp . our findings indicate that t cell immunity has a stronger genetic imprint than b cell immunity , while the latter might be driven by environmental factors . we also found eight genome - wide significant loci associated to cell levels , four of which were not reported previously . moreover , we were able to link immune cell count qtls to gwas snps associated with immune - mediated diseases . within the hfgp 500fg cohort , three complementary studies focus on a broader understanding of the variability in human cytokine responses . ( 2016 ) identified host and environmental factors that contribute to variation of cytokine responses , while li et al . ( 2016 ) mapped 17 new genetic variants and microbiome factors , respectively , that explain variability of cytokine responses ( li et al . , 2016 , schirmer et al . , 2016 ) . like immune cell counts and ig levels , cytokine responses were influenced by age and gender ( ter horst et al . , 2016 ) , and cytokine responses also revealed annual seasonal dependencies ( ter horst et al . , 2016 ) . together , these different hfgp 500fg studies provide important resources for understanding the human immune response . future studies using the hfgp cohorts will focus on assessing the effect of other factors ( e.g. , microbiome , infection , and immune - mediated inflammatory disease ) on the variation of immune cell counts and function . these studies will contribute to the goal of precision medicine in infections and inflammation by allowing for more accurate predictions of disease status and better treatment efficacy . the hfgp study was approved by the ethical committee of radboud university nijmegen ( no . 42561.091.12 ) . the study was performed in a cohort of 516 healthy individuals of western - european ancestry from the hfgp ( 500fg ; for inclusion criteria and further description see http://www.humanfunctionalgenomics.org ) . we measured myeloid and lymphoid immune cell levels by 10-color flow cytometry ( table s4 ) and serum ig ( sig ) concentrations by fluorescence enzyme immunoassay ( immunocap ) in 516 dutch individuals of western - european descent , aged 18 to 75 years , recruited over the years 20132014 as part of the 500fg study within the hfgp ( http://www.humanfunctionalgenomics.org ) . we focused on a set of 73 manually annotated immune cell subpopulations and seven different classes of igs ( figure s5 ) . to minimize biological variability , cells were analyzed within 23 hr after sample collection on a 10-color navios flow cytometer ( beckman coulter ) equipped with three solid - state lasers ( 488 nm , 638 nm , and 405 nm ) . calibration of the machine was performed once a week , and little adjustment to the machine setting had to be made during the inclusion period of the study . see supplemental experimental procedures for details on cell processing , reagents , gating , and analysis . volunteers from the 500fg cohort were genotyped using the illumina human omniexpress exome-8 v1.0 snp chip . the genotype was called with opticall 0.7.0 using the default settings , excluding samples with a call rate 0.99 . variants with hardy - weinberg equilibrium ( hwe ) 0.0001 , call rate 0.99 , and minor - allele frequency ( maf ) 0.001 were also filtered out . ethnic outliers were identified by multi - dimensional scaling plots of samples merged with 1000 genome data and excluded from further analysis . for further imputation of this dataset , we aligned the strands and variant identifiers to the reference genome of the netherlands ( gonl ) dataset using genotype harmonizer . the phasing was performed with shapeit2 version 2 with the gonl as a reference panel . finally , the data were imputed using impute2 with the gonl as the reference panel . only imputed variants with a quality score 0.8 were used for further cell count quantitative loci mapping . all statistical analysis were performed using the statistical programming language r ( r core team , 2012 ) . cell counts were normalized using an inverse rank transformation ( irt ) algorithm , shown in table s5 . ig levels were normalized using a log2 transformation . to properly ascertain cell count correlations , we first corrected the normalized cell counts for age , gender , and seasonal effects using a linear model . associations were then calculated using the normalized and corrected cell counts via spearman correlation analysis and clustered using these coefficients as distance by an unsupervised hierarchical clustering approach . the same methodology was applied to calculate the association between cell counts and igs . significance was declared after multiple testing correction ( fdr 0.05 ) ( benjamini and hochberg , 1995 ) . the euclidean distances used on the multi - dimensional scaling between cell types were obtained based on the spearman coefficients described above ( venables and ripley , 2002 ) . see supplemental experimental procedures for details regarding statistical analysis of the association of cell counts or ig levels with age , gender , and season . for 442 individuals , absolute cell count data and genotype information was available . for 407 individuals , ig levels and genotype data were available . we calculated parental and grandparental percentages , which are defined as the percentage of a certain cell type within the subpopulation of cells from which it was isolated . this was performed for cell counts of all measured cell types because it has been shown that these percentages tend to reduce inter - experimental noise and therefore increase statistical power for qtl mapping ( orr et al . , 2013 ) . absolute cell counts and percentages were transformed by irt ( orr et al . , 2013 ) . we then corrected the irt cell counts and log2 ig values using a linear model correcting for age , gender , and month of sample collection . lastly , qtl mapping was performed using a linear model as implemented in the matrix - eqtl r package ( shabalin , 2012 ) , where we associated immune traits to genotype information . a p value < 5e6 was considered to be genome - wide significant . composed of 627 healthy dutch volunteers , to test for possible eqtl effects of the ccqlts . for lldeep , both gene expression data ( obtained through rna - seq ) and genotype information are available . for this , we fitted a linear model using tmm - normalized ( robinson et al . , 2010 ) expression data to the genotype information . given that the number of tests depended on the ccqtl genomic location for each independent locus , a threshold of fdr ( 0.05 ) was used , depending on the number of tests performed in that specific window . to estimate the proportion of variance explained by genetics , we used a linear mixed model implemented in the gcta tool ( yang et al . , 2010 ) . we applied it to each of the cell counts and percentages and to ig levels using the complete set of genetic variants quantified in our cohort . the immune traits were pre - processed as described for qtl mapping using irt cell counts and log2 ig values corrected for age , sex , and month of sample collection . given the relatively small sample size , the confidence intervals for heritability estimation can be wide ( zaitlen and kraft , 2012 ) . the accession number for the raw flow cytometry data and analyzed data files are available upon request to the authors ( http://hfgp.bbmri.nl ) . , m.g.n . , and c.w . conceived and directed the study with input from all authors .

at the present time , synthetic meshes are frequently used in pelvic organ prolapse ( pop ) surgery with very good clinical outcomes . at the same time although rare , these complications can have serious consequences . during the past several years , many reports of complications associated with synthetic meshes have been published in the literature . the most frequent complications include vaginal mucosa erosion , mesh shrinkage , infections , pain , urinary tract disorders and a recurrence of prolapse . there have also been reports of rare complications , such as bowel , bladder , and blood vessel perforation during insertion . the recent randomized controlled trial published by altman et al . showed improved anatomical and functional outcomes following an anterior transvaginal mesh ( tvm ) procedure compared to native tissue repair . over the years , the transvaginal mesh kit has become a widespread treatment option in pop and has been entered in routine practice in majority pelvic floor surgery centers worldwide [ 2 , 3 ] . furthermore , when complications occur , the lack of knowledge and experience can cause a further severe morbidity if it is not diagnosed and treated in a proper way [ 47 ] . it is known that using mesh is associated with high rates of complications and repeat operations in genital surgery [ 8 , 9 ] . a systematic review by diwadkar et al . showed that women in the mesh kit group had a higher re operation rate than those who had a traditional surgery or sacrocolpopexy . different re interventions were done due to complications including mesh protrusion ( 5.8% ) , pain ( 2.5% ) and dyspareunia ( 2.2% ) . von theobald described 104 surgeries in 83 women who had complications of previous mesh surgery . the indications for the re interventions included mesh protrusion , infection , pain , granuloma , voiding problems and mesh shrinkage . altman et al . also reported more complications in the group of women who had undergone mesh repair . this included more bladder injury ( 3.5% ) , pelvic pain ( 2.5% ) and mesh exposure ( 3.2% ) . complications of vaginal mesh are often overestimated , sometimes overemphasized and often poorly described and managed . the aim of our study was to assess the complications , to determine the way of prevention and treatment of complications following vaginal mesh surgery interventions . this is a retrospective , multicenter study conducted in 7 referral pelvic floor repair centers . inclusion criteria for the study were patients operated for pelvic organ prolapse with vaginal meshes . the surgical procedure was standardized , according to local protocols , and limited to using a trocar guided transvaginal mesh kit ( prolift , gynecare , usa ) for repair of anterior , posterior and apical vaginal prolapse . we have retrospectively analyzed the intraoperative and early postoperative complication rates for these patients by reviewing patients files . according to the local practice , this data was extracted and analyzed as well . at the start of the study , all patients who considered themselves symptomatic were invited for thorough office check up that included vaginal exam , uroflowmetry and postvoid residual measurement followed by cystoscopy when appropriate . the baseline characteristics are presented using absolute and relative ratio with either means or standard deviations ( sd ) . comparisons of patient proportions were presented as odds ratio ( or ) with 95% confidence intervals ( cis ) . in total , 677 cases using mesh devices were analyzed from a period between 2006 to 2010 . overall , 303 patients have had anterior repair , 51 posterior repair , 232 vaginal vault repair and 91 patients had combined anterior and posterior surgery . only 86.5% ( 586/677 ) were available for phone interview , others were lost for follow up due mainly to failure to contact the patients . during comparative data analysis group 1 included 525 women without complications after mesh surgery with mean age of 60 ( 12.7 ) years . group 2 included 22.5% of patients ( 152/677 ) who have had any kind of complications , including intra operative and mesh related injuries . complications of pop repair with mesh prosthesis intraoperative and early postoperative complications were registered in 88 patients ( 12.9% ) . the most common injuries were related to bleeding and were limited to pelvic / perineal hematomas and severe intraoperative blood loss ( more than 500 cc ) . fifteen women ( 2.2% ) had blood loss over 500 ml , although three of these women were associated with vaginal hysterectomies . three women had serious vascular injuries : inferior gluteal vessels , obturator vessels , paraurethral venous plexus . in all these cases , main symptoms of vaginal , retropubic or perineal hematomas in the postoperative period were fever , significant decrease in the level of hemoglobin , buttock pain and urinary retention due to compression of bladder neck . moreover , ten major vaginal hematomas ( > 500 ml ) led to urinary retention or transformed into an abscess . bladder , urethral and even ureteral injuries occurred during dissection of the tissues , insertion of trocars of anterior mesh device , and fixation of the mesh to the bladder neck . all but two cases of urinary tract injuries were intraoperatively found on cystoscopy at the time of surgery . in one of the overlooked cases it led to stone formation after protrusion of the mesh into the bladder . another undetected bladder injury was confirmed on the 7 day after surgery when continuing hematuria increased . undetected bladder injury led to stone formation and later transformed to protrusion of the tape into the bladder in one case . that patient required laparotomy and removal of the mesh with resection of the bladder wall . a single case of ureteral injury happened to a 70year old woman with stage iii cystocele who underwent anterior transvaginal mesh implantation of a four armed anterior implant . it was inserted using four trocars passed through the obturator foramina . at the second day post her temperature was 37.2c , and the blood tests revealed normal white and red blood cell counts . a contrast ct scan showed urinary leakage on the right side ( figure 1 ) extending to the interfascial space of the right hip . a retroperitoneal approach revealed complete injury of the right ureter at the level of the right posterior arm of the mesh . . urine leakage extending to the interfascial space of the right hip ( b ) . five cases of rectal injuries occurred during the initial dissection of the rectum from the posterior vaginal wall due to presence of extensive scar tissue and therefore they were not related to mesh insertion . in three cases the injury was repaired before insertion of the mesh with no further postoperative complications . in all cases related complications were registered in 64 cases ( 9.4% ) and they included vaginal adhesions in 2 ( 0.3% ) women , vaginal extrusions in 32 ( 4.8% ) , bladder erosion in 1 ( 0.2% ) , urethro vaginal fistula in 2 ( 0.3% ) , mesh shrinkage in 7 ( 1% ) , buttock and perineal pain in 16 ( 2.2% ) , mesh infection in 4 ( 0.6% ) . all vaginal mucosa erosions with protrusion of the mesh were detected on vaginal examination between 2 and 24 weeks after surgery . all women with mesh extrusions had symptoms of offensive vaginal discharge and dyspareunia . in 20 ( 12% ) in general , some vaginal extrusions were less than 0.5 cm in size ( 18 cases ) and were managed conservatively with local estrogens , afterwards they all required partial excision of exposed mesh . two women had large mesh extrusions with signs of local infections and buttock phlegmonal abscess following anterior and posterior mesh implantation . moreover , we have registered a case of necrotizing fasciitis in a patient with stage iii pop . the patient opted for vaginal hysterectomy and prolapse repair with mesh kit ( prolift anterior , gynecare ) . morphological examination proved the presence of iud and found an endometrial cancer ( t1n0m0 ) . at day 6 after the surgery , the patient developed swelling and redness on the thighs , severe pain , high fever , hypotension and other signs of systemic toxicity . necrotizing fasciitis of both thighs was diagnosed . the patient was emergently taken to the surgical theater for fasciotomy and debridement . after the surgery , the patient was treated in an intensive care unit for 18 days and afterwards she died due to intoxication and fatal coagulopathy . it was registered in 5 cases in our study and was characterized by severe vaginal pain , dyspareunia , vaginal shortening , urethral obstruction , and prolapse recurrence . it involves mobilization of the mesh , dividing of the fixation arms , and excision of contracted mesh . the surgical intervention is potentially associated with an increased risk of visceral injury and hemorrhage . the goal of surgical management is to relieve the tension by dividing the central graft from the arms and excising all areas of contracted mesh . statistical analysis of the data revealed several risk factors associated with intraoperative , early postoperative and mesh related complications . the patients younger than 55 years have 3.3 times more chance to develop perioperative complications . the risk for mesh related complications is even higher and amounted to 8.2 , but this finding was compromised by a wide confidence interval that had made it statistically non significant . concomitant vaginal hysterectomy 2.8 times increases the risk of surgical complications and 2.4 times for mesh related ones . development of postoperative hematomas increases the risk of mesh related complications by 2.5 times . according to our data , patients with less prominent prolapse ( < iii pop q ) repaired by mesh have had 4 times more risk for mesh related problems . other factors such as bmi , high stage prolapse and others listed in table 2 do not influence the risk of complications . risk factors and complications following vaginal mesh surgery statistically significant statistically non significant statistically non significant because of large variance of ci the most common injuries were related to bleeding and were limited to pelvic / perineal hematomas and severe intraoperative blood loss ( more than 500 cc ) . fifteen women ( 2.2% ) had blood loss over 500 ml , although three of these women were associated with vaginal hysterectomies . three women had serious vascular injuries : inferior gluteal vessels , obturator vessels , paraurethral venous plexus . in all these cases , main symptoms of vaginal , retropubic or perineal hematomas in the postoperative period were fever , significant decrease in the level of hemoglobin , buttock pain and urinary retention due to compression of bladder neck . moreover , ten major vaginal hematomas ( > 500 ml ) led to urinary retention or transformed into an abscess . bladder , urethral and even ureteral injuries occurred during dissection of the tissues , insertion of trocars of anterior mesh device , and fixation of the mesh to the bladder neck . all but two cases of urinary tract injuries were intraoperatively found on cystoscopy at the time of surgery . in one of the overlooked cases it led to stone formation after protrusion of the mesh into the bladder . another undetected bladder injury was confirmed on the 7 day after surgery when continuing hematuria increased . undetected bladder injury led to stone formation and later transformed to protrusion of the tape into the bladder in one case . that patient required laparotomy and removal of the mesh with resection of the bladder wall . a single case of ureteral injury happened to a 70year old woman with stage iii cystocele who underwent anterior transvaginal mesh implantation of a four armed anterior implant . it was inserted using four trocars passed through the obturator foramina . at the second day post her temperature was 37.2c , and the blood tests revealed normal white and red blood cell counts . a contrast ct scan showed urinary leakage on the right side ( figure 1 ) extending to the interfascial space of the right hip . a retroperitoneal approach revealed complete injury of the right ureter at the level of the right posterior arm of the mesh . five cases of rectal injuries occurred during the initial dissection of the rectum from the posterior vaginal wall due to presence of extensive scar tissue and therefore they were not related to mesh insertion . in three cases the injury was repaired before insertion of the mesh with no further postoperative complications . in all cases mesh related complications were registered in 64 cases ( 9.4% ) and they included vaginal adhesions in 2 ( 0.3% ) women , vaginal extrusions in 32 ( 4.8% ) , bladder erosion in 1 ( 0.2% ) , urethro vaginal fistula in 2 ( 0.3% ) , mesh shrinkage in 7 ( 1% ) , buttock and perineal pain in 16 ( 2.2% ) , mesh infection in 4 ( 0.6% ) . all vaginal mucosa erosions with protrusion of the mesh were detected on vaginal examination between 2 and 24 weeks after surgery . all women with mesh extrusions had symptoms of offensive vaginal discharge and dyspareunia . in 20 ( 12% ) in general , some vaginal extrusions were less than 0.5 cm in size ( 18 cases ) and were managed conservatively with local estrogens , afterwards they all required partial excision of exposed mesh . two women had large mesh extrusions with signs of local infections and buttock phlegmonal abscess following anterior and posterior mesh implantation . moreover , we have registered a case of necrotizing fasciitis in a patient with stage iii pop . the patient opted for vaginal hysterectomy and prolapse repair with mesh kit ( prolift anterior , gynecare ) . morphological examination proved the presence of iud and found an endometrial cancer ( t1n0m0 ) . at day 6 after the surgery , the patient developed swelling and redness on the thighs , severe pain , high fever , hypotension and other signs of systemic toxicity . necrotizing fasciitis of both thighs was diagnosed . the patient was emergently taken to the surgical theater for fasciotomy and debridement . non clostridial streptococci were identified from the wound . after the surgery , the patient was treated in an intensive care unit for 18 days and afterwards she died due to intoxication and fatal coagulopathy . it was registered in 5 cases in our study and was characterized by severe vaginal pain , dyspareunia , vaginal shortening , urethral obstruction , and prolapse recurrence . it involves mobilization of the mesh , dividing of the fixation arms , and excision of contracted mesh . the surgical intervention is potentially associated with an increased risk of visceral injury and hemorrhage . the goal of surgical management is to relieve the tension by dividing the central graft from the arms and excising all areas of contracted mesh . statistical analysis of the data revealed several risk factors associated with intraoperative , early postoperative and mesh related complications . the patients younger than 55 years have 3.3 times more chance to develop perioperative complications . the risk for mesh related complications is even higher and amounted to 8.2 , but this finding was compromised by a wide confidence interval that had made it statistically non significant . concomitant vaginal hysterectomy 2.8 times increases the risk of surgical complications and 2.4 times for mesh related ones . development of postoperative hematomas increases the risk of mesh related complications by 2.5 times . according to our data , patients with less prominent prolapse ( < iii pop q ) repaired by mesh have had 4 times more risk for mesh related problems . other factors such as bmi , high stage prolapse and others listed in table 2 do not influence the risk of complications . risk factors and complications following vaginal mesh surgery statistically significant statistically non significant statistically non significant because of large variance of ci modern surgical techniques for the management of pelvic organ prolapse are presumed to be a minimally invasive with almost no complications . at the same time , it should be emphasized that the implantation of surgical mesh is permanent , that some complications associated with the implanted mesh may require additional surgery , and that even surgery may not correct the complication . small vessel injuries during extensive dissection , especially of paravesical and pararectal spaces were hardly avoidable . the blind insertion of the trocars into the obturator foramen , ischiorectal fossa , ileococcygeus muscle and the sacrospinous ligament exposes the patient to the potential injury to the rectum , bladder , inferior gluteal vessels , pudendal nerve and vessels , and the sciatic nerve . anatomical landmarks are essential in pelvic organ prolapse repair . during hands on training for mesh surgeries , it is necessary to pay special attention to safety landmarks and anatomical topography . the ureters are always in close proximity to the cervix , and ureteral injury should be considered as a potential risk in patients with extensive prolapse . despite the risk of mesh extrusion , which is a specific complication of using any graft , synthetic materials are preferred over autologous graft materials because synthetic graft materials are durable , largely available and relatively inexpensive . synthetic graft materials also reduce the risk of morbidity and operative time as fascia is not harvested . mesh exposure can be caused by the use of any type of synthetic graft material . it is not possible to identify the precise rate of mesh exposure due to a lack of long term follow up ; however , the overall rate based on short term follow up has been reported to be 0.816% [ 14 , 15 ] . polypropylene mesh is the preferred synthetic graft material because of the lower mesh exposure rate . in addition to mesh type , other factors , such as surgical technique , concomitant hysterectomy , cigarette smoking , hormonal status and a history of medical diseases , may be related to mesh exposure . according to the present data there are differences in the rates of mesh extrusions with different materials : 7.5% for braided polyester , 5.1% for polypropylene , 9.1% for porcine dermis , 0% for expanded polytetrafluoroethylene ( eptfe ) , 25% for eptfe plus synthetic graft and 19% for any eptfe . concomitant hysterectomy has been evaluated in a number of mesh exposure related studies and inconsistent findings have been obtained . another study in which laparoscopic asc was performed showed that previous or concurrent hysterectomy did not affect mesh exposure . densinger compared total abdominal and supracervical hysterectomy groups in patients undergoing asc and determined that mesh exposure was increased sevenfold ( 95% ci , 2.319.6% ) in the group in which total abdominal hysterectomies were performed . in our study , a 2.8fold increase in mesh exposure was observed in the concurrent hysterectomy group compared to the previous hysterectomy group . in our opinion , there is more than one factor involved in mesh exposure . this large retrospective cohort study included 677 patients suffering with pelvic organ prolapse and operated with trocar guided mesh kits . we have found that 22.5% of patients presented with any type of complication , either perioperative or mesh related . the retrospective design was the main limitation of the study , besides the fact that mesh related complications were investigated only in those cases when the patients were symptomatic . some patients were lost for follow up ( 23.5% ) . with a cross sectional evaluation of the patients , the rate of asymptomatic complications , such as mesh extrusions or mesh shrinkage might become more prominent . nevertheless , trocar guided mesh repair of the pop might be a cause of serious complications , that may become even life threatening . further studies with objective evaluation and long term follow up of the patients are urged in the nearest future . taking this into consideration , a thorough evaluation of mesh related complications is very important for proper planning of surgery and developing informed consent . according to our study we have found that younger age , less prominent prolapse and concomitant hysterectomies are associated with a higher risk of peri operative and mesh related complications .

dermatologists have witnessed the many new successes in psoriasis treatment that are documented by improvement of the psoriasis area and severity index ( pasi ) . it measures four aspects of disease activity , namely reduction of erythema , scaling and infiltration as well as area involved . all components of pasi can be documented by photography , except for infiltration , which until today has to be felt by touching the actual lesions . this information is lost on photography , which precludes retrospective pasi scoring based on pictures . moulage - making was an exclusive and sought - after art that was primarily used for teaching , but also to document clinical and laboratory research during the first half of the 20th century [ 1 , 2 ] . the largest collections of moulages remaining today are in paris , zrich ( www.moulagen.ch ) , vienna , bonn and athens . moulages were essentially utilized as life - size casts of actual patients ' skin lesions to demonstrate dermatoses to medical students . when photography became popular in the 19th century , moulages were eventually replaced by color slides that were quicker and cheaper to generate . it greatly depends on the type and direction of illumination , including flash , contains shadows , visual artefacts arising from perspective and distance from the subject , as well as distortion of colors . digital photography has added chromatic aberration at the picture edges , blooming ( indicating local overexposure ) and , most prominently , the usual curse when trying to publish a picture : jpeg compression artefacts . moulages , instead , are based on an actual cast of the dermatosis in question and can therefore more accurately reproduce the dimensions of skin lesions . also , in contrast to photography , some non - essential elements such as overgrowth of hair can be omitted . each example is expensive and unique , can reproduce only a limited area of the body and requires careful conservation . we applied the technique of making wax moulages to document a case of psoriasis improvement for posterity . in most places this technique was kept a secret and got lost over time . the museum of wax moulages in zurich and the deutsches hygienemuseum in dresden are the only institutions that succeeded in preserving the original technique , including the recipes for the wax mixtures . in zurich , the moulageuse elsbeth stoiber passed on her technique to michael geiges in 1998 . sabina carraro and johanna stierlin are using this same technique to form duplicates or new moulages . up to present , the museum of wax moulages is the only place where moulages are being fabricated again . as an exception , navena widulin from the berlin museum of medical history at the charit was introduced to the zurich technique by michael geiges in 2004 , which resulted in a small collection of forensic wax moulages exhibited on several occasions . the moulage is cast using a silicone negative formed directly from the patient 's back using a mixture of bleached bees wax , calcium carbonate and dammar resin . the wax replica is colored using oil - based paint ( cobalt blue , carmine dark , bitumen and gamboge ) , traditionally next to the patient who acts as a model . the coloring is very time - consuming . in our case , because of the outpatient setting , numerous differently illuminated photographic pictures served as templates . a 57-year - old architect presented with moderate - to - severe plaque - type psoriasis that had first appeared in 2008 . topical therapy with steroid , calcipotriol and salicylic acid , followed by phototherapy with narrow - band uvb and methotrexate therapy , were unable to reach pasi50 . therefore , at a pasi of 12.7 , therapy with adalimumab was initiated and the most prominent plaques on the lower back documented by wax moulage . the wax cast demonstrated the improvement of infiltration from an average elevation from the skin surface of > 1 mm to 0 mm ( fig . however , the patient had not reached the clinical threshold of pasi50 overall , as his pasi was still at 10.4 ( table 1 ) . our modern moulage shows both the science - driven improvement of psoriasis achievable nowadays with biologics , but also how exquisitely dependent on the choice of location the art of moulage really was with its restricted area of reproduction . this effect namely that the rest of the patient remains unseen is easily forgotten when admiring the surviving moulages in the museum . taken together , all available techniques to document skin diseases have advantages and limitations and for learning dermatology , nothing beats seeing actual live patients .

several clinical trials have investigated the effect of the long - acting anticholinergic bronchodilator , tiotropium bromide ( barnes et al 1995 ) , on lung function and dyspnea at rest ( van noord et al 2000 ; vincken et al 2002 ; casaburi et al 2002 ; celli et al 2003 ; brusasco et al 2003 ; verkindre et al 2006 ) and during exercise ( odonnell et al 2004 ; casaburi et al 2005 ; maltais et al 2005 ) , and assessed its influence on quality of life ( vincken et al 2002 ; casaburi et al 2002 ; brusasco et al 2003 ; casaburi et al 2005 ; verkindre et al 2006 ) or on the occurrence of exacerbations ( vincken et al 2002 ; niewoehner et al 2005 ; dusser et al 2006 ) in chronic obstructive pulmonary disease ( copd ) . most of these studies indicated significant and sustained improvements of the primary outcome parameters , and consistently reported improvements in forced expiratory volume in one second ( fev1 ) and/or inspiratory capacity ( ic ) after different tiotropium bromide treatment periods up to one year . given that tiotropium bromide is thought to act through its selective and prolonged binding to m3 receptors ( gross 2004 ) , which are predominantly located in the large and medium - sized airways ( barnes 2004 ) , it could a priori be expected that the improvement in airway function would be mainly located in these larger conductive airways . while the observed fev1 improvements could be brought in agreement with this proposition , the ic improvements have prompted several authors to hint at the implication of small airways in this anticholinergic bronchodilation process ( van noord et al 2000 ; verkindre et al 2006 ) . we have recently shown that copd patients are characterized by a combination of both large and small conductive airways ventilation heterogeneity , as opposed to smokers with similar smoking history but without copd in whom only small airways are affected ( verbanck et al 2004 ) . this was measured with a model - based analysis of the multiple breath washout test which delivers independent phase iii slope - derived indices of ventilation heterogeneity originating in the conductive and acinar lung zone ( scond and sacin , respectively ) ( verbanck et al 1997 , 1998 ) . in an earlier study , copd patients have shown no significant response of scond or sacin to 400 g of salbutamol with a small spacer inhalation aid ( verbanck et al 1999 ) . in the present study , we use scond and sacin measurements to test the hypothesis that the small airways situated in the conductive and/or proximal acinar lung zones could participate to some degree in the tiotropium bromide response and thus affect inspiratory capacity , which is linked to dyspnea and exercise limitation in copd patients ( odonnell et al 2004 ) . in particular , this mechanistic study aims to investigate whether the reported beneficial effects on pre - dose ( trough ) and peak values of fev1 and ic are paralleled by improvements in small air - ways heterogeneity . in order to assess whether any potential improvement in small airways ventilation heterogeneity was specifically linked to improvement in hyperinflation , we deliberately included copd patients with varying degrees of hyperinflation at baseline ( which is also representative of a real - life clinical situation ) . forty patients with a clinical diagnosis of copd for at least 2 years were recruited if they fulfilled the following inclusion criteria : aged over 40 years , smoking history of at least 15 packyears , fev1/forced vital capacity ( fvc ) < 70% and post bronchodilator fev1 < 80%pred . patients with a history of concomitant asthma or atopy or patients requiring supplemental oxygen therapy were excluded . none of the patients had suffered any exacerbation in the six month period prior to the study , nor any recent upper respiratory tract infection . the copd patients already on tiotropium bromide treatment were asked to discontinue tiotropium for at least 4 weeks before the screening visit . patients had been requested not to take any short- or long - acting bronchodilator medication for at least 8 h and 24 h , respectively , before each visit . there was 1 screening visit and 3 visits further referred to as study visits , ie , a baseline visit ( visit0 ) and two visits after respectively 3 and 6 weeks ( visit3wks and visit6wks ) of tiotropium bromide therapy ( 18 g dry powder inhaled via the handihaler device once daily in the morning ) . on visit3wks and visit6wks , on the screening visit , patients underwent lung function and ventilation distribution testing according to standardized procedures . lung function included spirometry and plethysmographic measurement of specific airway conductance ( sgaw ) and ic ( icpl ) . given the potential problems with plethysmographic volume determination in the case of obstructed patients ( rodenstein and stanescu 1982 ) , we also used the preferred method of ic computation ( ie , the difference between open circuit functional residual and total lung capacity volume levels ) . for the computation and interpretation of ventilation heterogeneity indices scond and sacin , we refer to a previous description ( verbanck et al 1997 ) , which is briefly summarized in the appendix . the screening visit served to characterize patients in terms of lung function and ventilation heterogeneity , and to assess their room for reversibility by the combination of a short - acting anticholinergic and a fast - acting 2-agonist drug . sixty minutes after inhalation of 80 g of ipratropium bromide , an intermediate spirometry was performed , and additional bronchodilation was elicited by inhaling 400 g of salbutamol . after another 30 min ( ie , cumulative post - bronchodilator time : 90 min),lung function and ventilation distribution testing was repeated . on the baseline study visit ( visit0 ) , patients first underwent lung function and ventilation distribution testing , and were then instructed to inhale 18 g of tiotropium bromide dry powder via the handihaler device for the first time . after 60 min , intermediate spirometry was performed , and after 90 min , the study visit was concluded with lung function and ventilation distribution testing . the next two study visits ( visit3wks and visit6wks ) , after respectively 3 and 6 weeks of daily tiotropium bromide treatment , involved exactly the same procedure as that on visit0 . given that for these two study visits , the last tiotropium bromide intake had been on the morning preceding the study day , the 90 min effect of tiotropium bromide could be assessed on trough values of lung function and ventilation heterogeneity in the laboratory . on each visit , patients rated their dyspnea on a visual analogue scale ( vas ) and by means of the medical research council ( mrc ) scale ( ranging 15 ) . finally , patients also filled out a symptom score sheet for wheezing , shortness of breath , cough , and chest tightness ranging not present,mild,moderate,severe ( scored 03 and summed to a total symptom score ranging 012 ) . two - way repeated measures analysis of variance ( anova ) ( statistica5.5 , statsoft , tulsa , ok ) was used to detect any differences between the 3 study visits prior to and 90 min after tiotropium bromide administration ( 2 factors : inter - visit and intra - visit tiotropium bromide effect ) . one - way repeated measures anova was used to detect any inter - visit differences on the symptom scores . spearman rank correlations were performed between changes in symptom or dyspnea scores and changes in lung function indices . forty patients with a clinical diagnosis of copd for at least 2 years were recruited if they fulfilled the following inclusion criteria : aged over 40 years , smoking history of at least 15 packyears , fev1/forced vital capacity ( fvc ) < 70% and post bronchodilator fev1 < 80%pred . patients with a history of concomitant asthma or atopy or patients requiring supplemental oxygen therapy were excluded . none of the patients had suffered any exacerbation in the six month period prior to the study , nor any recent upper respiratory tract infection . the copd patients already on tiotropium bromide treatment were asked to discontinue tiotropium for at least 4 weeks before the screening visit . patients had been requested not to take any short- or long - acting bronchodilator medication for at least 8 h and 24 h , respectively , before each visit . there was 1 screening visit and 3 visits further referred to as study visits , ie , a baseline visit ( visit0 ) and two visits after respectively 3 and 6 weeks ( visit3wks and visit6wks ) of tiotropium bromide therapy ( 18 g dry powder inhaled via the handihaler device once daily in the morning ) . on visit3wks and visit6wks , on the screening visit , patients underwent lung function and ventilation distribution testing according to standardized procedures . lung function included spirometry and plethysmographic measurement of specific airway conductance ( sgaw ) and ic ( icpl ) . given the potential problems with plethysmographic volume determination in the case of obstructed patients ( rodenstein and stanescu 1982 ) , we also used the preferred method of ic computation ( ie , the difference between open circuit functional residual and total lung capacity volume levels ) . for the computation and interpretation of ventilation heterogeneity indices scond and sacin , we refer to a previous description ( verbanck et al 1997 ) , which is briefly summarized in the appendix . the screening visit served to characterize patients in terms of lung function and ventilation heterogeneity , and to assess their room for reversibility by the combination of a short - acting anticholinergic and a fast - acting 2-agonist drug . sixty minutes after inhalation of 80 g of ipratropium bromide , an intermediate spirometry was performed , and additional bronchodilation was elicited by inhaling 400 g of salbutamol . after another 30 min ( ie , cumulative post - bronchodilator time : 90 min),lung function and ventilation distribution testing was repeated . on the baseline study visit ( visit0 ) , patients first underwent lung function and ventilation distribution testing , and were then instructed to inhale 18 g of tiotropium bromide dry powder via the handihaler device for the first time . after 60 min , intermediate spirometry was performed , and after 90 min , the study visit was concluded with lung function and ventilation distribution testing . the next two study visits ( visit3wks and visit6wks ) , after respectively 3 and 6 weeks of daily tiotropium bromide treatment , involved exactly the same procedure as that on visit0 . given that for these two study visits , the last tiotropium bromide intake had been on the morning preceding the study day , the 90 min effect of tiotropium bromide could be assessed on trough values of lung function and ventilation heterogeneity in the laboratory . on each visit , patients rated their dyspnea on a visual analogue scale ( vas ) and by means of the medical research council ( mrc ) scale ( ranging 15 ) . finally , patients also filled out a symptom score sheet for wheezing , shortness of breath , cough , and chest tightness ranging not present,mild,moderate,severe ( scored 03 and summed to a total symptom score ranging 012 ) . two - way repeated measures analysis of variance ( anova ) ( statistica5.5 , statsoft , tulsa , ok ) was used to detect any differences between the 3 study visits prior to and 90 min after tiotropium bromide administration ( 2 factors : inter - visit and intra - visit tiotropium bromide effect ) . one - way repeated measures anova was used to detect any inter - visit differences on the symptom scores . spearman rank correlations were performed between changes in symptom or dyspnea scores and changes in lung function indices . the 40 patients ( 36m/4f ) participating in this study were stable copd patients on maintenance treatment mostly involving inhaled steroids ( 37/40 ) in combination with long - acting 2-agonists ( 37/40 ) . all patients were also on a combination of short - acting 2-agonists and ipratropium bromide , but at study entry , ipratropium bromide was discontinued . figure 1 shows a histogram of the residual volume over total lung capacity ratio ( rv / tlc ) measured at the screening visit before bronchodilator administration , in the patient group under study ( n = 40 ) . the median rv / tlc being 54.8% , the group was split into a subgroup with a high degree of hyperinflation if rv / tlc 55% ( hyp ; n = 20 ) and a subgroup with a low degree of hyperinflation if rv / tlc < 55% ( hyp ; n = 20 ) . table 1 shows lung function , ventilation heterogeneity and symptom scores prior to tiotropium bromide administration on the study visits visit0 , visit3wks and visit6wks . for each parameter in table 1 , change from baseline ( ) was computed as the difference between the value of visit6wks and that of baseline visit0 in order to test for correlations of change . in the hyp group , none of the correlations between changes in symptom or dyspnea scores and changes in lung function parameters were significant ( p > 0.1 for all ) . in the hyp group , changes in total symptom or mrc score did not correlate with any lung function or ventilation heterogeneity changes ( p > 0.1 for all ) . however , vas did correlate with ic ( r = 0.54 ; p = 0.016 ) and also with sgaw ( r = 0.49 ; p = 0.03 ) ( figure 2 ) . figure 2b clearly identifies an outlier at sgaw = 0.055 cmh2o s ( open circle ) ; upon detailed inspection , this was due to the plethysmographic determination of lung volume but there was no objective reason for exclusion . panels a in figures 36 illustrate , for the hyp group , inter- and intra - visit changes in fev1 , ic , scond , or sgaw , ie , those parameters of table 1 showing any significant inter- or intra - visit change in this subgroup . panels b in figures 36 display the corresponding data for the hyp group . in the hyp group , fev1 ( figure 3a ) and ic ( figure 4a ) significantly increased 90 min after the first 18 g tiotropium bromide dose on visit0 ( p 0.001 for both ) , while no such intra - visit increases occurred during visit3wks and visit6wks ( p > 0.1 for both ) . intra - visit changes in sgaw ( figure 5a ) showed a similarity with fev1 and ic behavior , in that sgaw also increased significantly during the baseline visit0 ( p < 0.001 ) but not during visit3wks or visit6wks ( p > 0.1 ) . while figure 5a suggests an increase in trough sgaw value , this was not significant ( p > 0.05 for both ) , yet , when excluding the abovementioned outlier ( figure 2b ; open circle ) , trough sgaw did increase significantly from 0.038 cmh2o s ( visit0 ) to 0.045 cmh2o s ( visit3wks ; p = 0.007 ) and 0.044 cmh2o s ( visit6wks ; p = 0.04 ) . while figure 6a indicates tendencies for intra - visit scond decreases ( ie , improvements ) of the same order as that observed on the screening visit , averaging an 11% decrease over the three study visits , the scond decrease only reached statistical significance on the last visit ( p = 0.02 ) . the complementary indicators of small airway function , end - expiratory flow after 75% of forced expired vital capacity ( fef75 ) and sacin , did not present any inter- or intra - visit effect in this subgroup ( p > 0.1 for all ) . the hyp group showed no improvements in trough values of any parameter after 3 or 6 weeks of tiotropium bromide treatment . another striking difference of the hyp with respect to the hyp group is that fev1 ( figure 3b ) and sgaw ( figure 5b ) showed highly significant intra - visit increases ( p < 0.01 for both ) on all study visits . by contrast , ic ( figure 4b ) only increased significantly ( p = 0.03 ) on the baseline visit ( p > 0.1 on visit3wks and visit6wks . while there were tendencies for intra - visit scond decreases ( ie , improvements ) of the same order as that observed at the screening visit , averaging a 12% decrease over the three study visits , the scond decrease failed to reach statistical significance ( visit6wks : p = 0.053 ) . as in the hyp group , fef75 and sacin did not present any inter- or intra - visit effect of tiotropium bromide in the hyp subgroup ( p > 0.1 for all ) . two supplementary analyses were performed to verify whether the observed patterns , that appear to be specifically linked to the presence of hyperinflation , were not a consequence of ( a ) the expected difference in disease severity ( in terms of fev1 ) between hyp and hyp subgroups , and ( b ) the arbitrary choice of the median rv / tlc as a cut - off ( in casu : rv / tlc = 55% ) for the classification into hyp and hyp subgroups . hence we repeated the analysis of table 1 , but by considering fev1 matched hyp and hyp subgroups ( n = 15 ; table 2 ) , and by considering hyp and hyp subgroups ( n = 10 ; table 3 ) with only those patients corresponding to the 10 lowermost rv / tlc ( < 46% ) and 10 uppermost the outcome of both supplementary analyses of tables 2 and 3 was very similar to that observed for the entire copd population in table 1 . this study shows a very distinct pattern of tiotropium bromide - induced improvement of airflow ( fev1 ) and ic between copd patients with a high degree of hyperinflation ( hyp ) and copd patients with low degree of hyperinflation ( hyp ) , in that only the hyp group showed significant increases in trough fev1 and ic values . in fact , reduction in ventilation heterogeneity was confined to the conductive airway compartment , was of limited magnitude , and followed a very similar pattern across both copd groups . the discrepancy between the behavior of ic and ventilation heterogeneity in the hyperinflated copd subgroup , makes it unlikely that small airways are implicated in the sustained improvements of ic observed by us and others . the modest intra - visit scond decreases ( reaching p = 0.05 significance on only one occasion ) imply that tiotropium bromide action is either affecting heterogeneity of the larger conductive airways ( thus not affecting small airways heterogeneity ) or acting in a very homogeneous way at the level of the small conductive airways ( thus not affecting small airways heterogeneity ) . however , the absence of any fef75 change with tiotropium bromide makes the latter possibility highly unlikely . rather , the difference in patterns of sgaw improvements between hyp and hyp groups , similar to that observed for ic and fev1 , suggests that larger airways or large lung units subtended by these large airways play a major role in the short- and long - term effect of tiotropium bromide . the sustained improvement of fev1 and ic after 3 and 6weeks of tiotropium bromide treatment only occurred in copd patients with considerable hyperinflation ( hyp ) . nevertheless , while the hyp patients showed no long - term fev1 or ic effects , their short - term fev1 increases ( 90 min after tiotropium bromide ) on each study visit were of the same magnitude as those obtained at baseline visit in hyp patients . no such similarity between short - term improvements across both copd subgroups was observed for ic . this indicates that the mechanism and site of bronchodilatation of tiotropium bromide ( reflected in fev1 ) are essentially the same in both copd groups , but that when considerable hyperinflation is present , a continued bronchodilating action has the additional benefit of relieving hyperinflation ( reflected in an ic improvement ) . the similar degree of bronchodilation in both groups suggests that any potential effect of the baseline state of hyperinflation on pulmonary distribution of the tiotropium bromide aerosol was negligible . in addition , the observed large airway effect suggests that either tiotropium bromide deposition was effectively confined to the large conductive airways , or that this was the only effective site of tiotropium bromide action on m3 receptors . the trough ic and fev1 values in the hyp group after 3 and 6 weeks of tiotropium bromide treatment were indistinguishable from the respective peak values seen at the baseline visit 90 min after tiotropium bromide . this contrasts with previous studies ( van noord et al 2000 ; vincken et al 2002 ; brusasco et al 2003 ; celli et al 2003 ; odonnell et al 2004 ; maltais et al 2005 ; niewoehner et al 2005 ; verkindre et al 2006 ) where trough fev1 and/or ic generally increased to a value intermediate between baseline and peak value , such that study visits after several weeks of tiotropium bromide treatment still showed room for fev1 or ic improvement . firstly , over 90% of the copd patients under study here were on a combination of a long - acting 2 agonist and inhaled steroid treatment , with tiotropium bromide as an add - on study medication , as opposed to most previous studies where long - acting 2 agonists were not permitted . this may account for some quantitative differences of tiotropium bromide effects with respect to previous studies ( here , typical changes in fev1 trough value are 10% baseline in hyp patients ) . secondly , in those previous studies where hyperinflation was not part of the inclusion criteria , the copd groups under study may have been a mixture of patients with a range of hyperinflation . one would then expect a pattern which is intermediate to that seen in our hyp and hyp groups , corresponding to a partial increase in trough fev1 and ic values between baseline and treatment visits , and an additional intra - visit increase from trough to peak fev1 or ic value . thirdly , the method of ic measurement varied across previous tiotropium bromide studies , using the difference between spirometric volume levels corresponding to frc and tlc ( corresponding to ic here ) or that derived from plethysmographic frc and tlc measurements ( corresponding to icpl here ) . the pitfalls inherent to plethysmographic volume determination in copd have been documented before ( rodenstein and stanescu 1982 ) and the difference between ic and icpl behavior across tables 1 to 3 emphasizes the interpretation problems with plethysmographically determined lung volumes . in conclusion , we have observed a pattern of improved airflow ( fev1 ) and inspiratory lung capacity ( ic ) with tiotropium bromide in copd patients , which was linked to their degree of hyperinflation at baseline . in patients with a low degree of hyperinflation , fev1 and by contrast , in copd patients with considerable hyperinflation at baseline , 3 weeks of tiotropium bromide treatment elicited improvements in trough ic and fev1 , close to the maximum obtainable value . the concomitant sgaw changes suggest that the main therapeutic effect is elicited in the large conductive airways . ventilation heterogeneity of the acinar airways was unaffected by tiotropium bromide , and the modest improvements in ventilation heterogeneity of the conductive airways were unrelated to changes in hyperinflation . the ventilation distribution test , ie , the multiple breath washout ( mbw ) , was carried out using a computer - controlled bag - in - box breathing assembly . tidal volume was targeted at 1 l and after a period of air breathing with stable end - expiratory lung volume at functional residual capacity , inspired air was switched to the test gas mixture . the test gas mixture consisted of pure o2 , and 1 l tidal breathing continued for 2025 breaths depending on the subject s lung volume ( dilution ) ; exhaled n2 concentration tracings were analyzed . from the mbw n2 tracings , indices scond and sacin were derived to represent the conductive and acinar components of ventilation heterogeneity , respectively , using an analysis that can be summarized as follows . during each expiration , the n2 phase iii slope is computed and normalized by the mean expired n2 concentration . this leads to a normalized n2 slope ( sn ) which increases as a function of breath number or lung turnover ( figure a1 ) ; lung turnover is determined as the cumulative expired volume divided by ventilated functional residual capacity , which is computed from a mass balance of the mbw test . on theoretical grounds it can be shown that : ( a ) the rate of rise of the sn curve is due to the convective flow asynchrony between lung units larger than acini , thus , due to heterogeneity originating in the conductive airways ; ( b ) the offset of the sn curve is mainly determined by diffusion - convection dependent heterogeneity generated in the acinar airways . hence , scond is simply computed as the rate of sn increase as a function of lung turnover , between 1.5 and 6 lung turnovers . then , sacin is computed as the sn value of the first mbw expiration minus a correction term to discard any conductive lung zone contribution ; this correction term equals the lung turnover corresponding to the first breath , multiplied by scond . illustrated in figure a1 are two selected copd patients who show a similar sacin ( 0.39 l and 0.41 l ) and markedly different scond ( open and closed circles correspond to respectively scond = 0.049 a normalized slope curve corresponding to a typical normal subject ( dotted line ) corresponds to sacin and scond values of 0.072 l and 0.029 l , respectively . the theory of mbw phase iii slope analysis leading to indices scond and sacin implies that ventilation heterogeneity can be attributed to different lung depths , ie , to the conductive and acinar lung zone , respectively , and that scond and sacin are intrinsically independent and are not comparable between each other ( despite having the same dimension , l ) . finally , since scond and sacin are derived from phase iii slopes , their value increases when ventilation heterogeneity increases . in particular , sacin will increase if ventilation heterogeneity is increased in the acinar lung zone , due to an alteration of the intra - acinar asymmetry ( eg , unequal narrowing of affected respiratory bronchioles ) . on the other hand , scond will increase when heterogeneous narrowing of conductive airways induces an alteration in the specific ventilation and/or flow asynchrony between the lung units subtended by these airways proximal to the terminal bronchioles . normalized n2 phase iii slopes ( mean sd ) as a function of lung turnover obtained in two patients participating in this study ( open and closed circles ) ; also represented is an idealized curve corresponding to normal normalized n2 phase iii slopes ( dotted line ) . an increase in scond reflects uneven narrowing of either the large or the small conductive airways , or both . a disproportionate scond increase with respect to the decrease of large airway parameters such as fev1 or specific airway conductance can be used to identify the small airways component of conductive lung zone heterogeneity . an increase in sacin represents a change in intra - acinar structural asymmetry , due to uneven narrowing of respiratory bronchioles or due to partial destruction of parenchyma , or both . again , a disproportionate sacin increase with respect to a decrease of diffusion capacity enables the identification of the nonemphysematous component of acinar lung zone heterogeneity .

the mechanism of transmission of the agent or agents causing sars is not yet fully understood but is probably mainly by droplet secretions , fomites , or person - to - person contact , as much of the transmission in hong kong has been limited to healthcare workers and family members . to date , no evidence of airborne transmission exists . in the amoy gardens outbreak in hong kong , aerosolization of fecal waste contaminated with the sars agent the virus has been reported to be stable in feces and urine at room temperature for at least 12 days , and up to 4 days in stool from patients experiencing diarrhea ( 5 ) . after drying on plastic surfaces , the virus can survive for up to 48 hours , although commonly used disinfectants and fixatives are effective against it ( 5 ) . for instance , the hong kong index patient is thought to have infected persons who transmitted the virus worldwide , subsequently resulting in outbreaks of > 300 patients in amoy gardens in hong kong and > 60 cases in singapore ( 610 ) . another hemodialysis patient has been involved in the transmission of sars in toronto ; therefore , such patients , who may have a relatively depressed immune system with associated high viral loads , may be unduly facilitating transmission of the virus . a more direct role of hemodialysis patients in the spread of viral infections has been previously observed in edinburgh , scotland , in the late 1960s , where transmission of hepatitis b was associated with mortality rates of 24% and 31% in renal patients and staff members , respectively ( 11 ) . the mechanism of transmission of the agent or agents causing sars is not yet fully understood but is probably mainly by droplet secretions , fomites , or person - to - person contact , as much of the transmission in hong kong has been limited to healthcare workers and family members . to date , no evidence of airborne transmission exists . in the amoy gardens outbreak in hong kong , aerosolization of fecal waste contaminated with the sars agent the virus has been reported to be stable in feces and urine at room temperature for at least 12 days , and up to 4 days in stool from patients experiencing diarrhea ( 5 ) . after drying on plastic surfaces , the virus can survive for up to 48 hours , although commonly used disinfectants and fixatives are effective against it ( 5 ) . for instance , the hong kong index patient is thought to have infected persons who transmitted the virus worldwide , subsequently resulting in outbreaks of > 300 patients in amoy gardens in hong kong and > 60 cases in singapore ( 610 ) . another hemodialysis patient has been involved in the transmission of sars in toronto ; therefore , such patients , who may have a relatively depressed immune system with associated high viral loads , may be unduly facilitating transmission of the virus . a more direct role of hemodialysis patients in the spread of viral infections has been previously observed in edinburgh , scotland , in the late 1960s , where transmission of hepatitis b was associated with mortality rates of 24% and 31% in renal patients and staff members , respectively ( 11 ) . procedures , such as the use of ventilators and nebulized bronchodilators , have been reported to have lead to spread by droplet transmission and aerosolization of virus - containing particles ( 12,13 ) . similarly other procedures , such as cardiopulmonary resuscitation , use of positive airway pressure devices , bronchoscopy , endotracheal intubation , airway suction , and sputum suction are thought to increase risk for infection ( 7 ) . although the use of such equipment in the treatment of most pneumonias , except influenza , presents no risk to staff , the emergence of sars has thrown into sharp focus the general safety of such routine practices , particularly when dealing with novel infectious agents . the sars outbreak is unique in its propensity to infect healthcare workers ; for instance , in china approximately 20% of cases are in healthcare workers , and early in the outbreak the rate was closer to 90% ( 14,15 ) . simple measures such as hand washing after touching a patient , the use of an appropriate and well - fitted facemask , and the introduction of infection control measures at an early stage , as well as quarantine of patients , may have reduced transmission ( 12 ) . restricting visitors to the hospital would further reduce the risk for transmission into the community . however , despite stringent use of full infection control procedures , breakthrough cases of sars still occurred in healthcare workers . coronavirus infections have been reported to infect lymphocytes , reducing their numbers in the hong kong patients by 30% ( 13 ) . immune - mediated cellular damage to the lungs has been reported ( 7 ) and has prompted the use of steroids in these patients . given the role of super - spreading patients ( 10 ) , who have relatively depressed immune systems , steroid use may further increase the viral load and prolong shedding of viable viral particles past the 12 weeks after symptoms disappear , potentially increasing the transmission of the disease and the duration of infectivity of the patient . two overlapping sets of disease signs and symptoms have been reported , with some patients having varying degrees of enteric disease . patients from china and at a number of hong kong hospitals have had relatively low rates of diarrhea ( 10% to 20% ) ( 3,13 ) , whereas patients from amoy gardens and canada have had higher rates , 50% to 70% ( 9,16,17 ) . some of these differences may result from the timing of data collection , with collection of data later in the course of the patient s illness including symptoms of diarrhea that may be associated with antibiotic therapy . however , these data suggest that possible differences in the mode of transmission , such as respiratory droplet compared to fecal - oral , or differences in the specificity of the organism to the respiratory or gastrointestinal tracts may also be present . mutations in isolates from respiratory or gastrointestinal tracts from the same cattle infected with coronavirus have been previously reported ( 18 ) ; such mutations may contribute to the observed differences in symptoms . the nonspecific disease signs and symptoms , long mean incubation period ( 6.4 days ) , long time between onset of symptoms and hospital admission ( from 3 to 5 days ) ( 6 ) , and lack of a reliable diagnostic test in the early phase of the illness ( 19 ) can lead to potential transmission to frontline healthcare workers and the community . similarly , the signs and symptoms in elderly patients , in whom the primary disease phase may be muted without evident fever , may further contribute to the spread of sars . additionally , as with other diseases , misdiagnosis can have fatal consequences . for example , in 2001 , an airline cabin crew member infected with malaria was misdiagnosed by two physicians , who did not identify the fact that she had recently traveled to a malaria - endemic area ( 20 ) . she was treated for common cold and died within 1 week of a malaria diagnosis by a tropical medicine specialist . misdiagnosis of a case of sars , particularly in a super - spreader in whom the disease symptoms may differ , could lead to rapid dissemination through the population . the nonspecific features and lack of an early diagnostic test have also led to difficulty of diagnosis with a potential threat to the community if such patients are discharged . the accuracy and timeliness of the reporting and dissemination of data relating to sars are important issues affecting public perception , and hence , fear , as well as the implementation of programs to limit spread of the disease . the initial inadequate reporting of cases in china ( 21 ) has tarnished the country s reputation , led to mistrust as to the magnitude of the outbreak , and may have hindered implementation of preventative measures . similarly , media attention , which plays a major role in the widespread dissemination of information , has a tendency to sensationalize information , leading to misconceptions over community preventative strategies , government and institutional procedures , and the magnitude of the outbreak . on the other hand , lack of information led to the development of public myths , with people in guangdong believing that boiling white vinegar would protect them from infection and leading to carbon monoxide poisoning from charcoal burning to heat the vinegar ( 22 ) . the accuracy and timeliness of the reporting and dissemination of data relating to sars are important issues affecting public perception , and hence , fear , as well as the implementation of programs to limit spread of the disease . the initial inadequate reporting of cases in china ( 21 ) has tarnished the country s reputation , led to mistrust as to the magnitude of the outbreak , and may have hindered implementation of preventative measures . similarly , media attention , which plays a major role in the widespread dissemination of information , has a tendency to sensationalize information , leading to misconceptions over community preventative strategies , government and institutional procedures , and the magnitude of the outbreak . on the other hand , lack of information led to the development of public myths , with people in guangdong believing that boiling white vinegar would protect them from infection and leading to carbon monoxide poisoning from charcoal burning to heat the vinegar ( 22 ) . sars presents formidable challenges to the healthcare community with medical , social , political , legal , and economic implications . all countries have to be prepared at a number of levels to deal with the threat posed by the sars epidemic and any other novel infectious disease . the healthcare sector should consider a few issues : 1 ) sars has emphasized the need for stringent infection control measures in hospitals on a regular basis , in anticipation of the next epidemic . while the measures may be in place , are we sure that they are being properly implemented at all times ? 2 ) healthcare workers should always follow simple , but stringent hygienic practices ( e.g. , washing hands before and after seeing a patient , even when no epidemic is apparent ) . 3 ) appropriate history taking , to obtain important information , such as recent travel history or contacts with possibly infected persons , when a patient with a fever is seen , could help to quickly identify persons at risk and reduce spread . 4 ) given the association with a number of super - spreaders and renal dialysis patients , strict quarantine procedures should be implemented if such persons are suspected of having sars . 5 ) the concepts of specificity and sensitivity need to be widely understood and applied . although the need for rapid diagnostic tests is important , introducing tests with inadequate sensitivity and unknown specificity should be prevented , as the data can not be interpreted . a negative test does not always exclude a disease , and discharging patients later diagnosed and readmitted could have serious consequences . 6 ) the use of high - risk medical procedures that may inadvertently spread the disease through aerosolization of the agent should be evaluated with potential new diseases in mind . other high - risk procedures should also be reconsidered with regard to infection control to limit risk from the use of intubation , cardiopulmonary resuscitation , and positive airway pressure devices . 7 ) quarantine and isolation procedures and contact tracing need to be instituted early in the outbreak , and access to hospitals treating such patients needs to be restricted to limit spread into the community . 8) environmental hygiene needs to be maintained . in the wake of the sars outbreak , the hong kong government has introduced a number of measures to improve public hygiene , including closely monitoring the integrity of sewage disposal systems ( deficiencies that were a possible source of the amoy gardens outbreak ) . the government has increased penalties for spitting , which still remains a commonplace habit . as with the outbreak of avian influenza , in which humans became infected through the purchase of live poultry , a process that still continues , the sars virus appears to have been contracted from an animal source ( possibly civet cats ) used for human consumption . close contact between humans and animal vectors in the southern china region has been responsible for a number of epidemics , including influenza a. a reduction in exposure to animal viral reservoirs should reduce the occurrence of such events . to that end , the chinese government has increased implementation of laws that prevent the consumption of wild animals . difficulties in obtaining information from all relevant sources could delay appropriate analyses , reporting of the situation , and implementation of necessary actions . the data collected should be two - tiered to include essential information required to control the outbreak , such as clinical details and contact information , as well as more detailed data that will enable ongoing or retrospective evaluation to determine , for instance , mode of transmission , which remains unconfirmed . leadership is essential to coordinate activities and information dissemination in order to minimize confused messages and public panic . coordination should be maintained with all relevant sectors including the health professionals , policymakers , community leaders , media , and the public . early detection and handling systems need to be consolidated to prepare for future epidemics . to this end , the hong kong government has announced the allocation of hk$1 billion ( us$1=hk$7.8 ) to fund a center for disease control . the role for the center remains to be clarified but should include monitoring for novel infections and research into existing agents . the center should also include an outbreak response unit that can be called on to spearhead coordinated action in a timely manner . the team should include infectious disease and public health specialists , epidemiologists , media spokespersons , administrators with suitable connections to frontline healthcare units , and other statutory bodies to enable collation and dissemination of important information and risk communication to relevant stakeholders . the unit will require legislative power to enable the rapid initiation of control measures both in the hospitals and the community . as no prophylaxis vaccination or specific proven treatment is yet available against sars , prevention is the only measure that one can take to prevent epidemics . communicating the risks and preventive measures in an effective and acceptable manner sars has had a significant impact on the local healthcare system ; a high proportion of patients require intensive care , coupled with prolonged hospitalization , overloading the system . similarly , the ready transmission to hospital care workers reduced the availability of knowledgeable healthcare workers to treat other patients and colleagues , and this further limited the ability of the hospitals to cope with the current outbreak . in summary , the current sars outbreak provides a timely reminder of the importance of maintaining basic healthcare practices at all times so that , when the next new disease strikes , we are well prepared to deal with it . establishing an outbreak response unit within the healthcare sector should be a priority with appropriate resources .

pheochromocytoma is rare but a critical tumor of chromaffin cells , which is frequently considered in the evaluation of hypertension , arrhythmia , or panic disorder and in the follow - up of patients with particular genetic diseases ( 1 , 2 ) . pheochromocytoma is a one of the potentially fatal causes of childhood hypertension and accounts for about 1% of pediatric hypertensive patients ( 3 , 4 ) . because pheochromocytoma is more common in adults than in children , most of the data available on its behavior and management have been based on adults , and thus the etiopathogenesis and management of pediatric pheochromocytoma remains obscure ( 1 ) . however , differences in the behavior of this tumor in children require special considerations to ensure optimal management of such pediatric population . this study aims to review one of the big pediatric series ever studied at a single medical center , and to make a brief review on the literature concerning advances in the genetics , biochemical diagnosis , localization , and management of pheochromocytoma . between 1984 and 2002 we treated 15 patients with pheochromocytoma : 11 males and 4 females with an average age at surgery of 11.7 yr ranging from 6 yr 9 months to 15 yr 7 months . we retrospectively evaluated the information available on each patient , including age , sex , clinical characteristics , diagnostic methods , treatment , pathologic finding , and outcome . tumors were localized by ultrasonography , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and by mibg scintigraphy . localized disease was defined as the case without a pathologic finding of surrounding tissue invasion , regional disease as disease with a pathologic finding of surrounding tissue invasion . the findings that are disclosed by pathologic examination are as follows : surrounding tissue invasion , capsular invasion , vascular invasion , necrosis , pleomorphism , atypical mitosis , cyst formation , and lymphoid reaction . in this study , the mean follow - up after resection was 95 months ( 5 - 223 months ) . statistical evaluations were performed using the spss package ( chi - squre test and fisher 's exact test ) . this patient had a father with a history of pheochromocytoma , which was treated by surgical resection when he was 45 yr old . their overall mean arterial blood pressures ( abps ) were 160 mmhg ranging from 140 to 190 mmhg and 100 mmhg ranging from 70 to 120 mmhg for systolic and diastolic pressure respectively . mean time between symptom onset and diagnosis was 11.9 months ranging from 1 month to 48 months . abnormal electrocardiography ( left ventricular hypertrophy ; n=7 , tachycardia ; n=2 , prolonged qt interval ; n=4 ) , hypertensive retinopathy ( n=5 ) , cardiomegaly at chest radiography ( n=6 ) , and hypertensive encephalopathy ( n=2 ) were also found . the average 24-hr daily urinary vanillylmendelic acid ( vma ) levels were 28.8 mg/24 hr ( range 0.6 - 52.8 mg/24 hr ) : all patients had a value higher than the upper limit of normal range ( normal value 0.7 - 6.9 mg/24 hr ) . elevated levels of serum epinephrine were found in one of the 15 patients only , and elevated levels of norepinephrine in three . i - metaiodobenzylguanidine ( mibg ) scintigraphy was performed in ten patients and intravenous pyelography in four . bone scans were performed in six patients to detect bone metastasis , and brain mri and eeg done in two patients who appeared symptoms of hypertensive encephalopathy . preoperative treatment with the phenoxybenzamine , nonselective -adrenergic antagonist , was performed in all patients for a median of 14 days ( range 7 - 21 days ) . the mean dose administered was 1.4 mg / kg / day ( range 0.2 - 2.44 mg / kg / day ) . prazocine , hydralazine , nifedipine , digoxine , lasix , or propranolol was also used intermittently . the transperitoneal approach was adopted in all patients , as we preferred a subcostal incision . fourteen patients had localized disease and one patient regional disease . none had metastatic disease . the tumors were in the right side adrenal gland in 3 patients , in the left side gland in 4 , bilaterally in 4 , and extra - adrenally in 4 . three of the four extra - adrenal pheochromocytomas were located in the organ of zuckerkandle and one was at the left renal hilum . seven patients with ipsilateral adrenal pheochromocytoma underwent ipsilateral adrenalectomy , including cortex sparing adrenalectomy ( n=2 ) , and contralateral adrenal gland biopsy ( n=1 ) . four patients with bilateral pheochromocytoma underwent bilateral adrenalectomy , including cortex - sparing adrenalectomy ( n=1 ) . four patients with an extra - adrenal pheochromocytoma received total excision , which included ipsilateral nephrectomy in one case with regional disease . the mean size of the removed masses was 4 cm ranging from 1.2 to 6 cm . there were several complications including operation site bleeding , ureter injury , lugol induced hypothyroidism and hypocortisonism . there was no difference of these pathologic results between recurred and non - recurred cases or between regional disease and localized disease ( table 4 ) . the mean follow up period to date is 95 months ( 5 - 223 months ) . after tumor removal , four patients recurred with the duration range of 2 yr 9 months to 14 yr ; three patients with localized disease and one patient with regional disease ( table 5 ) . the recurrent sites include lung parenchyme , pancreas , adrenalectomy site , and the organ of zukerkandle . one patient with pancreatic recurrence was given mibg therapy and was lost to follow up later . one patient with bilateral adrenalectomy site recurrence also received mibg therapy and subsequently mass excision for hypertension control but expired due to hypertensive encephalopathy . thus , mibg therapy was administered to two recurred patients but its effect was meager . of the four recurrent cases , one patient with regional disease had three separate recurrences . the first and second recurrent masses were surgically excised but the third recurrent mass has been kept under observation with anti - hypertensive medication , without surgical excision . rare as it is , pheochromocytoma is a chromaffin cell tumor that must be considered in patients with hypertension , autonomic disturbances , panic attacks , adrenal incidentalomas , or familial disease featuring a predisposition to develop pheochromocytoma ( 1 , 2 ) . about 5 - 20% of these extremely rare tumor are found in the pediatric population , and with an incidence of two per million , pheochromocytoma accounts for secondary hypertension in 0.5% of children , and is more common in preadolescent boys and teenage girls ( 3 - 6 ) . most studies have confirmed male preponderance and greater tendency for familial occurrence and multi - focality ( 43% in children vs. 30% in adults ) , an increased incidence of extra - adrenal tumors ( 30 - 40% in children vs. 10% in adults ) , and similar / lower risks of a malignant pheochromocytoma ( 3.5% ) ( 7 , 8) . our experience revealed a male predominance ( 73% ) , a preadolescent tendency ( mean age : 11.7 yr ) , an extra - adrenal location tendency ( 26.6% ) , and lower malignant development ( 0% ) , but the incidence of multifocal locations ( 26.6% ) . and a familial tendency ( 6.6% ) was lower than that found in the literatures ( 7 , 8) . the presentation of pheochromocytoma is variable , and patients may often be asymptomatic ( 9 ) . some symptoms of pheochromocytoma include sweating , visual complaints , nausea , vomiting , weight loss , polydipsia and polyuria and these are more common in children than in adults ( 7 , 10 ) . in our series , symptoms such as hypertension , sweating , headache , palpitations and vomiting tended to agree with findings commonly presented in the literature . although children with pheochromocytoma usually present with sustained hypertension , 80% of our patients presented with paroxysmal hypertension . the most important consideration when choosing an initial biochemical test is the reliability of the test in terms of excluding pheochromocytoma . the diagnosis of pheochromocytoma usually requires biochemical data indicating excessive catecholamine production by the tumor , which is performed by measuring vanillylmendelic acid ( vma ) and metanephrine 24-hr urinary excretion , giving a sensitivity and specificity of 80 - 100% ( 10 - 13 ) . a positive correlation between the size of a tumor and the urinary vma level has been reported ( 10 ) . in our series , all patients had an elevated level of urine vma , but we can not confirm a positive correlation between tumor size and the urinary vma level ( p>0.05 ) . further advances , measurements of plasma free metanephrines , normetanephrine , metanephrine , and the respective o - methylated metabolites of ne or epinephrine represent recently developed . preoperative imaging procedures are essential to the accurate and noninvasive localization of the number , site , size , and extension of these lesions for successful surgical management . in a recent study of 282 patients in which mr imaging , ct , and mibg were compared , mr imaging was found to be the most sensitive technique for localization of adrenal and extra - adrenal pheochromocytomas . computed tomography and magnetic resonance imaging i - metaiodobenzylguanidine ( mibg ) scanning and positron emission tomography have been used in identifying primary , recurrent and metastatic tumors ( 2 , 7 ) . ct and/or ultrasonography were used in all patients and allowed the visualization of the tumor site , the tumor size and of tumor extension . mibg scanning was also used to detect recurrent and metastatic tumors in 10 patients and recurrent tumor were detected in four patients . the most important aspect of preoperative preparation is the identification and treatment of hypertension and of other sequelae of catecholamine - induced vasoconstriction . most centers support to place patients on adrenergic blockades such as phenoxybenzamine and upon a positive fluid balance preoperatively ( 2 , 7 , 13 - 16 ) . current guidelines recommend phenoxybenzamine dosages of 160 mg at the maximum per day , divided into four equal doses every 6 hr , which should be gradually tapered over a 10- to 14-day period ( 13 ) . surgical excision of pheochromocytoma has been performed using a transabdominal approach , with palpation of the contralateral adrenal gland and sympathetic chain to detect additional tumors ( 2 ) . compared to open operations , laparoscopic surgery has the advantages of less postoperative pain , reduced postoperative stay , and a reduced need for analgesics and of improved functional and cosmetic results . therefore , laparoscopic surgery is associated with reduced morbidity versus open surgery in experienced hands ( 2 , 6 , 7 , 14 , 17 - 21 ) . despite many adult studies , few reports have described the results of this laparoscopic technique in pediatric patients , and more data is needed to confirm benefits of the laparoscopic approach in pediatric patients . because recurrent pheochromocytomas may develop by 20% to 33% of patients with familial syndrome undergoing partial adrenalectomy , bilateral adrenalectomy is recommended in bilateral pheochromocytoma , especially associated with hereditary syndromes . however , the range of 25% and 33% of patients undergoing bilateral adrenalectomy develop adisonism crisis at certain point of time , and the attendant mortality rates are high . moreover , 30% of patients develop clinically significant fatigue , and 48% consider themselves handicapped ( 22 , 23 ) . therefore , a minimally invasive approach , such as bilateral adrenal cortex - sparing tumor enucleation , certainly presents as an intriguing alternative to bilateral adrenalectomy , especially in those associated with hereditary syndromes . given the greater likelihood of a metachronous recurrence in children , consideration should be given adrenalsparing surgery in selected patients ( 2 , 7 , 13 ) . we performed cortex - sparing adrenalectomy in two ipsilateral adrenal pheochromocytomas and in one bilateral adrenal pheochromocytoma . the three patients that underwent cortex - sparing adrenalectomy survived without recurrence or hypocortisonism ( follow - up periods : 13 yr 5 months , 11 yr 5 months , and 5 months ) , but we were not able to confirm a significant difference between the cortex spared adrenalectomy group and the cortex no - spared adrenalectomy group in terms of the postoperative cortisol level . malignant pheochromocytoma is rare in childhood and the prognosis of this tumor in children is not well known . presenting signs , the localizations of the primary tumor or of metastases , and the course of malignant pheochromocytoma in children do not seem to be significantly different from those in adults ( 24 ) . in cases of malignant pheochromocytoma , multimodality treatments can be used . however , the benefits of chemotherapy , i - mibg therapy , external radiation and octreotide remained undetermined . the mean 5-yr survival rate for malignant pheochromocytomas is about 40% ( 13 , 24 ) . ten percent of pheochromocytoma are associated with familial cancer syndromes , such as multiple endocrine neoplasm type 2 , von hippel - lindau , neurofibromatosis type 1 , and familial carotid body tumors in which germ - line mutation of the ret , vhl , or nf1 genes have been found ( 2 , 6 , 7 ) . patients with a positive family history , bilateral adrenal pheochromocytoma , recurrent or multiple pheochromocytoma , and symptoms at a younger age should undergo clinical or genetic testing mutations of the von hippel - lindau or ret genes . succinate dehydrogenase mutation has been described previously in five families with familial paraganglioma syndrome ( 25 - 27 ) . in both the sporadic and familial forms of pheochromocytoma , allelic losses at 1p , 3p , 17p , and 22q have been reported , yet the molecular pathogenesis of these tumors is largely unknown ( 28 ) . in one patient with familial pheochromocytoma , recurrence in pheochromocytoma has been reported to range from 10% to 40% ( 10 , 29 - 31 ) , which concurs with our recurrence rate ( 26.6% ) . in the current study , however no significant difference was observed between localized and regional tumors in this regard . in three of four patients with recurrence , surgical resection has produced good results in terms of the relief of hypertension , but mibg therapy , which was performed for 2 patients , has produced no symptom relief . our conclusion is that early diagnosis and total excision constitute proper management in pheochromocytoma , and that the treatment of choice in recurred pheochromocytoma is surgical excision , if possible . furthermore , since the interval between resection and recurrence varies , long - term follow - up should be performed in pediatric pheochromocytoma .

with the increasing availability of completely sequenced genomes , comparative genomic approaches are becoming more important to decipher the functions of genes . methods , which are powerful , using the conservation of gene proximity on genomes ( i.e. determining potential operons ) can understand functional associations between genes ( 13 ) . genes in an operon are functionally associated with each other in prokaryotes ; thus , various kinds of operon prediction methods have been developed to understand the functional relationships and to annotate genes ( 413 ) . databases that accumulate the experimentally verified operon information should be useful to validate such prediction methods and also to understand the functional association between genes . however , databases providing genome - wide operon information have been limited to certain specific genomes ( 14,15 ) . although the string database was developed to identify functional associations between genes for multiple genomes , it uses gene neighborhood based on genome context methods ( 16 ) . here , we introduce the database called odb ( operon database ) , which provides operon data documented in the literature and putative operons that are conserved in terms of known operons . furthermore , to characterize operons , it integrates genome context , gene co - expression obtained from microarray data , functional links in biological pathways and data on the conservation of gene order across genomes . odb also provides operon prediction based on these various types of data as an application of our database . these datasets are fully pre - computed so that all information can be quickly accessed . in addition , it provides an operon prediction tool , which makes the system useful for both bioinformatics researchers and experimental biologists . we note that the experimentally verified operons , which we have collected , have been verified by a variety of means , from direct measurements such as primer extension and northern blots to less direct methods such as gene knock - out experiments . our database represents an ongoing effort to increase the coverage of operons . the current version of our database contains about 2000 known operon information in more than 50 genomes obtained from a total of 825 literatures ( table 1 ) . transcriptional unit individually as opposed to the generally understood usage of the term that may include multiple overlapping transcriptional units . operon structures are often observed in prokaryotes , but nematodes also have similar transcriptional systems ( 17,18 ) . note that the operons from bacillus subtilis contain operons obtained from transcriptional maps stored in bsorf ( ) . because these maps were derived from the results of northern blotting experiments , we added these operons into our database . note that these entries can be distinguished from the operons obtained from the literature , as the origin of the source ( bsorf ) is annotated in the database . table 1 also shows putative operons that are conserved in terms of known operons . when we calculated these conservations , we used kegg oc as the ortholog gene set ( 19 ) , which is ortholog gene clustering based on smith waterman sequence similarity scores . if genes in a known operon have ortholog genes in another genome and these ortholog genes are consecutively located on the same strand of the genome , we regarded them as a putative but highly reliable operon . operon for the eukaryotic gene clusters , but we use this term operationally in our database . as a result , odb uses a relational database management system ( mysql , ) to store and manage all information including not only known and putative operons but also primary data , such as gene location and definition , and associations between genes . this system contains four types of associations between genes that determine an operon : ( i ) intergenic distances , ( ii ) functional links in biological pathways , ( iii ) gene co - expression obtained from microarray data and ( iv ) the conservation of gene order across multiple genomes . these four types of associations are considered indicators and that the genes linked by them can organize an operon . therefore , we pre - calculated these associations among all genes in all available genomes to characterize operons . genes in an operon are often closely located on the genome compared with those between non - operons . intergenic distances are defined as the number of bases between the end position of a gene and the start position of the next gene on the genome . for example genes appearing in a metabolic pathway are often clustered on the genome to be co - transcribed ( 20 ) . the number of steps indicates that when two genes are linked across a compound , the number of steps is one . in this way , we calculated the number of steps not only in the same pathway map but also across different pathway maps . the kegg expression database contains the gene expression data derived from microarrays of four organisms , b.subtilis , escherichia coli k-12 w3110 , synechocystis sp . pcc6803 and saccharomyces we calculated the pearson 's correlation coefficients between gene expression profiles obtained from these microarray data . because it is considered that microarray data reflect actual gene transcription and that they are powerful tools to predict operons , co - expressed gene clusters on the genome are possible operons . however , the limitation of experimental conditions and quality of the experiments still leave the issue that certain operons are not transcribed and that the level of gene co - expression is not homogeneous . therefore , there are cases where genes are not co - expressed even if they are genes in a known operon . gene order in an operon is often shuffled and collapsed in evolutionary history ( 21,22 ) . therefore , conservation of gene order across genomes is rather rare , especially in distantly related genomes . if such conservation is observed , they are probably related to a physical interaction such as a molecular complex ( 23 ) . that is , given a gene pair , we took each of their ortholog genes from all genomes , calling this ortholog gene pair. then we calculated the step number between these two ortholog genes . when the gene pair is adjacently located on the genome , the step number is regarded as one . here , we ignore the genomes included in the same taxonomic group , which are defined in kegg ( ) . all - against - all runs of these associations between genes were performed . pre - computed results are stored in a table , allowing quick retrieval against the query specified by users . each table in our system corresponds to a particular genome to facilitate efficient access and retrieval of the information . when users search a gene or an operon of interest , the gene cluster including it can be identified by its name and identifier . then , the user is presented with a summary of genes and associations between genes in the region on the genome ( figure 1 ) . primary data such as gene names , gene ids , definitions , ko ids as functional classes , kegg pathway ids and ec numbers are presented . this view includes graphical symbols of operons , genes , pathways and ec numbers and each symbol is also linked to the kegg database . for the biological pathway table , the shortest step numbers between genes are presented . for the ortholog gene table , the shortest step numbers between the ortholog gene pairs are shown . in these tables , additional pages are accessible which show the detail of the information . for the gene expression table , the correlation coefficients between gene expression profiles are shown , and the strength of co - expression is illustrated by a color gradient ranging from blue to red . because the conditions to determine putative operons are very strict and are not genome - wide , odb also provides a system to predict operons , using the four associations . given a specific species , predicted operons that may exist within that species are returned . there are two options that are available : simple and advanced prediction mode . for a simple mode , users can obtain prediction results based on default parameter values that have been validated by known operons . however , in advanced prediction mode , users can freely change these parameter values , which are based on the four types of associations described above . when genes linked by these associations are clustered on the genome , they are likely to be an operon . thus , we benchmarked the accuracy of the predictions based on combinations of various values of intergenic distances , step numbers between ortholog genes and the number of the genomes having conserved ortholog genes that are linked within a specific range of step numbers . therefore , the optimal values that predict the largest number of operons while keeping the accuracy high is provided as default values in simple prediction mode ( supplementary data ) . when there is little or no known operon information in a genome , the default values of another genome in the same taxonomic group and having sufficient operon information is used as an alternative to the genome . if such genomes are also unavailable , we used the values of b.subtilis ( see supplementary data for details ) . odb provides a platform for searching known operons and consequent putative operons and for predicting operons with high accuracy validated by literature - based operon data . it includes about 2000 literature - based operons in over 50 genomes and about 13 000 putative operons in over 200 genomes . in addition , the data from kegg pathway and related resources that are provided allow analyses not only based on a specific genomic context but also across genomes . thus , it is the first of its kind to integrate operon data from a variety of genomes , providing a wide - ranging coverage of operons . this integrated system of both known literature - based and genomic data is a useful system for bioinformatics researchers and experimental biologists .

hemolysis , elevated liver enzymes , and low platelet count ( hellp ) syndrome is frequently associated with severe form of pre - eclampsia . however , the relationship between 2 symptoms is controversial , and the cause of hellp syndrome is still unknown . but there is a growing consensus that it has a familial tendency . at present , 2 classification systems are used for the diagnosis of hellp syndrome . diagnostic criteria developed at the university of tennessee are high lactate dehydrogenase ( ldh ) ( > 600 u / l ) , high aspartate aminotransferase ( ast ) ( 70 the mississippi hellp system further classifies the disorder by the plt counts : 1 ) class i if plts count less than 50,000/l ; 2 ) class ii if between 50,000/l and 100,000/l ; and 3 ) class iii if greater than 100,000/l . hellp syndrome is often regarded as acute rejection reaction to embryo implantation but the etiology is not fully understood . hellp syndrome has an incidence rate ranging from 0.5%0.9% in all pregnancies , and the rate increases up to 10%20% in pregnant women with preeclampsia . serious symptoms requiring dialysis and/or mechanical ventilation necessitate admission to an intensive care unit ( icu ) . inadequate treatment of hellp symptom may contribute to serious complications such as disseminated intravascular coagulation ( dic ) syndrome , hepatic hematoma , cardioplegia , myocardial ischemia , adult respiratory distress syndrome , pulmonary edema , acute kidney failure , intra - uterine growth retardation ( iugr ) , and infant respiratory distress syndrome . corticosteroid can be administered for the treatment of hellp , still , parturition is the most effective method to avoid complications . nutritional management of inpatients has been shown to be important in recovery from various diseases . also , requirements of energy , protein and micronutrients are elevated in critically ill patients due to abnormal nutrient loss from diarrhea and vomiting , increased metabolic rate , electrolyte abnormality , infection , surgery , and trauma . respiratory muscle wasting leads to prolonged use of mechanical ventilation and icu stay , complications related to infection and subsequently higher mortality . therefore , nutrition support using enteral and parenteral nutrition ( pn ) is critical to prevent malnutrition in hospitalized patients with inadequate dietary intake . goals of nutrition support for the critically ill patients include maintenance of lean body mass , enhancement of immune function , and prevention of metabolic complications . tertiary hospitals in korea are equipped with nutrition support team ( nst ) and strive for nutrition support for the hospitalized patients . we report the case of rare hellp syndrome patient who was admitted in icu for 20 days and was under nutritional management focused on total parenteral nutrition ( tpn ) , enteral nutrition ( en ) and then successfully switched to oral feeding . we observed that nst care allowed stabilization of the symptoms of the patient and led to discharge without complications . a 30-year - old female patient ( gestational age : 18 weeks ) , with a history of pain in the right shoulder , nausea , and dyspepsia for 3 days , was admitted to hospital complaining severe right upper quadrant pain . after medical evaluation , the patient was diagnosed with intraperitoneal hemorrhage due to rupture of liver hematoma and taken to the operating room for an emergency operation . on the surgery , post - surgery blood test results showed that levels of ast / alanine aminotransferase ( alt ) , ldh , total bilirubin , direct bilirubin , and c - reactive protein ( crp ) were above normal range . on the contrary , levels of plt , albumin , and cholesterol were lower than normal . despite the absence of high blood pressure , the patient was diagnosed with hellp syndrome based on lab test results , liver damage , and proteinuria . on day 2 after admission , the patient was initially fasted and was supported by central pn at day 4 . on day 9 , second surgery was performed to remove hematoma and then referred to nst . on referral to nst , the patient 's nutritional status was moderate malnutrition based on over 7 days of nil per os ( nothing by mouth [ npo ] ) and moderate accumulation of fluids . and the patient was on tpn ( combiflex lipid 1,000 inj [ central ] [ jw pharmaceutical , seoul , korea ] ; infusion rate 40 ml / hr ) and was given 1,080 kcal energy and 36 g protein daily . this is far less than required doses of 1,5401,650 kcal and 7282 g protein , calculated on the basis of initial body weight and hyper - metabolism ( 55 kg , 2830 kcal / kg , 1.31.5 g protein / kg ) . the progression of nutritional support and clinical aspects of the patient during admission was shown in figure 1 . hod , hospital onset of day ; nst consult , nutrition support team consult ; nsvd , normal spontaneous vaginal delivery ; tpn , total parenteral nutrition . it was judged that tpn in use ( combiflex lipid 1,000 inj [ central ] ; jw pharmaceutical ) could not meet the high protein demand induced by severe infection and high body fluid output from gastric drainage and hemovac drain . however , the patient could not receive large amount of tpn because she was on fluid restriction for the management of severe edema and liver failure . to resolve this situation , nst suggested alternative tpn ( smofkabiven inj 1,500 [ central ] [ fresenius kabi ab , uppsala , sweden ] ; 0.8 bag / daily ) which could provide higher amount of protein and micro minerals such as zinc , which is necessary for immune function and wound healing . in addition , nst recommended adding an ample of vitamin mixture ( tamipool inj ; celltrion pharm inc . , cheongju , korea ) to tpn . , the patient 's body weight was estimated to be 6366 kg due to edema . on day 15 ( 5 days after second surgery ) , the patient was referred to nst for en . nst evaluated the patient 's nutritional status and classified the status into moderate malnutrition state on the basis of poor energy supply below 75% of the requirement for > 7 days and the edema . since the patient had edema , nutrition requirement was calculated based upon ideal body weight , not actual body weight . at this point , estimated energy requirement to avoid overfeeding was 1,3281,593 kcal ( 2530 kcal / kg ) and protein requirement was 6480 g ( 1.21.5 g / kg ) . again , protein composition of standard enteral formula was not sufficient to meet the demand , so nst advised to use high protein enteral formula . from day 15 , the amount of enteral formula gradually increased to reach 1,500 kcal/1,500 ml at day 20 . also , edema has subsided , and the patient weighed down to 50 kg . the energy and protein deliveries during hospital stay were shown in figures 2 and 3 . during the first 2 weeks after icu admission , the tpn amount was far less than the requirement , and thereafter the tube feeding amount was approached to the minimum requirement . on day 22 , the patient was transferred to general ward . on day 24 , oral feeding of clear liquid diet was initiated . the patient experienced 10% weight loss from her initial body weight and weighed 49.5 kg . nst classified the patient into severe malnutrition state and calculated nutrition requirement based upon standard body weight ( 53.1 kg ) to promote rapid recovery . nst estimated that energy requirement was 1,4341,699 kcal ( 2732 kcal / kg ) and protein 5864 g ( 1.11.2 g / kg ) . nst suggested using oral nutrition supplement ( encover 200 ml [ 200 kcal , 7 g ] ; jw pharmaceutical ) to increase energy intake but this was not included in the diet regimen . the patient was successfully switched to regular diet and was discharged without significant complications . at discharge , two months after discharge , thorough checkup was performed at outpatient clinic and found improvements in liver function without specific symptoms other than mild fatty liver . hod , hospital onset of day ; crp , c - reactive protein ; alk . phos , alkaline phosphatase ; ast , aspartate aminotransferase ; got , glutamic oxaloacetic transaminase ; alt , alanine aminotransferase ; gpt , glutamic pyruvic transaminase ; t. bilirubin , total bilirubin ; d. bilirubin , direct bilirubin ; bun , blood urea nitrogen ; plt count , platelet count ; ldh , lactate dehydrogenase . albumin level is a sensitive marker of visceral protein and may not represent whole body protein . as shown in figure 4 , plasma cholesterol and albumin levels were below normal range at the time of admission but have improved significantly during hospitalization . in early stage of hospital stay , crp level was severely aberrant but returned to near normal range on discharge . the patient was under intensive care in icu for 20 days . while in icu , for majority of the surgical patients in icu , npo is recommended due to intraperitoneal surgery , enteroplegia , inflammation , and gastrointestinal hemorrhage . and these conditions necessitate proper pn for patients . the american society for parenteral and enteral nutrition ( aspen ) guideline advises to deliver early nutrition support therapy , primarily by the enteral route , and start pn as early as possible when en is not feasible in poorly nourished patients . the european society of clinical nutrition and metabolism ( espen ) also states that all patients who are not expected to be on normal nutrition within 3 days should receive pn within 2448 hours if they can not tolerate en . the espen guideline indicates that icu patients should receive at least 25 kcal / kg / day to target over the next 23 days . they also should receive enough protein to meet their requirement from day 1 of icu . as for the patient in this case report , pn was commenced on day 4 of hospitalization and lasted about 10 days . during that period , energy and protein supply was below 70% of the requirement and therefore could not meet the high metabolic demand after surgery . critically ill patients are prone to mineral deficiency due to higher requirement following massive loss from the body . deficiencies of zinc , iron , selenium and vitamins a , b , c are quite common among the critically ill and these deficiencies can increase the risk of multiple organ failure , muscle wasting , delayed wound healing and impaired immune function . when compared to guideline of american society of parenteral and enteral ( aspen ) and recommendation from our own nst , administered tpn to the subject did not contain enough minerals and electrolytes ( table 3 ) . on the other hand , multivitamin injection ( tamipool inj 5 ml / v ; celltrion pharm inc . ) suggested by nst could provide various vitamins to meet aspen guideline also ( table 4 ) . it is estimated that the subject could benefit from effective nutrition support if the tpn was adjusted according to nst recommendation from the early stage . the information for each nutritional supplement are follow as : combiflex lipid 1,000 inj ( central ) , jw pharmaceutical , seoul , korea ; smofkabiven inj ( central ) , fresenius kabi ab , uppsala , sweden . pn , parenteral nutrition ; na , sodium ; k , potassium ; mg , magnesium ; ca , calcium ; p , phosphorus ; zn , zinc ; cu , copper ; se , selenium ; mn , manganese ; cr , chromium . the information for tamipool inj is follow as : tamipool inj , celltrion pharm inc . , meanwhile the patient suffered severe weight loss ( 13.8% of initial body weight ) during hospital stay . after considering the weight loss incurred by stillbirths , the weight loss was 7.1% when compared to the weight before pregnancy . medical team and nst should take into consideration that severe edema can obscure accurate body weight measurement . in this patient , sufficient tpn supply was impossible in the early stage due to the presence of severe edema and liver failure . when clinical symptoms started to improve , the patient should have been given ample amount of nutrition to replenish early deficit . this indicates the necessity of intense nutrition intervention including oral nutrition supplement during oral feeding period . in conclusion , in difficult situations where the patient was suffering from increased nutrition demand after surgery and insufficient tpn supply in early stage , monitoring and intervention of nst in line with proper treatment of the medical team , continuous nutrition care of nst led to the significant improvements in clinical indices of disease and nutritional status . for the management of critically ill patients , accurate requirement estimation of macronutrients but therefore , inquisitive monitoring of patient followed by elaborate nutrition care of nst team is essential for the excellent clinical outcome .

two major pathophysiologic abnormalities underlie most cases of type 2 diabetes ( t2d ) : 1 ) insulin resistance and 2 ) defects in pancreatic -cell function . the current consensus is that both are essential components in disease pathogenesis even if their relative importance , the precise temporal sequence of events , and underlying mechanisms vary considerably in different populations and individual patients . in october 2013 , the global partnership to accelerate diabetes research , cosponsored by the endocrine society and the american diabetes association , assembled international experts ( see appendix ) to inform the global health research agenda by reviewing the state of the science and identifying pressing research needs related to -cell dysfunction in t2d . powerful evidence for a genetic component to t2d ( 1 ) has driven extensive efforts to identify genetic variants contributing to risk . monogenic forms of diabetes such as maturity - onset diabetes of the young ( mody ) have proven to be natural models for understanding mechanisms underlying insulin secretion defects ; genes discovered through family - based approaches are important regulators of insulin secretion and -cell development . findings that neonatal diabetes is most commonly due to activating mutations in genes encoding the atp - sensitive potassium channel subunits kir6.2 or sur1 and can be treated with high - dose sulfonylureas despite being insulin dependent also provide a compelling case for genetic evaluation of monogenic diabetes with therapeutic and prognostic implications ( 26 ) . exome - wide and whole - genome sequencing approaches will expand current capacity to study these disorders . genome - wide association studies ( gwas ) using high - density genotyping arrays have transformed understanding of the genetic architecture of t2d ( 7 ) . at the time of writing , over 60 genetic loci have been convincingly associated with t2d , the great majority in some way involved in -cell biology , underscoring the importance of -cell dysfunction in t2d pathogenesis ( 8,9 ) . gwas data must be interpreted with great caution until the precise genes in the loci associated with t2d have been identified and the impact of specific variants on -cell function is more clearly understood , as exemplified in a recent enigmatic study on the possible protective effects against t2d of loss - of - function mutations in slc30a8 ( encoding the islet zinc transporter znt8 ) ( 10 ) . in any case , these variants explain only a small proportion of total genetic risk ( 11 ) . studies based on exome and whole - genome sequencing technology are under way to identify low - frequency , high - impact variants accounting for a greater component of risk ( 12 ) . other novel approaches have been proposed , but genetic discovery models to date have largely been simple case - control studies of this complex metabolic disorder ( 13 ) . it is furthermore evident that other factors that influence gene expression contribute toward the complexity of t2d , specifically epigenetic mechanisms and micrornas ( mirnas ) . epigenetic mechanisms refer to functional changes to the genome that do not involve any alteration in nucleotide sequence . such mechanisms ( e.g. , dna methylation and histone modifications ) can be active during fetal as well as postnatal and adult life and impact the level of expression of select genes associated with t2d ( 14 ) . while the epigenome may be dynamic and change due to environmental exposure , modifications may also be stable and inherited , making epigenetics a potentially important pathogenic mechanism . the possibility that the environment can alter the pancreatic islet epigenome and subsequently affect -cell function and diabetes pathogenesis is specifically reflected in human and animal studies linking an impaired intrauterine environment and resulting low birth weight to an increased risk for postnatal metabolic disease , with decreases in -cell proliferation , mass , and insulin secretion in the face of documented epigenetic modifications in key -cell genes ( 15,16 ) . in addition , a low - protein diet in utero alters the epigenetic profile of hnf4a in rodent islets , associated with impaired islet function ( 17,18)findings supported by human studies . studies of pancreatic islets from nondiabetic donors ( 19,20 ) and patients with t2d ( 21 ) have identified epigenetic modifications in genes that potentially affect -cell function . such studies of human pancreatic islets together with in vitro studies of clonal -cells further suggest that hyperglycemia alters dna methylation of pdx1 and ins ( 2225 ) . dna methylation mainly occurs on cytosines in cpg dinucleotides , and approximately 50% of single nucleotide polymorphisms ( snps ) associated with t2d introduce or remove a cpg site . these cpg - snps are associated with differential dna methylation , gene expression , alternative splicing events , and hormone secretion in human pancreatic islets , suggesting strong genetic - epigenetic interactions ( 26 ) . it has also been suggested that histone modifications in human islets contribute to reprogramming -cells to -cells , possibly due to the large number of bivalent marks in -cells ( 27 ) . lipid treatment also alters the activity of enzymes responsible for histone modifications in clonal -cells , in parallel with decreased glucose - stimulated insulin secretion ( 28 ) . other recent studies indicate that histone deacetylases ( hdacs ) contribute to cytokine - mediated -cell damage , suggesting hdac inhibition as a possible diabetes treatment ( 29 ) . mirnas are a class of small noncoding rna molecules that modulate gene expression by binding to specific target messenger rnas to prevent their translation and/or to promote degradation . it has been suggested that altered mirna expression may contribute toward -cell failure in t2d and that these molecules may serve as biomarkers for the disease ( 30 ) . individual mirnas are highly represented in -cells ( 31 ) , impacting function and mass both positively and negatively . for example , knockout of mir-375 promotes progressive hyperglycemia in mouse models due to decreased insulin content and progressive loss of -cell mass ( 32 ) . there is increasing interest in the modulation of mirna expression , and a recent study has revealed an epigenetic mechanism in islets from patients with t2d ( 33 ) . . this will require insight and expertise from investigators in a wide range of fields that complement the specific skills of diabetes - focused researchers . clearly , extensions of conventional gwas are highly desirable , including evaluation of genetic models beyond single genes , coupled with more sophisticated quantitative measures of -cell function . a major barrier is the lack of large - scale , population - based samples with high - quality metabolic measurements of -cell function and the dearth of explanations for how discovered t2d genes actually mediate diabetes risk . studies on epigenetics and mirnas are distinctive in going beyond statistical associations to integrate multiple pathways to identify function , but they are in their infancy . understanding factors altering the expression of mirnas and the epigenome in pancreatic islets and -cells from prediabetic and t2d subjects and , further , developing selective small molecules that target epigenetic enzymes to improve -cell function and/or treat diabetes is essential . environmental modification could influence both mirnas and epigenetics , and a provocative question is whether such modifications can be used to predict -cell failure risk . finally , combining epigenetic and genetic research to integrate the entire body of data . this will require insight and expertise from investigators in a wide range of fields that complement the specific skills of diabetes - focused researchers . clearly , extensions of conventional gwas are highly desirable , including evaluation of genetic models beyond single genes , coupled with more sophisticated quantitative measures of -cell function . a major barrier is the lack of large - scale , population - based samples with high - quality metabolic measurements of -cell function and the dearth of explanations for how discovered t2d genes actually mediate diabetes risk . studies on epigenetics and mirnas are distinctive in going beyond statistical associations to integrate multiple pathways to identify function , but they are in their infancy . understanding factors altering the expression of mirnas and the epigenome in pancreatic islets and -cells from prediabetic and t2d subjects and , further , developing selective small molecules that target epigenetic enzymes to improve -cell function and/or treat diabetes is essential . environmental modification could influence both mirnas and epigenetics , and a provocative question is whether such modifications can be used to predict -cell failure risk . finally , combining epigenetic and genetic research to integrate the entire body of data will be necessary to explain the missing -cell loss in response to nutrient excess and stress was traditionally felt to occur exclusively via -cell death . although -cell death might be a final common pathway in the natural history of t2d , more recent evidence indicates a more complex situation in which -cells can initiate several alternative responses to avert irreversible loss , suggesting the potential for earlier intervention . mouse studies led by accili and colleagues ( 34 ) have shown that -cell dysfunction due to foxo1 deficiency during pregnancy and aging is primarily associated with -cell dedifferentiation rather than death . this finding revisits an earlier one ( 35 ) suggesting -cell dedifferentiation during disease development , although additional experimentation is needed to determine whether cells lose their defining -cell characteristics temporarily , revert to an immature fetal or neonatal - like state with impaired glucose - stimulated insulin secretion that reinitiates expression of fetal hormones , or indeed revert to an undifferentiated progenitor state . -cells that temporarily stop responding to glucose and storing normal amounts of insulin ( 36,37 ) . emerging evidence in mice also shows considerable plasticity within islets , allowing intraislet cell conversions , but only in the face of extreme -cell destruction ( 38 ) . the similarity between -cell and -cell transcriptomes in mice and humans supports this model , as does the discovery that hormone gene promoters in different islet cell types present similar methylation patterns . a challenge in understanding -cell failure is elucidating key elements responsible for their function and survival , including their apparently unique vulnerability to environmental changes . here , clonal cell lines selected for characteristics such as secretory defects after high - glucose exposure or susceptibility to cytokine - induced death have proved useful ( 39 ) . insights have also come from studying the unique substrate metabolism of -cells , chiefly focusing on the link between pyruvate cycling and glucose - induced insulin secretion ( 40 ) . studying the transcriptional control of replication is yet another way in which basic models may provide valuable translatable information and ultimately generate hypotheses for evaluation in primary -cells in vitro and in vivo ( 41 ) . many physiologic stressors impact -cell function in the environment of metabolic overload and insulin resistance commonly found in human obesity - linked t2d ( fig . while -cells initially respond by activating compensatory pathways to improve the insulin secretory response , eventually they initiate several pathologic programs that synergistically promote -cell dysfunction and , ultimately , death . to understand and intervene in disease progression , ongoing investigations are exploring which of the following pathologic conditions , or -cell stressors , are initiated first and which might represent the most effective intervention points . stressors on the -cell in the pathogenesis of t2d . in the excessive nutritional state found in obesity , hyperglycemia and hyperlipidemia develop , increasing metabolic load coupled with concurrent inherent insulin resistance and chronic inflammation . the pancreatic islet response to this new environment is likely variable among individuals with differing genetic susceptibility but may include inflammatory stress , er stress , metabolic and oxidative stress ( e.g. , glucotoxicity , lipotoxicity , and glucolipotoxicity ) , amyloid stress , and loss of islet cell integrity . if untreated , these interrelated stressors increase with time , promoting -cell dysfunction ( coupled with increased glucagon secretion ) and ultimately loss of -cell mass and possibly dedifferentiation that mark the onset of t2d . endoplasmic reticulum ( er ) stress appears to arise when markedly increased insulin production to meet metabolic demand necessitates increasing flux through the rough er , with stress evident in the unfolded protein response that chaperones newly synthesized proinsulin along the secretory pathway . while er stress may play a key role in the pathogenesis of certain forms of monogenic diabetes , its role in common t2d remains unclear . metabolic and oxidative stress , primarily from obesity s excessive nutritional state , leads to -cell damage associated with glucotoxicity , lipotoxicity , and glucolipotoxicity ( 42 ) . an emerging concept is a link between oxidative stress and observed dna damage leading to altered transcription factor expression . although -cells are uniquely geared for efficient oxidative metabolism both to provide energy via atp production and to generate secondary signaling mechanisms markedly increased glycolytic flux in hyperglycemia may underlie dysfunction . moreover , because -cells lack certain antioxidant enzymes that dispose of reactive oxygen species ( ros ) , increased ros production may promote dysfunction and even apoptosis . mishandling of excessive cholesterol commonly seen in t2d , with accumulation in -cells , could perhaps impair secretion ( 43 ) . amyloid plaques , which characterize islets in t2d , consist mainly of islet amyloid polypeptide ( iapp ) . in chronic hyperglycemia / hyperlipidemia , ( pro)iapp synthesis increases in -cells , parallel to proinsulin , and can reach threshold levels that allow proapoptotic iapp oligomers to form ( 44 ) that induce interleukin ( il)-1 release to recruit macrophages and enhance local islet inflammation ( 45 ) . whether increased local islet inflammation , well established in t2d pathogenesis ( 46 ) , results from a janitorial macrophage infiltration to clear damaged islet -cells and/or an innate inflammatory response remains unresolved . what has become clear , however , is that anti - inflammatory therapies ( i.e. , il-1 antagonism ) can preserve some -cell functional mass in t2d ( 47 ) . this may contribute to poorly regulated secretion of insulin and glucagon , perhaps contributing toward hyperglucagonemia that exacerbates hyperglycemia in t2d . it remains unclear which pathway is disrupted first , and this may actually depend on the individual . regardless of the initiation event , a feed - forward loop becomes induced that is difficult to stop . given the likely molecular cross talk and convergence between the pathways , targeting a single molecule could have beneficial effects by blocking multiple stressor pathways . recent studies have offered novel insight into pathways activated within -cells to cope with stress . novel therapies that exploit these natural defense mechanisms to prevent or reverse -cell failure in t2d may be possible . it is postulated that if -cell dysfunction is not ameliorated by effective therapy , with time there is loss of -cell identity through dedifferentiation and ultimately death . this sequence suggests the need to intervene as early as possible in the course of disease . endoplasmic reticulum ( er ) stress appears to arise when markedly increased insulin production to meet metabolic demand necessitates increasing flux through the rough er , with stress evident in the unfolded protein response that chaperones newly synthesized proinsulin along the secretory pathway . such changes may promote -cell secretory dysfunction and , under chronic challenge , apoptosis . while er stress may play a key role in the pathogenesis of certain forms of monogenic diabetes , its role in common t2d remains unclear . metabolic and oxidative stress , primarily from obesity s excessive nutritional state , leads to -cell damage associated with glucotoxicity , lipotoxicity , and glucolipotoxicity ( 42 ) . an emerging concept is a link between oxidative stress and observed dna damage leading to altered transcription factor expression . although -cells are uniquely geared for efficient oxidative metabolism both to provide energy via atp production and to generate secondary signaling mechanisms markedly increased glycolytic flux in hyperglycemia may underlie dysfunction . moreover , because -cells lack certain antioxidant enzymes that dispose of reactive oxygen species ( ros ) , increased ros production may promote dysfunction and even apoptosis . mishandling of excessive cholesterol commonly seen in t2d , with accumulation in -cells , could perhaps impair secretion ( 43 ) . amyloid plaques , which characterize islets in t2d , consist mainly of islet amyloid polypeptide ( iapp ) . in chronic hyperglycemia / hyperlipidemia , ( pro)iapp synthesis increases in -cells , parallel to proinsulin , and can reach threshold levels that allow proapoptotic iapp oligomers to form ( 44 ) that induce interleukin ( il)-1 release to recruit macrophages and enhance local islet inflammation ( 45 ) . whether increased local islet inflammation , well established in t2d pathogenesis ( 46 ) , results from a janitorial macrophage infiltration to clear damaged islet -cells and/or an innate inflammatory response remains unresolved . what has become clear , however , is that anti - inflammatory therapies ( i.e. , il-1 antagonism ) can preserve some -cell functional mass in t2d ( 47 ) . this may contribute to poorly regulated secretion of insulin and glucagon , perhaps contributing toward hyperglucagonemia that exacerbates hyperglycemia in t2d . it remains unclear which pathway is disrupted first , and this may actually depend on the individual . regardless of the initiation event , a feed - forward loop becomes induced that is difficult to stop . given the likely molecular cross talk and convergence between the pathways , targeting a single molecule could have beneficial effects by blocking multiple stressor pathways . recent studies have offered novel insight into pathways activated within -cells to cope with stress . novel therapies that exploit these natural defense mechanisms to prevent or reverse -cell failure in t2d may be possible . it is postulated that if -cell dysfunction is not ameliorated by effective therapy , with time there is loss of -cell identity through dedifferentiation and ultimately death . this sequence suggests the need to intervene as early as possible in the course of disease . impaired insulin secretion assessed by oral or intravenous glucose tolerance testing and hyperglycemic clamp studies is seen in prediabetes as well as early in the disease , with reduced secretion negatively correlated with glycemia ( 48 ) . although declines in -cell mass and function are not well correlated , presumably because function depends greatly on diabetes milieu , climbing glucose levels and deteriorating -cell function are tightly correlated ( 49 ) . while -cell function appears to decline progressively , insulin secretion defects seem at least partially reversible , especially early in the disease . family history and obesity are major risk factors for both youth and adults . in youth , a family history of t2d is also associated with a high risk for decreased insulin sensitivity and response , and increased proinsulin - to - insulin ratio ( 50 ) . impaired glucose tolerance ( igt ) in youth is characterized by -cell dysfunction manifested in impaired first - phase , but preserved second - phase , insulin secretion relative to sensitivity . however , youth with igt are no more insulin resistant than peers with normal glucose tolerance ( ngt ) if matched for body composition and fat topography ( 51 ) . once treated with metformin after progressing to t2d , youth show a greater rate of treatment failure than adults ( 52 ) . -cell mass increases during the first decade of life due to proliferation during the first 5 years ( 53,54 ) , stabilizing during adolescence with considerable variation ( three- -cell mass in t2d likewise varies , overlapping with that of normal individuals ( 5557 ) , although current data suggest a 2060% reduction ( 58 ) . currently , measurements of human -cell mass rely entirely on postmortem , cross - sectional assessments ; inability to assess mass noninvasively via imaging or biomarkers impedes determining temporal changes . furthermore , histomorphometric measurement of -cell area , volume , and mass is often imprecisely defined and complicated by technical inconsistencies ( 59 ) . importantly , too , because insulin sensitivity may vary as much as 10-fold in humans , variations in -cell mass may be linked to individual insulin sensitivity . thus , an individual with low insulin sensitivity and t2d may have a higher than normal -cell mass that is functionally reduced , suggesting that de facto variation might be considerably reduced if corrected for insulin sensitivity . marked variation in degree of -cell loss in t2d could potentially be reconciled by adopting standardized approaches to quantification using area , volume , and/or mass in available pancreases . it would be enormously helpful to be able to measure -cell mass in humans with noninvasive techniques . many approaches have been and are being examined , as has recently been reviewed ( 60 ) . one promising approach is to use fluorescent exendin-4 derivatives , which should bind preferentially to -cells ( 61,62 ) . however , a major issue concerns how much sensitivity and precision can be achieved by any noninvasive approach . a desirable goal might be the ability to measure changes in -cell mass as small as 5% because such changes are expected to be important for -cell function . given the difficulty of studying human -cells , well - defined clinical phenotypes correlated with -cell defects and/or noninvasive imaging or biomarkers defining proliferation and death in pancreatic samples could help elucidate the discordance between functional and morphometric assessments as well as help identify changes indicating early -cell failure in high - risk individuals ( 63 ) . standardizing methodologies measuring -cell function and defining approaches for specific clinical questions may also facilitate cross - study comparisons . currently , -cell function is variably assessed using fasting indices derived from insulin and glucose values , dynamic testing with oral glucose or standardized meals , and responses following intravenous glucose infusions ( 64 ) . interpreting -cell function in the context of glycemia and concurrent insulin resistance is critical in clinical studies evaluating therapeutic efficacy . other features of potential interest include sustainability and durability of treatment effects during and after therapy . marked variation in degree of -cell loss in t2d could potentially be reconciled by adopting standardized approaches to quantification using area , volume , and/or mass in available pancreases . it would be enormously helpful to be able to measure -cell mass in humans with noninvasive techniques . many approaches have been and are being examined , as has recently been reviewed ( 60 ) . one promising approach is to use fluorescent exendin-4 derivatives , which should bind preferentially to -cells ( 61,62 ) . however , a major issue concerns how much sensitivity and precision can be achieved by any noninvasive approach . a desirable goal might be the ability to measure changes in -cell mass as small as 5% because such changes are expected to be important for -cell function . given the difficulty of studying human -cells , well - defined clinical phenotypes correlated with -cell defects and/or noninvasive imaging or biomarkers defining proliferation and death in pancreatic samples could help elucidate the discordance between functional and morphometric assessments as well as help identify changes indicating early -cell failure in high - risk individuals ( 63 ) . standardizing methodologies measuring -cell function and defining approaches for specific clinical questions may also facilitate cross - study comparisons . currently , -cell function is variably assessed using fasting indices derived from insulin and glucose values , dynamic testing with oral glucose or standardized meals , and responses following intravenous glucose infusions ( 64 ) . interpreting -cell function in the context of glycemia and concurrent insulin resistance is critical in clinical studies evaluating therapeutic efficacy . other features of potential interest include sustainability and durability of treatment effects during and after therapy . interventions to prevent and treat diabetes by improving -cell function are based on the premise that -cell dysfunction can be reversed . this appears true for at least some portion of the pathogenesis spectrum , but limits of reversibility remain unexplored . available data suggest that in established diabetes effects on -cell function are not sustained following withdrawal of active therapies ; whether this is true at earlier stages of diabetes or in prediabetes is unknown . in the diabetes prevention program ( dpp ) , greater baseline -cell function and insulin sensitivity contributed independently to the restoration of ngt following lifestyle changes ( 65,66 ) . improvements in -cell function also appear to play a role in pharmacologic approaches to prevention and treatment . interventions in the troglitazone in prevention of diabetes ( tripod ) and pioglitazone in prevention of diabetes ( pipod ) and actos now for prevention of diabetes ( act now ) ( pioglitazone ) studies , for example , significantly improved the oral disposition index . moreover , changes such as regression to igt , maintenance of ngt , or progression to diabetes were proportional to insulin secretion response ( 67 ) . glp-1based therapies magnify glucose - stimulated insulin secretion , as seen with significantly improved -cell function in t2d following short - term infusions of glp-1 ( 68,69 ) and longer - term treatments with dipeptidyl peptidase-4 inhibitors ( 70 ) and glp-1 receptor agonists ( 71,72 ) . however , while augmented insulin production as assessed with glucose and arginine stimulation continues during active treatment and is of clinical value , none of these agents have produced a meaningful , persistent change following therapy withdrawal ( 71,73 ) . new evidence suggests that roux - en - y gastric bypass surgery ( rygb ) exerts antidiabetes effects in part via -cell functional improvements . rygb is unique among weight - loss surgery approaches to diabetes for producing marked improvements in metabolic status , including disease remission in at least 80% of patients ( 74 ) . the overall benefit clearly has a basis in the direct effects of weight loss , with attendant reduction in insulin resistance , and acute caloric restriction ( 75,76 ) . however , many lines of evidence demonstrate glycemic benefits independent of weight ( 77 ) , including changes in -cell function ( 78 ) . although studies to date have not clearly dissected changes in insulin sensitivity from changes in -cell function , augmented glp-1 secretion amplifies rygb s antidiabetes effects on pancreatic islets ( 79 ) , and post - rygb hyperinsulinemic hypoglycemia may involve -cell hypertrophy or neogenesis as well ( 80,81 ) . the observation that the strongest predictor of diabetes remission is duration of diabetes and insulin use prior to surgery ( 82 ) rather than weight regain also suggests underlying -cell health as a limiting factor for rygb s antidiabetes benefits . novel mechanisms of action and targets in -cells , such as fatty acid receptor activation , glucokinase activators , fractalkine , betatrophin , and -secretase 2 inhibitors , continue to emerge , adding to the potential approaches to alter the natural history of diabetes . for decades it has been known that relieving hyperglycemia can itself improve insulin secretion and restore metabolic control , at least temporarily . recently , early interventions with insulin therapy in newly diagnosed t2d ( 83 ) , with targeted anti - inflammatory therapy using an il-1 antagonist ( 84 ) , and with glp-1 receptor agonists ( 85 ) have demonstrated that -cell dysfunction can be reversed temporarily . however , durability of these effects following therapy withdrawal remains challenging . to date , these responses have been evaluated only in established diabetes ; whether they can alter the natural history of -cell failure earlier in disease progression is unknown . numerous pathophysiologic pathways contributing to progressive -cell failure have been identified as viable targets for intervention . however , no study has established whether one pathogenic pathway dominates or can serve as a single major target . moreover , different pathophysiologic processes may be active at different stages of progression , and optimal targets may shift accordingly . for example , early hyperglycemia may activate multiple pathophysiologic processes , including inflammation , amyloid accumulation , dedifferentiation , apoptosis , and genetic alterations ; whether any one target is optimal or sufficient at this stage remains undetermined . further , there may also be a stage of pathogenesis beyond which therapeutic interventions can not sufficiently enhance -cell function , making treatments targeting other aspects of metabolic dysregulation relatively more valuable . interventions targeted by stage of pathogenesis or combinatorial approaches may be required to preserve or restore -cell function . numerous pathophysiologic pathways contributing to progressive -cell failure have been identified as viable targets for intervention . however , no study has established whether one pathogenic pathway dominates or can serve as a single major target . moreover , different pathophysiologic processes may be active at different stages of progression , and optimal targets may shift accordingly . for example , early hyperglycemia may activate multiple pathophysiologic processes , including inflammation , amyloid accumulation , dedifferentiation , apoptosis , and genetic alterations ; whether any one target is optimal or sufficient at this stage remains undetermined . further , there may also be a stage of pathogenesis beyond which therapeutic interventions can not sufficiently enhance -cell function , making treatments targeting other aspects of metabolic dysregulation relatively more valuable . interventions targeted by stage of pathogenesis or combinatorial approaches may be required to preserve or restore -cell function . deterioration of -cell function antedates and predicts diabetes onset and progression , is genetically determined , and can be predicted with accuracy even though current tests are cumbersome and not well standardized . there is , however , continued debate surrounding the relative contributions of decreased function or mass to clinically manifest -cell dysfunction . this leads to confusion that extends beyond semantics and that will not be resolved until there are precise noninvasive methods to relate changes in -cell mass and function over time . multiple pathways underlie decreased -cell function and mass , some of which may be shared , with reduction in mass , perhaps a consequence of processes that initially caused dysfunction . in addition , the concept of -cell dedifferentiation in t2d has regained favor . even in the late stages of the disease , residual -cells remain , and their number is possibly underestimated due to absence of markers of -cell identity in dedifferentiated cells ( 86 ) . to date , most genes suggested by gwas as associated with t2d are also associated with reduced -cell function in the nondiabetic population and are known to be expressed in -cells and implicated in their development , function , or survival . however , many of these genes are also expressed in other tissues where their dysfunction may disturb glucose homeostasis and thereby , indirectly , -cell function . other genetic variation associated with diabetes , both common and less common , will be identified as the power of genetic studies increases ; the challenge will be to turn this information into new biological insights . the study of epigenetic changes in the -cell in t2d is likely to provide important new insight as well , but these studies are severely limited by the small number of islets available from patients with t2d and the difficulties separating cause and effect in hyperglycemia . a variety of interventions including weight loss , insulin , thiazolidinediones , and anti - inflammatory drugs can improve -cell function temporarily with improved glucose control , and these outcomes are certainly of value to patients . however , the limited number of clinical studies with appropriate protocols indicates that existing therapy does not arrest progression of -cell dysfunction in t2d let alone reverse it , with the possible exception of gastric bypass surgery . the group of experts identified areas for future research that would improve our understanding of -cell failure in t2d , hopefully leading to more effective prevention and the development of treatment with more durable beneficial effects on -cell function than is possible today .

meningiomas are the second most common central nervous system neoplasm in adults and account for 15 - 20% of all primary brain tumors . although most meningiomas are benign , -10% of meningiomas demonstrate more aggressive clinical behavior , as non - benign meningiomas . the term " chordoid meningiomas " was first coined by kepes et al . as a histopathological classification of tumors removed in seven patients ( aged 8 - 19 years ) with castleman 's syndrome12 ) . in the 2007 revision of the who classification of tumors of the cns , chordoid meningioma ( cm ) joined clear cell meningioma and atypical meningioma in the grade ii category , due to its high rate of recurrence , particularly following subtotal resection13 ) . most previous studies have described the pathological features of cm precisely , but have lacked data on its clinical course or treatment outcomes . furthermore , one recent study suggested that cm should be downgraded because of its relatively benign course25 ) . thus , we analyzed the clinical features and treatment outcomes of 16 patients with cm and reviewed the literature . we performed surgical removal for 2599 cases of initially diagnosed intracranial meningioma in our hospital between january 1999 and december 2012 . we reviewed the pathology data of these patients and 16 patients with pathologically confirmed cm were identified . all cases were reevaluated to confirm the histopathological diagnoses and to classify the tumors according to the 2007 who classification . we included all 16 patients in this study and retrospectively evaluated their medical records , radiological findings , and pathological findings . all patients underwent preoperative computed tomography ( ct ) scans and magnetic resonance imaging ( mri ) . ct scans were reviewed to check for bony invasion , defined as the existence of a focal osteolytic area adjacent to the tumor . the extent of resection was determined by the operation records and postoperative mris , and was graded according to simpson 's classification22 ) . incomplete resections were defined as simpson grade ii , iii , or iv resections . in our treatment strategy , the first post - treatment mri examination was done 3 - 6 months after the end of treatment and on an annual basis thereafter . regardless of clinical symptoms , tumor recurrence or progression were defined as any newly detected enhancement after total resection or as any increased area of the residual tumor after subtotal resection , respectively . all documented cases of local recurrence were diagnosed radiographically and follow - up periods were measured from the time of the primary surgery . to evaluate the proliferative capacity of individual tumors , the degree of ki-67 labeling was determined using mib-1 immunostaining in selected sections of excised specimens . the ratio of the mib-1-positive cells to the total cell number was defined as the mib-1 labeling index ( % ) . there was no clear cut - off value that was defined as " high mib-1 labeling index " ; however , several previous studies have revealed that an mib-1 labeling index score of > 2 - 4% at initial surgery was associated with a high incidence of recurrence of meningiomas10,15,16,17,18,19 ) . in the present study as there was no consensus on adjuvant radiation therapy for who grade ii meningiomas , adjuvant radiation therapy was performed according to the individual physician 's discretion for patients with incomplete resections . the median dose of radiation was 56.5 gy ( range : 54 - 60 gy ) in 27 to 35 fractions . progression - free survival ( pfs ) was determined on the basis of the length of time from the date of surgery to the appearance of radiological evidence of recurrence on follow - up mri . statistical analyses for pfs were performed by comparing computer - generated curves estimated by the kaplan - meier method . to evaluate the risk factors for recurrence follow - ups were conducted in the outpatient clinic , and the median follow - up period was 56.5 ( range : 3 - 170 ) months . the median follow - up period of patients with recurrence was 55.5 ( range : 3 - 170 ) months and patients with no recurrence was 56.5 ( range : 14 - 89 ) months . we performed surgical removal for 2599 cases of initially diagnosed intracranial meningioma in our hospital between january 1999 and december 2012 . we reviewed the pathology data of these patients and 16 patients with pathologically confirmed cm were identified . all cases were reevaluated to confirm the histopathological diagnoses and to classify the tumors according to the 2007 who classification . we included all 16 patients in this study and retrospectively evaluated their medical records , radiological findings , and pathological findings . all patients underwent preoperative computed tomography ( ct ) scans and magnetic resonance imaging ( mri ) . ct scans were reviewed to check for bony invasion , defined as the existence of a focal osteolytic area adjacent to the tumor . the extent of resection was determined by the operation records and postoperative mris , and was graded according to simpson 's classification22 ) . incomplete resections were defined as simpson grade ii , iii , or iv resections . in our treatment strategy , the first post - treatment mri examination was done 3 - 6 months after the end of treatment and on an annual basis thereafter . regardless of clinical symptoms , tumor recurrence or progression were defined as any newly detected enhancement after total resection or as any increased area of the residual tumor after subtotal resection , respectively . all documented cases of local recurrence were diagnosed radiographically and follow - up periods were measured from the time of the primary surgery . to evaluate the proliferative capacity of individual tumors , the degree of ki-67 labeling was determined using mib-1 immunostaining in selected sections of excised specimens . the ratio of the mib-1-positive cells to the total cell number was defined as the mib-1 labeling index ( % ) . there was no clear cut - off value that was defined as " high mib-1 labeling index " ; however , several previous studies have revealed that an mib-1 labeling index score of > 2 - 4% at initial surgery was associated with a high incidence of recurrence of meningiomas10,15,16,17,18,19 ) . in the present study as there was no consensus on adjuvant radiation therapy for who grade ii meningiomas , adjuvant radiation therapy was performed according to the individual physician 's discretion for patients with incomplete resections . the median dose of radiation was 56.5 gy ( range : 54 - 60 gy ) in 27 to 35 fractions . progression - free survival ( pfs ) was determined on the basis of the length of time from the date of surgery to the appearance of radiological evidence of recurrence on follow - up mri . statistical analyses for pfs were performed by comparing computer - generated curves estimated by the kaplan - meier method . to evaluate the risk factors for recurrence follow - ups were conducted in the outpatient clinic , and the median follow - up period was 56.5 ( range : 3 - 170 ) months . the median follow - up period of patients with recurrence was 55.5 ( range : 3 - 170 ) months and patients with no recurrence was 56.5 ( range : 14 - 89 ) months . there were 6 men and 10 women ( male - to - female ratio of 1 : 1.7 ) . the mean age at the time of diagnosis was 40 ( range , 14 - 74 ) years . the locations of tumors were as follows : convexity , 3 ; falx , 2 ; sphenoid ridge , 3 ; parasagittal , 1 ; intraventricular , 1 ; tuberculum sellae , 2 ; olfactory groove , 1 ; optic nerve sheath , 1 ; pineal area , 1 ; and tentorium , 1 . based on the mri findings , preoperatively , three patients were suspected to have langerhans cell histiocytosis ( n=1 ) , malignant glioma ( n=1 ) , and subependymal giant cell astrocytoma ( n=1 ) . mib-1 labeling index data were available for 12 patients and showed widely varying values ( median : 5.04% , range : 1 - 26.6 ) . a high mib-1 labeling index ( > 4% ) was seen in 7 of 12 ( 58.3% ) specimens . one pediatric patient showed clinical features of castleman 's disease8 ) with a cerebellar cm and intestinal lymphangiectasia . patient data , including initial symptoms and tumor size , are detailed in table 1 . simpson grade i , ii , and iii resections were performed in four , nine , and three patients , respectively . in contrast , 5 of the 12 patients with incompletely resected tumors showed recurrence during the follow - up period ( odds ratio , 2.14 ) . adjuvant radiation therapy was associated with longer pfs in the incompletely resected group . among the 12 patients with incompletely resected tumors , eight underwent adjuvant radiation therapy . patients who underwent surgery with adjuvant radiation therapy showed significantly longer pfs ( 121 months , 95% ci=82.1 - 159.9 ) than those who underwent surgery alone ( 40.5 months , 95% ci=9.6 - 71.3 ) by the log - rank test ( p=0.027 ) ( fig . , four showed local recurrence around the primary site , one showed distant intracranial recurrence , and one showed distant intracranial recurrence with spinal leptomeningeal seeding . the average mib-1 labeling index was 11.56% in cases with recurrence and 5.95% cases without recurrence . focusing on the incompletely resected cases , it was 14.82% and 5.57% . however , it failed to reach statistical significance ( mann - whitney u - test , p=0.731 , p=0.451 ) the treatments for recurring cm wereas as follows : gamma knife radiosurgery ( gks ) , 4 ; gks+radiation therapy , 1 ; and endoscopic transnasal tumor resection , 1 ( table 1 ) . the median follow - up period of these patients after recurrence was 23.5 months ( range : 11 - 36 ) . in this series , we had two previous mris free of meningiomas , which were taken at 20 and 28 months before the cm diagnoses . in these patients , 2 ) ( table 1 ) visited the emergency department with severe headache and vomiting . axial t1-weighted contrast - enhanced mri showed small enhancing mass at the pineal area with mild ventriculomegaly ( fig . the patient underwent endoscopic biopsy and third ventriculostomy ( etv ) , and cm was confirmed pathologically . an mri was conducted 3 weeks after surgery and it showed slightly decreased ventricular size ( fig . four weeks after surgery , we performed simpson grade iii resection with an occipitotranstentorial ( ott ) approach . three months after resection surgery , a contrast - enhanced ct was obtained for the follow up study , and it revealed the recurrencd of enhancing masses at the pineal area and right foramen of monro ( fig . foramen of monro : tumor volume 1100 mm , prescription dose 25 gy/50% , pineal area : tumor volume 322 mm , prescription dose : 25 gy/50% ) was performed for recurred lesion and followed by radiation therapy ( intensity - modulated radiation therapy , 40 gy/20 fractions ) . six months after resection surgery , intractable back pain developed , and contrast - enhanced spinal mri showed multiple epidural enhancing masses at the thoracolumbar spine ( fig . fourteen months after resection surgery , the patient expired due to uncontrolled cns metastasis of cm . 2 ) ( table 1 ) visited the emergency department with severe headache and vomiting . axial t1-weighted contrast - enhanced mri showed small enhancing mass at the pineal area with mild ventriculomegaly ( fig . the patient underwent endoscopic biopsy and third ventriculostomy ( etv ) , and cm was confirmed pathologically . an mri was conducted 3 weeks after surgery and it showed slightly decreased ventricular size ( fig . three months after resection surgery , a contrast - enhanced ct was obtained for the follow up study , and it revealed the recurrencd of enhancing masses at the pineal area and right foramen of monro ( fig . foramen of monro : tumor volume 1100 mm , prescription dose 25 gy/50% , pineal area : tumor volume 322 mm , prescription dose : 25 gy/50% ) was performed for recurred lesion and followed by radiation therapy ( intensity - modulated radiation therapy , 40 gy/20 fractions ) . six months after resection surgery , intractable back pain developed , and contrast - enhanced spinal mri showed multiple epidural enhancing masses at the thoracolumbar spine ( fig . fourteen months after resection surgery , the patient expired due to uncontrolled cns metastasis of cm . the ultimate goal of surgery is a complete resection to fully remove the tumor and the involved dura mater and skull . however , the complete resection of a tumor is sometimes difficult because of its locatior . in this study , many cms were located in the skull base or deep - seated in the brain , which would make their complete removal difficult . because of the insufficient number of cases , it is difficult to verify the meaning of a complete resection statistically except for the odds ratio . interestingly , the pattern of the resective procedure ( i.e. , en bloc vs. piecemeal resection ) could be related with iatrogenic metastasis . piecemeal resection was inevitable in a few cases depending on the location of the tumor particularly for intra - ventricle tumors , or those deep - seated in the brain . in fact , the primary locations of the cm were intra - ventricular and the pineal area in two cases of remote recurrence . although there has been no previous literature about iatrogenic metastasis of who grade ii meningiomas during surgery , few cases of iatrogenic metastasis during surgery have been reported . ldemann et al.14 ) reported a case of the iatrogenic seeding of malignant meningioma along a surgical trajectory , and sadahira et al.20 ) reported on the implantation of malignant meningioma to the abdominal wall during resection surgery . further clinical experience is required to clarify the importance of the pattern of the resective procedure in the case of who grade ii meningiomas , including cm . however , clinical benefits have been reported , especially for the treatment of residual tumors , recurrence , and both who grade i and iii meningiomas2,4,6,7 ) . however , there s have been no randomized or prospective studies . we performed adjuvant radiation therapy fon eight patients with simpson grade ii and iii resections , at the physician 's discretion . because of its rarity , in patients with cm , clinical experience with adjuvant radiation therapy has not yet been accumulated . recently , wang et al.25 ) reported on the benefits of adjuvant radiation therapy after incomplete cm resection . however , in cases of simpson grade i resection , recurrence only occurred in only one of four patients and it was a remote - site recurrence . based on this result , adjuvant radiation therapy after simpson grade i resection for local tumor control may not always be recommended . the mib-1 labeling index increases with who grade : 1.00 - 1.35% for grade i , to 1.90 - 9.30% for grade ii or atypical , and 5.60 - 19.5% for grade iii1,3,5,21 ) . moreover , a higher mib-1 labeling index is related to meningioma recurrence5,21,24,26 ) . however , other studies have refuted these relationships between mib-1 labeling index and the clinical features of meningiomas11,23,25 ) . in this series , the average mib-1 labeling index in cases with recurrence was higher than cases without recurrence , but failed to reach statistical significance . focusing on incompletely resected cases , the gap of mib-1 labeling index between the groups with recurrence and without recurrence widened , but it also failed to reach statistical significance . however , the only case with the development of leptomeningeal seeding after resection surgery was the case with the highest mib-1 labeling index . because of the small number of cases , it is hasty to conclude the clinical meaning of mib-1 labeling index in this study . accumulation of more clinical experience is demanded to so that this issue can be clarified . the mib-1 labeling index for cm in this series was quite variable , at 1.0 - 26.6% . this indicates that biological nature may be diverse even within the single pathological disease entity of cm . as a result , the mib-1 labeling index may provide more information about biological features and the prognosis for each cm case . we made inferences from the mris and the minimum growth rates in these two patients were faster than the previously reported growth rates for benign or incidentally found meningiomas9,27 ) . recently , some have suggested downgrading cm in the who classification unless poor prognostic factors are found . in our experience , this suggestion seems to result from the wide spectrum of biological characteristics within this single disease entity . however , regarding our series , with the high recurrence rate , especially in the non - adjuvant radiation therapy group , the possibility of leptomeningeal seeding , the relatively high growth rate , and high mib-1 labeling index in some cases , it is difficult to argue that cm is a benign meningioma . there are some limitations to our study ; the number of patients was not sufficient to determine biological or clinical characteristics . additionally , potential biases may exist , because of the collected clinical data being reviewed retrospectively . because of the rarity of cm , a multicenter prospective randomized study is required to clarify the various controversies that have been noted previously . the cm is difficult to manage and has a high recurrence rate . in our series ,

respiratory tract infections , such as community - acquired pneumonia ( cap ) , acute exacerbation of chronic bronchitis ( aecb ) , or acute bacterial sinusitis , are important causes of morbidity and mortality worldwide . the most frequent causative organism in cap is streptococcus pneumoniae or , less commonly , one of the atypical organisms mycoplasma pneumoniae , chlamydophila pneumonia , or legionella pneumophilia.1 in aecb , the primary organisms are haemophilus influenzae , s. pneumonia , and moraxella catarrhalis.2 the antibiotic used to treat any of these respiratory tract infections should be chosen to maximize bacterial eradication and minimize the risk of antimicrobial resistance development . of particular concern is the emergence of the multidrug - resistant s. pneumonia.3,4 moxifloxacin is an 8-metoxifluoroquinolone with a broad spectrum of antibacterial activity . it is effective against gram - positive pathogens and gram - negative pathogens , atypical pathogens , and anaerobic bacteria . among respiratory pathogens , most penicillin- and macrolide - resistant and -lactamase - producing strains are sensitive to moxifloxacin.5 this study was designed to obtain data on the effectiveness and safety of moxifloxacin ( administered intravenously [ iv ] or sequentially [ iv followed by oral ] ) under daily treatment conditions in china in a large number of patients with diagnosed bacterial respiratory tract infections . this was a prospective , noninterventional , noncontrolled , multicenter , postmarketing surveillance trial conducted between february 2006 and march 2007 in china in patients presenting at a hospital as part of a global study . the study was carried out within an approved indication in accordance with us food and drug administration and european medicines agency guidelines and applicable local law(s ) and regulation(s ) . doctors were instructed to prescribe moxifloxacin for treatment of a respiratory tract infection in the usual manner in accordance with the chinese marketing authorization as far as possible . the physician documented data at an initial visit and an end of therapy ( eot ) visit and/or , in the case of sequential therapy , an interim visit when the patient switched to oral treatment . observation parameters were fever , cough , dyspnea , and sputum , which were documented to assess symptom development . effectiveness parameters included course of severity of infection , course of patient s general condition , course of clinical signs and symptoms , duration until improvement and recovery , and overall assessment of effectiveness by patient and physician . safety parameters were adverse events ( aes ) , including adverse drug reactions ( adrs ) , and global assessment of tolerability . descriptive and explorative analyses were conducted with the software system sas for windows , ( version 9.1 ; sas institute , lans , nc ) according to confidentiality guidelines , each patient was assigned an individual five - digit number ( pseudonymization ) , and only the physicians knew which patient had which number and could identify the patient . the analysis was based on the intent - to - treat ( itt ) population . for qualitative variables , absolute and relative frequencies were calculated . for stratified analyses , row percents ( row% ) and column percents ( col% ) were calculated . for numeric data , the following distribution parameters were calculated : median , mean , standard deviation ( sd ) , minimum , maximum , and 1% , 5% , 25% , 75% , 95% , and 99% percentiles . for coding of verbatim documentation , the following coding systems were applied : concomitant therapies : world health organization dd 2005/q3 ; adverse events : meddra version 10.0 . the definitions for the safety and the itt populations were identical in this study . patients were included in the itt population if at least one dose of moxifloxacin was taken during the observational period . seventy - four of 2027 enrolled patients were excluded from itt analysis due to one or more of the following reasons : i ) patient double entry , ii ) first visit in more than 2 days before the start of the study ( retrospective documentation ) , and iii ) no symptoms at initial visit . thus , for the itt population as well as in the safety analysis , data of 1953 patients were analyzed . this was a prospective , noninterventional , noncontrolled , multicenter , postmarketing surveillance trial conducted between february 2006 and march 2007 in china in patients presenting at a hospital as part of a global study . the study was carried out within an approved indication in accordance with us food and drug administration and european medicines agency guidelines and applicable local law(s ) and regulation(s ) . doctors were instructed to prescribe moxifloxacin for treatment of a respiratory tract infection in the usual manner in accordance with the chinese marketing authorization as far as possible . the physician documented data at an initial visit and an end of therapy ( eot ) visit and/or , in the case of sequential therapy , an interim visit when the patient switched to oral treatment . observation parameters were fever , cough , dyspnea , and sputum , which were documented to assess symptom development . effectiveness parameters included course of severity of infection , course of patient s general condition , course of clinical signs and symptoms , duration until improvement and recovery , and overall assessment of effectiveness by patient and physician . safety parameters were adverse events ( aes ) , including adverse drug reactions ( adrs ) , and global assessment of tolerability . descriptive and explorative analyses were conducted with the software system sas for windows , ( version 9.1 ; sas institute , lans , nc ) according to confidentiality guidelines , each patient was assigned an individual five - digit number ( pseudonymization ) , and only the physicians knew which patient had which number and could identify the patient . the analysis was based on the intent - to - treat ( itt ) population . for qualitative variables , absolute and relative frequencies were calculated . for stratified analyses , row percents ( row% ) and column percents ( col% ) were calculated . for numeric data , the following distribution parameters were calculated : median , mean , standard deviation ( sd ) , minimum , maximum , and 1% , 5% , 25% , 75% , 95% , and 99% percentiles . for coding of verbatim documentation , the following coding systems were applied : concomitant therapies : world health organization dd 2005/q3 ; adverse events : meddra version 10.0 . the definitions for the safety and the itt populations were identical in this study . patients were included in the itt population if at least one dose of moxifloxacin was taken during the observational period . seventy - four of 2027 enrolled patients were excluded from itt analysis due to one or more of the following reasons : i ) patient double entry , ii ) first visit in more than 2 days before the start of the study ( retrospective documentation ) , and iii ) no symptoms at initial visit . thus , for the itt population as well as in the safety analysis , data of 1953 patients were analyzed . the definitions for the safety and the itt populations were identical in this study . patients were included in the itt population if at least one dose of moxifloxacin was taken during the observational period . seventy - four of 2027 enrolled patients were excluded from itt analysis due to one or more of the following reasons : i ) patient double entry , ii ) first visit in more than 2 days before the start of the study ( retrospective documentation ) , and iii ) no symptoms at initial visit . thus , for the itt population as well as in the safety analysis , data of 1953 patients were analyzed . a total of 1953 patients treated with moxifloxacin were documented and were valid for effectiveness and safety evaluation . the vast majority of patients were of asian origin ( 99.8% ) ; one patient was white ( 0.1% ) and one patient was black ( 0.1% ) . for two patients , a total of 1284 patients were male ( 65.7% ) , and 669 patients were female ( 34.3% ) . the age ranged from 14.0 to 105.0 years with a mean age of 59.6 17.5 years . a total of 46.9% ( n = 915/1953 ) of the patients suffered from cap , and 40.1% ( n = 783/1953 ) of the patients examined were diagnosed with aecb . combined infections ( cap and aecb , cap and other , aecb and other , cap and aecb and other ) were diagnosed in 3.8% ( n = 75/1953 ) of all cases , and 15.9% ( n = 310/1953 ) of patients were affected by other infections . most frequent other infections were respiratory infection ( 7.4% , n = 23/310 ) , cross - infection in hospital ( 4.8% , n = 15/310 ) , and inhalation pneumonia ( 4.8% , n = 15/310 ) . of the patients , 22.4% were smokers and 19.3% past smokers , which is known to be an important etiological factor with regard to chronic bronchitis ; 57.5% of the patients were nonsmokers . in terms of alcohol consumption , mild in 205 patients ( 10.5% ) , moderate in 1217 patients ( 62.3% ) , and a total of 721 ( 36.9% ) patients ( 38.2% of aecb patients and 30.7% of the cap group ) had already received at least one antibiotic treatment for the current bacterial infection before the start of moxifloxacin therapy , whereas the majority of 1186 patients ( 60.7% ) had not yet received any antibiotic pretreatment ( table 1 ) . the route of moxifloxacin application was exclusively iv in 78.3% of patients , whereas moxifloxacin sequential therapy was administered in 21.6% of the patients . in the case of sequential therapy , 415 out of 421 patients ( 98.6% ) started with moxifloxacin iv therapy and then switched to oral treatment . a total of 99.6% of all patients treated with iv ( patients treated exclusively with iv and treated by sequential therapy ) received the recommended daily dose of 400 mg , six patients ( 0.3% ) were administered 800 mg moxifloxacin treatment , and in one case ( 0.1% ) information on moxifloxacin iv dose was missing . oral therapy was administered as a 400 mg daily dose regimen in 99.8% of the patients , and in only one case ( 0.1% ) was information missing . the mean duration of moxifloxacin treatment was 9.1 days ( sd 3.6 days ) with a median of 8.0 days . exclusive iv therapy was applied for , on average , 8.1 days ( sd 2.9 days ) . the total treatment duration varied with the patients diagnoses : aecb therapy was slightly shorter ( 8.9 days , sd 3.7 days ) compared with cap therapy ( 9.2 days , sd 3.4 days ) and the therapy for other diagnoses ( 9.0 days , sd 3.7 days ) , respectively . combined infections were treated for an average of 8.4 days ( sd 2.8 days ) . the majority of all documented patients treated by sequential therapy ( 53.2% , 224/421 ) received moxifloxacin treatment for a time period of 1014 days , whereas duration for most patients treated exclusively with iv therapy was 79 days ( 41.2% , 631/1530 ) . a small proportion of patients was treated for less than 5 days ( sequential therapy : 0.5% ; iv therapy : 2.8% ) . a total of 26.8% treated by sequential therapy and 3.3% treated by exclusive iv therapy , respectively , were treated for more than 14 days . at each visit , the patients condition and severity of infection were assessed and recorded by the attending physician . additionally , fever and the three most prevalent symptoms ( dyspnea , cough , and sputum ) were reported ( figure 1 ) . the patients condition and severity of infection at the start and end of therapy are displayed in figures 2 and 3 . in total , there were 1949 patients recorded with data on improvement and 1951 patients recorded with data on recovery . a total of 82.5% ( n = 1607/1949 ) of the patients had improved after 5 days of treatment ; 68.9% ( n = 1345/1951 ) patients recovered after 10 days . at the end of the observational period , an improvement was observed in 98.1% ( n = 1911/1949 ) of the respiratory tract infection patients treated with moxifloxacin , and recovery was documented for 89.9% ( n = 1754/1951 ) of the participating patients ( figure 4 ) . moxifloxacin iv treatment , as well as sequential therapy ( moxifloxacin iv / oral ) , was very effective in the treatment of respiratory tract infections . for 93.3% ( n = 1822/1953 ) of the documented patients , effectiveness was rated as very good ( 56.4% , n = 1102/1953 ) and good ( 36.9% , n = 720/1953 ) by the attending physician . for sufficient and insufficient , 5.3% ( n = 103/1953 ) and 1.3% ( n = 25/1953 ) were rated individually . stratification by diagnosis revealed best ratings ( very good and good ) in the patient group suffering from cap ( 94.5% ) . of the aecb patients , 92.6% had very good and good effectiveness ratings , compared with 88.1% of patients with other infections . the physicians final assessment of effectiveness was also stratified by severity of the infection at the initial visit : effectiveness was assessed as very good and good in 95.1% of patients with a mild infection , in 94.6% of patients with a moderate infection , and in 89.8% of patients with a in multimorbid patients with at least two concomitant diseases , effectiveness was assessed as very good and good in 91.3% of patients compared with 95.0% of patients with fewer than two concomitant diseases . physicians recorded that 96.8% ( n = 1890/1953 ) of patients were very satisfied not satisfied was rated in only 2.9% ( n = 56/1953 ) of patients . during this open - label moxifloxacin postmarketing surveillance study of 1953 = 1115/1953 ) or good ( 36.5% , n = 712/1953 ) in 93.5% ( n = 1827/1953 ) of all patients . sufficient in 5.1% and as insufficient in 0.8% of the patients individually ( figure 6 ) . pretreatment ( 95.6% very good or good ) as well as lower morbidity ( fewer than two concomitant diseases , 94.4% very good or good ) revealed better results regarding tolerability compared with patients without pretreatment ( 90.6% very good or good ) and patients with higher morbidity ( two or more concomitant diseases , 92.5% ) . the incidence rates of aes and adrs were 0.72% ( n = 14/1953 ) and 0.67% ( n = 13/1953 ) , respectively . symptoms such as nausea and vomiting , which are well known in antibiotic treatment and also with quinolones , were predominant ( each with 0.15% ) . one serious ae occurred ( 0.05% ) , which was also defined as a serious adr . in this case , an 82-year - old male patient encountered falling white blood cell count , which led to hospitalization necessary or prolonged , but the event finally resolved . in total , there were 33 aes . out of those , twenty - eight of the aes ( 24 of 29 adrs ) were resolved or improved by the end of the observational period . a total of 1953 patients treated with moxifloxacin were documented and were valid for effectiveness and safety evaluation . the vast majority of patients were of asian origin ( 99.8% ) ; one patient was white ( 0.1% ) and one patient was black ( 0.1% ) . for two patients , a total of 1284 patients were male ( 65.7% ) , and 669 patients were female ( 34.3% ) . the age ranged from 14.0 to 105.0 years with a mean age of 59.6 17.5 years . a total of 46.9% ( n = 915/1953 ) of the patients suffered from cap , and 40.1% ( n = 783/1953 ) of the patients examined were diagnosed with aecb . combined infections ( cap and aecb , cap and other , aecb and other , cap and aecb and other ) were diagnosed in 3.8% ( n = 75/1953 ) of all cases , and 15.9% ( n = 310/1953 ) of patients were affected by other infections . most frequent other infections were respiratory infection ( 7.4% , n = 23/310 ) , cross - infection in hospital ( 4.8% , n = 15/310 ) , and inhalation pneumonia ( 4.8% , n = 15/310 ) . of the patients , 22.4% were smokers and 19.3% past smokers , which is known to be an important etiological factor with regard to chronic bronchitis ; 57.5% of the patients were nonsmokers . in terms of alcohol consumption , mild in 205 patients ( 10.5% ) , moderate in 1217 patients ( 62.3% ) , and a total of 721 ( 36.9% ) patients ( 38.2% of aecb patients and 30.7% of the cap group ) had already received at least one antibiotic treatment for the current bacterial infection before the start of moxifloxacin therapy , whereas the majority of 1186 patients ( 60.7% ) had not yet received any antibiotic pretreatment ( table 1 ) . the route of moxifloxacin application was exclusively iv in 78.3% of patients , whereas moxifloxacin sequential therapy was administered in 21.6% of the patients . in the case of sequential therapy , 415 out of 421 patients ( 98.6% ) started with moxifloxacin iv therapy and then switched to oral treatment . a total of 99.6% of all patients treated with iv ( patients treated exclusively with iv and treated by sequential therapy ) received the recommended daily dose of 400 mg , six patients ( 0.3% ) were administered 800 mg moxifloxacin treatment , and in one case ( 0.1% ) information on moxifloxacin iv dose was missing . oral therapy was administered as a 400 mg daily dose regimen in 99.8% of the patients , and in only one case ( 0.1% ) was information missing . the mean duration of moxifloxacin treatment was 9.1 days ( sd 3.6 days ) with a median of 8.0 days . exclusive iv therapy was applied for , on average , 8.1 days ( sd 2.9 days ) . the total treatment duration varied with the patients diagnoses : aecb therapy was slightly shorter ( 8.9 days , sd 3.7 days ) compared with cap therapy ( 9.2 days , sd 3.4 days ) and the therapy for other diagnoses ( 9.0 days , sd 3.7 days ) , respectively . combined infections were treated for an average of 8.4 days ( sd 2.8 days ) . the majority of all documented patients treated by sequential therapy ( 53.2% , 224/421 ) received moxifloxacin treatment for a time period of 1014 days , whereas duration for most patients treated exclusively with iv therapy was 79 days ( 41.2% , 631/1530 ) . a small proportion of patients was treated for less than 5 days ( sequential therapy : 0.5% ; iv therapy : 2.8% ) . a total of 26.8% treated by sequential therapy and 3.3% treated by exclusive iv therapy , respectively , were treated for more than 14 days . at each visit , the patients condition and severity of infection were assessed and recorded by the attending physician . additionally , fever and the three most prevalent symptoms ( dyspnea , cough , and sputum ) were reported ( figure 1 ) . the patients condition and severity of infection at the start and end of therapy are displayed in figures 2 and 3 . in total , there were 1949 patients recorded with data on improvement and 1951 patients recorded with data on recovery . a total of 82.5% ( n = 1607/1949 ) of the patients had improved after 5 days of treatment ; 68.9% ( n = 1345/1951 ) patients recovered after 10 days . at the end of the observational period , an improvement was observed in 98.1% ( n = 1911/1949 ) of the respiratory tract infection patients treated with moxifloxacin , and recovery was documented for 89.9% ( n = 1754/1951 ) of the participating patients ( figure 4 ) . moxifloxacin iv treatment , as well as sequential therapy ( moxifloxacin iv / oral ) , was very effective in the treatment of respiratory tract infections . for 93.3% ( n = 1822/1953 ) of the documented patients , insufficient , 5.3% ( n = 103/1953 ) and 1.3% ( n = 25/1953 ) were rated individually . the overall effectiveness assessment made by the physicians stratification by diagnosis revealed best ratings ( very good and good ) in the patient group suffering from cap ( 94.5% ) . of the aecb patients , 92.6% had very good and good effectiveness ratings , compared with 88.1% of patients with other infections . the physicians final assessment of effectiveness was also stratified by severity of the infection at the initial visit : effectiveness was assessed as very good and good in 95.1% of patients with a mild infection , in 94.6% of patients with a moderate infection , and in 89.8% of patients with a effectiveness was assessed as very good and good in 91.3% of patients compared with 95.0% of patients with fewer than two concomitant diseases . physicians recorded that 96.8% ( n = 1890/1953 ) of patients were very satisfied not satisfied was rated in only 2.9% ( n = 56/1953 ) of patients . during this open - label moxifloxacin postmarketing surveillance study of 1953 patients in china , moxifloxacin was principally well tolerated . ( 57.1% , n = 1115/1953 ) or good ( 36.5% , n = 712/1953 ) in 93.5% sufficient in 5.1% and as insufficient in 0.8% of the patients individually ( figure 6 ) . pretreatment ( 95.6% very good or good ) as well as lower morbidity ( fewer than two concomitant diseases , 94.4% very good or good ) revealed better results regarding tolerability compared with patients without pretreatment ( 90.6% very good or good ) and patients with higher morbidity ( two or more concomitant diseases , 92.5% ) . the incidence rates of aes and adrs were 0.72% ( n = 14/1953 ) and 0.67% ( n = 13/1953 ) , respectively . symptoms such as nausea and vomiting , which are well known in antibiotic treatment and also with quinolones , were predominant ( each with 0.15% ) . one serious ae occurred ( 0.05% ) , which was also defined as a serious adr . in this case , an 82-year - old male patient encountered falling white blood cell count , which led to hospitalization necessary or prolonged , but the event finally resolved . in total , there were 33 aes . twenty - eight of the aes ( 24 of 29 adrs ) were resolved or improved by the end of the observational period . in other cases , acute respiratory tract infections are the most common illnesses in humans and are associated with significant morbidity and mortality . antibiotics are commonly prescribed empirically for respiratory tract infections in adults and children in primary care . the inappropriate prescribing of antibiotics has the potential to cause drug - related aes , escalate the prevalence of antibiotic - resistant organisms in the community , and increase primary care consultation rates for minor illness ( standing medical advisory committee 1998 ) . how to use antibiotics in the treatment of respiratory tract infection fortunately , there are national and international guidelines for the treatment of respiratory tract infections , eg , the european respiratory society s lower respiratory tract infections guidelines.6 in china , guidelines were also established to guarantee the appropriate use of antibiotics in respiratory tract infection patients , especially cap and aecb patients . moxifloxacin is recommended in both chinese guidelines7,8 for treating cap and aecb . for cap , moxifloxacin is recommended for preliminary empirical use in the following patients : young to middle - aged adults without underlying disease , elderly people or those with underlying disease , patients who have been hospitalized but not admitted to the intensive care unit ( icu ) , those using a combination with other antibacterials in icu for pseudomonas aeruginosa infection risk , and those with aspiration factor . for aecb , moxifloxacin is recommended for empirical use in grade iii and iv patients without p. aeruginosa infection risk . more and more physicians have experience in using moxifloxacin in china ; nevertheless , there is still the need to collect and analyze local data , especially with a large sample size in clinical practice , in order to gather the newest efficacy data in treatment with moxifloxacin . in our study , a patient age over 45 years should be considered a major factor for defining the presence of chronic bronchitis . indeed , aecb patients were more frequently older patients ( 80.8% of them were aged 50 years and older ) , whereas cap patients were nearly evenly distributed in the age range of 3079 years . the overall effectiveness assessment showed that for 1822 ( 93.3% ) of 1953 documented patients , effectiveness was rated as very good and good by the attending physician . liu and landen reported in 2007 that in their postmarketing surveillance of moxifloxacin in treating respiratory tract infection , improvement was observed in 69.1% of the patients after 3 days of moxifloxacin treatment and in 90.4% after 5 days.9 full recovery had occurred in 71.3% by 7 days and in 86.8% by 10 days . physicians rated moxifloxacin as very good or good in 92% of patients for effectiveness . the results are similar in both studies , which demonstrated moxifloxacin to be of good effectiveness in treating respiratory tract infection.9 for safety consideration , moxifloxacin treatment was well tolerated , with physicians rating overall tolerability as very good and good in 93.5% of patients . the physicians rated tolerability to be sufficient in 5.1% and to be insufficient in 0.8% of the patients . pretreatment ( 95.6% ) as well as lower morbidity ( fewer than two concomitant diseases , 94.4% ) corresponded to better results regarding tolerability compared with no pretreatment ( 90.6% ) and higher morbidity ( two or more concomitant diseases , 92.5% ) . symptoms such as nausea and vomiting , which are well known in antibiotic treatment , were predominant ( 0.15% , respectively ) . similar results were acquired from liu and landen s study.9 physicians rated moxifloxacin as very good or good in 90.8% of the patients for tolerability . a total of 129 aes occurred in 74 ( 1.9% ) patients and mainly involved either the gastrointestinal or nervous system . all events were mild or moderate , and most resolved or improved after stopping treatment.9 this study involved 150 investigators and enrolled a large number of patients in china . although this study lacked bacteriological testing and did not evaluate bacterial outcome , in medical practice , antibiotics are evaluated on the basis of clinical signs and symptoms . nevertheless , this study is a single - arm , open - label study , and bias is inevitable when evaluating final effectiveness . stricter studies in clinical practice are needed to further investigate this area . in general , the data of this study provide a large sample of evidence from china for rational antibiotic usage in treating respiratory tract infections in the future . in this observational study , moxifloxacin was generally well tolerated and highly effective in the treatment of different respiratory tract infections requiring initial iv therapy . the safety and tolerability information collected in this study confirms the clinical safety profile of moxifloxacin and its benefit as a valuable choice of antibiotic treatment for respiratory tract infections .

crowns and fixed partial dentures ( fpds ) are the major prosthodontic treatment modalities for past several decades with factors like esthetics , contact points and pontics playing an important role in varying their design . however , one property of cement retained fixed restorations , retention , has been of prime importance in all designs . it is proposed to be influenced by factors such as height and diameter of the preparation , luting cement and predominantly by one of the operator controlled factors , convergence angle ( ca ) . the length and diameter of the preparations are often limited by the existing dental anatomy ; and improved retention due to cement depends upon mechanical interlocking of cement , surface area of cement coverage , durability of cement , resistance to mechanical breakdown and dissolution . thus , ca , the angle between two opposing axial walls that equals the sum of the taper ( angle between one axial wall and the long axis of the preparation ) of two opposing axial walls of a preparation , is considered as a prime variable . it gives marginal and internal adaptation for the crowns and can be controlled by the operator . ca is inversely proportional to the retention , with greater angles reducing the retention of crowns . recommended a 6 degree ( ) taper , which gives a convergence of 12 as ideal ; tylman et al . state that the ideal taper is 2 on anterior teeth and 25 on the posterior teeth . johnston suggested 57 ( 1014 ca ) and kronfeld accepted a range that includes all these figures . to know the ideal and/or the recommended ca , a tooth preparation should possess for attaining maximum retention , led to the proposal of guidelines on preparation taper and convergence , both for individual and group of teeth , depending on height to base diameter ratio . studies conducted on dental students , general dentists and specialists to determine the clinically achievable angles , depicted values outside the recommended range with the mean value between 14.3 and 20.1. interestingly , there was no apparent correlation to the operator level of education or experience ; some practitioners were even preparing teeth with a ca between 30 and 50. assessment of ca achieved in preparations of fpds was not significant , compared to those of individual preparations . nonetheless , increasing the ca of fpds to achieve a common path has been found to be a common practice . among several reasons for failure of fpds , lack of retention however , all the existing literature is on the retention of single crowns , leaving behind the effect of these angles on retention when planning for multiple unit castings ; thus creating a major lacuna in the field of cas . hence , the present study was carried out to verify the retention of nickel - chromium alloy single crowns as well as fpds casted for dies machined with two different degrees of convergence , 12 and 20 . a total of 110 aluminum dies were machined ( at hebic industrial training institute , mangalore , india ) to stimulate complete veneer crown preparations with either a ca of 12 ( n = 55 ) or 20 ( n = 55 ) , each group containing canines ( n = 25 ) , premolars ( n = 15 ) and molars ( n = 15 ) . the dimensions of simulating dies were as follows : canines 5 mm of gingival diameter ( d ) and 6 mm of occlusogingival height ( h ) ; premolars 5 mm of d and 5 mm of h ; molars 6 mm of d and 4 mm of h. each die had a 1 mm 90 shoulder finish line and all the above dimensions were recorded using an optical measuring microscope ( nikon model mm-11u ) . five dies each , simulating canine , premolar and molar from both ca groups were used for testing retention of single unit and remaining dies used for the preparation of 3 , 6 or 9 units fpds . each 3 units fpd was made using a premolar and a molar die of same convergence threaded on rectangular metal platform with the help of a retainer shaft in the position of first molar and first premolar , thus making a total of 5 units in each ca group . for each 6 units fpd , two canine dies of the same convergence were threaded in position of canines on both sides of the arch , making a total of 5 units in each group . each 9 units was prepared with canine , premolar and molar dies on one side of the arch and a canine in cross arch , making a total of 5 units in each group . a die spacer ( pico - fit , renfert , usa ) was applied for all the dies using a unidirectional application technique to obtain a uniform film thickness of 1 mm over which aluminum molds were fabricated . using these molds , wax patterns of multiple units were made by attaching the single patterns with the help of a 2.5 mm diameter wax connector . each wax pattern was immediately invested with hygroscopic technique after marginal refinement to minimize distortion . the conventional lost wax technique was carried out for complete elimination of wax from the molds and all castings were carried out in induction casting machine ( fornax t , bego , usa ) to make cobalt - chromium copings and fpds . the intaglio surface and margins were checked for blebs , air abraded with 50 m aluminum oxide particles under a pressure of 60 psi and cemented using zinc phosphate luting cement ( de trey zinc cement , dentsply , germany ) ( mixed according to manufacturer 's recommendations and consistency standardized to american dental association specification no . the vent on the occlusal surface of the casting enabled the easy escape of excess cement through it , thus ensuring a proper adaptation at the margins of the copings to the die . the excess cement was removed from the margins before it was fully set and all the cemented units were stored in 100% humidity for 24 h ; then secured in an instron universal testing machine and adjusted such that the casting was in line with the central axis of the machine arm . an aluminum rod with hooks at either end was attached to the loop on the occlusal surface of the casting at one end and to the instron machine on the other end . in case of the multiple units fpd castings , double sided hooked rods of equal length were attached to the loops on all the abutments and the platform adjusted under the instron machine arm such that equal force was directed to all the abutments . the machine was adjusted to apply an initial load of 50 newtons ( n ) for 20 s , followed by a sustaining pressure until the casting was pulled free from the die . the peak load values in n were recorded , tabulated and analyzed statistically using unpaired t - test . with the level of significance set at 0.05 , power of 80% to detect a force difference of 2 n ; and force required to dislodge the crown / fpd as primary outcome measure , a minimal sample size of five was required . with the level of significance set at 0.05 , power of 80% to detect a force difference of 2 n ; and force required to dislodge the crown / fpd as primary outcome measure , a minimal sample size of five was required . with the level of significance set at 0.05 , power of 80% to detect a force difference of 2 n ; and force required to dislodge the crown / fpd as primary outcome measure , a minimal sample size of five was required . with the level of significance set at 0.05 , power of 80% to detect a force difference of 2 n ; and force required to dislodge the crown / fpd as primary outcome measure , a minimal sample size of five was required the mean , standard deviation , and range of tensile forces necessary to dislodge the castings for all the test groups are listed in table 1 and graph 1 . mean tensile force in newton for single and multiple unit removal mean force in newtons for single and multiple unit removal it can be appreciated from the results that 12 units needed greater force to dislodge irrespective of the type of the tooth compared to 20 convergence . however , the difference between the two cas is statistically significant for canines and molars ( p < 0.01 ) while nonsignificant for premolar preparations ( p > 0.5 ) . for all the multiple units , 12 units needed statistically significant ( p < 0.01 ) greater force , compared to 20 ca and irrespective of angle , there was a simultaneous increase in the amount of force required to dislodge the fpd as the number of units increased . however , this increase from 3 to 6 units was less and statistically insignificant in units with 20 angle ( p > 0.5 ) . it can be appreciated from the results that 12 units needed greater force to dislodge irrespective of the type of the tooth compared to 20 convergence . however , the difference between the two cas is statistically significant for canines and molars ( p < 0.01 ) while nonsignificant for premolar preparations ( p > 0.5 ) . for all the multiple units , 12 units needed statistically significant ( p < 0.01 ) greater force , compared to 20 ca and irrespective of angle , there was a simultaneous increase in the amount of force required to dislodge the fpd as the number of units increased . however , this increase from 3 to 6 units was less and statistically insignificant in units with 20 angle ( p > 0.5 ) . the ca established during tooth preparation predominantly influences the retention , resistance and marginal fit of the prosthesis . the first study comparing the relationship between retention and ca was done on cemented complete veneer crowns with angles ranging between 5 and 45 , in increments of five , which concluded that the retention at 5 will be far superior to its counterpart at 45. to find the ideal ca led to further observations , based on which heading educators have recommended a range of ideal values that tooth preparations should possess . in the present study , 12 was selected for various reasons ; the first and foremost being , the maximum angle in the range of ideal values , to accommodate optimal thickness of cement , which results in lesser variation in marginal discrepancy as well as higher retention values . second being a proven fact that , preparations up to 12 angle produce no effect on retention , irrespective of the type of luting agent used , and the marginal and internal adaptation was also found to be good . additionally , clinical advantages comprise the ability to see the preparations to be free of undercuts with the naked eye , and ease to prepare a tooth to house a ca of 12 by the human hand . however , the average achieved in practice by the dental students , general dentists and specialists , with no apparent correlation to their level of education or experience , was 2021. hence , in the present study , 20 was chosen as another angle for comparison . thus , the degrees of convergence were selected for specific reasons ; 12 being the maximum ideal limit taught in dental schools and 20 the mean of achievable angles . for cas in the range of 1020 , the minimal occlusocervical ( oc ) as molars have a greater diameter and bear greater occlusal forces , they are prepared with greater convergence than anterior teeth , with 4 mm projected as the minimal oc dimension . these dimensions were maintained in the present study along with the recommended oc / fl ratio of 0.4 or higher for both single and multiple unit preparations . it can be appreciated from the results of the present study that , among single units , premolars have no significant difference in retention even on increasing the ca , whereas canines and molars had significant decrease ; that cautions us about the care to be taken while preparing canines and molar . however , it is reported that , in practice , premolar preparations will have smaller angles than anteriors , which in turn will have smaller angles than molar preparations . the limitation in visual assessment of ca and anatomy of the tooth were reported as reason for this . additionally , mandibular tooth will be prepared with greater angles than maxillary , which was ascribed to difficulty in preparing ideal angle due to poor access . hence , we highlight the importance of minimizing the ca during molar tooth preparations using small headed handpieces and while preparing anterior teeth suggest periodical assessment for better retention . usage of high magnification loupes or surgical operating microscope was also suggested to facilitate minimal abutment ca . during multiple preparations , increasing the ca to achieve a common path of draw is a common practice , leading to greater angle for fpd abutments than individual crowns . thus , these overtapered abutments sacrifice the retentive form , which places the completed prosthesis in jeopardy . also , removal of too much tooth structure from the axial surfaces can damage the pulpal integrity of these abutments . hence , the form of prepared teeth and the amount of tooth structure removed are important contributors to the mechanical , biologic and esthetic success of even fpds . the results of the present study prove the hypothetical concept that , as the number of units increase or when there is cross arch stabilization the retention of fpds will increase , irrespective of the ca angle . when the two ca groups were compared , irrespective of the number of units , 12 ca had significantly better retention than 20 ; among which 6 units fpds showing marked difference . hence , we suggest concentrating more on achieving ideal ca when planning for a 6 units fpd . besides ca , arch curvature has its effect on the stresses occurring in fpds that have pontics outside the interabutment axis line , as for 6 units fpds that replace the four incisors . for these , additional retention can be gained from the lever arm in opposite direction and at a distance from interabutment axis equal to the length of the lever arm . for 9 units fpds , eliminating the common inaccuracies caused by traditional techniques and visually assessing the finished preparations for unobstructed uniform path of insertion is also very important for the good prognosis of the prosthesis besides maintaining the ca . the decrease noted in 3 units with 20 can be attributed mainly to increase in the ca of molars rather than premolars , which is clearly noticed in the retention of single units of the present study . casting should be well - fitted before cementation ; however , the successful long - term performance of a restoration can also be ascribed to the role of luting agent . it is proposed that , the role of cement comes into play when the teeth are conical or having short crowns or when the convergence is > 20. the type of cement does not affect the retention of crowns when the convergence is 12 or less ; thus the choice of cement for crowns in this range of angles has limited clinical significance with the luting ability of the cement considered to have only a secondary role . though in the present study zinc phosphate was used as luting agent , we suggest to concentrate on the selection of luting agent when the minimal ca of 12 can not be maintained . the limitation of the present study is continuous force was delivered until their dislodgement being this an in vitro study , whereas the forces in the mouth will be repetitive and mild . crowns in a clinical situation might function despite ca much greater than recommended which is due to the fact that intraoral forces and loads interact in a manner that are more complex than can be reproduced in the laboratory . all the single crowns and fpd prostheses showed more retention when the ca was 12 ; and as the number of units increased in fpds there was a better retention irrespective of ca . however , this study highlights the importance of emphasizing on ca when preparing canines and molars for single units and also while preparing canines for a 6 units fpd , as there is marked decrease in the retention when ca was increased from 12 to 20. in summary , customizing the focus placed on ca during preparation can be emphasized , depending on the tooth and the design of fpd . whenever ideal preparation can not be achieved , we stress on considering the auxiliary retention methods .

pulmonary rehabilitation ( pr ) has become an integral component in the treatment of chronic obstructive pulmonary disease ( copd ) patients . the short - term effects on improving symptoms , exercise tolerance , and health - related quality of life ( hrqol ) were demonstrated by numerous randomized studies and meta - analyses.1 this was confirmed by a recent study from our group , showing positive short - term results of a comprehensive pr program ( prp ) in a large group of copd patients with severely impaired health status.2 we were able to show statistically significant and clinically relevant positive effects on 6-minute walking distance ( 6mwd ) , muscle force , cycle exercise endurance time , and hrqol . although the majority of studies reported positive results on exercise performance and hrqol , only half of the studies that investigated exacerbation rate and/or use of health care resources reported statistically significant positive effects.3 the studies published varied considerably in sample size , severity of disease , type and duration of the program offered , follow - up time , and whether or not patients participated in a maintenance program . reducing exacerbation frequency and subsequent health care utilization is considered to be an important outcome target in the treatment of copd.4,5 exacerbations lead to increased complaints , reduced hrqol , and reduced physical activity.6 this can lead to reduced exercise tolerance and muscle dysfunction.7,8 furthermore , frequent exacerbations are related to accelerated lung function decline and are associated with increased mortality.913 pr might decrease exacerbation and hospitalization rates by improving physical condition and physical activity in daily life . recent studies show that less active persons have a higher risk for hospital admission.14,15 moreover , the improvement of knowledge of the disease , awareness of body symptoms , and self - management skills might lead to earlier recognition and treatment of exacerbations.16 the purpose of this study was , therefore , 1 ) to evaluate the effect of a prp on self - reported exacerbation and hospitalization frequency during 1 year after pr compared with 1 year before pr , and 2 ) to explore determinants ( at baseline and after completing a prp ) that might predict a change in exacerbation and hospitalization frequency . a prospective , observational study of patients enrolled in a comprehensive prp in a tertiary referral center in the netherlands was performed . the study was institutional review board exempt because all procedures and measurements were part of the usual care in our institute , and only deidentified , preexisting data were used . all patients with copd who participated in the comprehensive program of the schoondonck center for pulmonary rehabilitation , breda , the netherlands , between june 2006 and december 2012 referral criteria and contraindications for participation in the program have been described and discussed extensively in a previous article.2 patients were referred by pulmonologists from the southwest region of the netherlands to this tertiary referral center for pr . reasons for referral were a high impact of the disease on health status , with no satisfactory response to prior medical and nonmedical treatment , including rehabilitation in primary care and hospital - based rehabilitation . the patients who were referred and enrolled typically had at least three of the following features : 1 ) severe to very severe airway obstruction , 2 ) a markedly limited exercise performance with a maximal power output on a cycle ergometer ( wmax ) of less than 50% predicted , 3 ) a severely impaired quality of life as measured with the st george s respiratory questionnaire ( sgrq ) total score of more than 50% , and 4 ) a relatively high annual , self - reported exacerbation rate ( more than three in the preceding year ) and markedly high health care utilization ( more than two exacerbation - related hospitalizations in the preceding year ) . the indication for participation in a comprehensive prp was based on evaluation of the impact of the chronic respiratory disorder on different dimensions of health status , that is , physiological functioning , symptoms , activities , quality of life , and health care utilization during an assessment.17 the measurement instruments are consistent with markers that have previously been identified as appropriate for measuring clinically relevant outcomes in copd.18 pulmonary function tests , incremental exercise test , and 6mwd were obtained according to american thoracic society / european respiratory society guidelines.1921 cycle exercise endurance was determined with a constant work rate test on a cycle ergometer at a work rate equal to 75% of wmax obtained in a preceding incremental test performed in the same week.22 the medical research council scale for dyspnea was recorded , and the bode index ( body mass index , airway obstruction , dyspnea , and exercise capacity ) was calculated.23,24 hrqol was measured with the sgrq and the medical outcomes study short - form survey , version 2 ( sf-36 , version 2).25,26 patient characteristics , program components , and outcome measurements have been described extensively earlier.2 the initial assessment was followed by an individually tailored comprehensive prp of 12 weeks duration , which was carried out in fixed groups of 810 persons.27 patients participated in the program 5 days a week , for a weekly average of 2025 hours , either as an in- or outpatient . besides group sessions , patients received interventions on an individual basis : for example , counseling by psychologist or social worker , art therapy sessions , and educational conversations with a dietician or respiratory nurse . indication for individual therapy sessions was determined by the healthcare professionals during the assessment week . to improve self - management skills , all patients made an individualized written action plan for exacerbations , under the supervision of a respiratory nurse and pulmonary physician . this was done by offering educational group sessions covering exacerbation management ( three sessions , 30 minutes each ) and individual counseling by a respiratory nurse ( up to five sessions , 30 minutes each ) . weekly group sessions of 1 hour on training in body awareness were an important instrument to teach the participants how to correctly recognize symptoms of exacerbation . at discharge , the participants were instructed to continue training with a local physiotherapist for 3060 minutes , twice weekly , for which the participants received written training instructions . participants were instructed to make an appointment with their referring pulmonary physician or respiratory nurse or both in the first 23 months after discharge . exacerbation frequency in the year preceding participation in the prp was recorded by asking the participant about the exacerbation frequency in the previous year . an exacerbation was defined as an increase in or new onset of more than one symptom of copd ( cough , sputum , wheezing , dyspnea , or chest tightness ) , with at least one symptom lasting 3 days or more and leading the patient s attending physician to initiate treatment with systemic glucocorticoids , antibiotics , or both.28 participants were also asked about the frequency of hospitalizations as a result of exacerbations in the previous year . exacerbations that were initially treated outside the hospital but resulted eventually in hospitalization , without interruption of the treatment , were considered as hospitalizations . the total number of exacerbations consisted of both exacerbations in and out of hospital . during the year following pr , questionnaires were sent to the participants at fixed intervals ( 3 , 6 , and 12 months after completing pr ) . patients were asked to record the number of exacerbations and exacerbation - related hospitalizations during the previous period . data were collected by an administrative assistant who tried to contact the participants by phone if data were missing to ensure a maximal retrieval of data . infrequent exacerbations were defined as zero to two exacerbations per year , and frequent exacerbations were defined as more than two exacerbations per year.29 the percentage of patients having infrequent or frequent exacerbations 1 year before and 1 year after pr was reported . four patterns of change were defined : unchanged infrequent , unchanged frequent , frequent to infrequent ( decreased ) , and infrequent to frequent ( increased ) . baseline characteristics and changes in exacerbation and hospitalization frequency were assessed for normality using visual inspection of normal quantile differences in baseline characteristics between completers , dropouts , and deaths were analyzed with an anova test or a kruskal changes in exacerbation and hospitalization frequency 1 year before compared with 1 year after were tested with a paired - samples t - test . change in exacerbations frequency type before and after pr was analyzed with a mcnemar test . a hierarchical logistic regression analysis was performed to ascertain effects of baseline characteristics on the likelihood to change from frequent exacerbation type 1 year before pr to infrequent exacerbation type 1 year after pr ( 1 = change from frequent to infrequent ; 0 = unchanged or change from infrequent to frequent ) . four blocks of baseline parameters were tested : age , sex , body mass index ( block one ) ; forced expiratory volume in 1 second ( percent predicted [ % pred ] ) , quadriceps force ( % pred ) , wmax ( % pred ) ( block two ) ; sgrq total score ( block three ) ; and exacerbation frequency in the year preceding pr ( block four ) . these parameters were chosen because they are commonly tested before pr and because they represent different domains of the disease . another similar hierarchical logistic regression analysis was done , with the same baseline characteristic but with reduction in exacerbation frequency ( at least one exacerbation less in the following pr compared to the year preceding pr ) ( = 1 ) or no reduction in exacerbation frequency as dependent variable ( = 0 ) . finally , a logistic regression analysis was performed with change from frequent to infrequent exacerbation pattern as dependent variable and change in sgrq total score , constant work rate test , and 6mwd after pr as independent variables . a prospective , observational study of patients enrolled in a comprehensive prp in a tertiary referral center in the netherlands was performed . the study was institutional review board exempt because all procedures and measurements were part of the usual care in our institute , and only deidentified , preexisting data were used . all patients with copd who participated in the comprehensive program of the schoondonck center for pulmonary rehabilitation , breda , the netherlands , between june 2006 and december 2012 were included in this study . referral criteria and contraindications for participation in the program have been described and discussed extensively in a previous article.2 patients were referred by pulmonologists from the southwest region of the netherlands to this tertiary referral center for pr . reasons for referral were a high impact of the disease on health status , with no satisfactory response to prior medical and nonmedical treatment , including rehabilitation in primary care and hospital - based rehabilitation . the patients who were referred and enrolled typically had at least three of the following features : 1 ) severe to very severe airway obstruction , 2 ) a markedly limited exercise performance with a maximal power output on a cycle ergometer ( wmax ) of less than 50% predicted , 3 ) a severely impaired quality of life as measured with the st george s respiratory questionnaire ( sgrq ) total score of more than 50% , and 4 ) a relatively high annual , self - reported exacerbation rate ( more than three in the preceding year ) and markedly high health care utilization ( more than two exacerbation - related hospitalizations in the preceding year ) . the indication for participation in a comprehensive prp was based on evaluation of the impact of the chronic respiratory disorder on different dimensions of health status , that is , physiological functioning , symptoms , activities , quality of life , and health care utilization during an assessment.17 the measurement instruments are consistent with markers that have previously been identified as appropriate for measuring clinically relevant outcomes in copd.18 pulmonary function tests , incremental exercise test , and 6mwd were obtained according to american thoracic society / european respiratory society guidelines.1921 cycle exercise endurance was determined with a constant work rate test on a cycle ergometer at a work rate equal to 75% of wmax obtained in a preceding incremental test performed in the same week.22 the medical research council scale for dyspnea was recorded , and the bode index ( body mass index , airway obstruction , dyspnea , and exercise capacity ) was calculated.23,24 hrqol was measured with the sgrq and the medical outcomes study short - form survey , version 2 ( sf-36 , version 2).25,26 patient characteristics , program components , and outcome measurements have been described extensively earlier.2 the initial assessment was followed by an individually tailored comprehensive prp of 12 weeks duration , which was carried out in fixed groups of 810 persons.27 patients participated in the program 5 days a week , for a weekly average of 2025 hours , either as an in- or outpatient . besides group sessions , patients received interventions on an individual basis : for example , counseling by psychologist or social worker , art therapy sessions , and educational conversations with a dietician or respiratory nurse . indication for individual therapy sessions was determined by the healthcare professionals during the assessment week . to improve self - management skills , all patients made an individualized written action plan for exacerbations , under the supervision of a respiratory nurse and pulmonary physician . this was done by offering educational group sessions covering exacerbation management ( three sessions , 30 minutes each ) and individual counseling by a respiratory nurse ( up to five sessions , 30 minutes each ) . weekly group sessions of 1 hour on training in body awareness were an important instrument to teach the participants how to correctly recognize symptoms of exacerbation . at discharge , the participants were instructed to continue training with a local physiotherapist for 3060 minutes , twice weekly , for which the participants received written training instructions . participants were instructed to make an appointment with their referring pulmonary physician or respiratory nurse or both in the first 23 months after discharge . exacerbation frequency in the year preceding participation in the prp was recorded by asking the participant about the exacerbation frequency in the previous year . an exacerbation was defined as an increase in or new onset of more than one symptom of copd ( cough , sputum , wheezing , dyspnea , or chest tightness ) , with at least one symptom lasting 3 days or more and leading the patient s attending physician to initiate treatment with systemic glucocorticoids , antibiotics , or both.28 participants were also asked about the frequency of hospitalizations as a result of exacerbations in the previous year . exacerbations that were initially treated outside the hospital but resulted eventually in hospitalization , without interruption of the treatment , were considered as hospitalizations . the total number of exacerbations consisted of both exacerbations in and out of hospital . during the year following pr , questionnaires were sent to the participants at fixed intervals ( 3 , 6 , and 12 months after completing pr ) . patients were asked to record the number of exacerbations and exacerbation - related hospitalizations during the previous period . data were collected by an administrative assistant who tried to contact the participants by phone if data were missing to ensure a maximal retrieval of data . infrequent exacerbations were defined as zero to two exacerbations per year , and frequent exacerbations were defined as more than two exacerbations per year.29 the percentage of patients having infrequent or frequent exacerbations 1 year before and 1 year after pr was reported . four patterns of change were defined : unchanged infrequent , unchanged frequent , frequent to infrequent ( decreased ) , and infrequent to frequent ( increased ) . baseline characteristics and changes in exacerbation and hospitalization frequency were assessed for normality using visual inspection of normal quantile differences in baseline characteristics between completers , dropouts , and deaths were analyzed with an anova test or a kruskal changes in exacerbation and hospitalization frequency 1 year before compared with 1 year after were tested with a paired - samples t - test . a p - value of less than 0.05 was considered significant . change in exacerbations frequency type before and after pr was analyzed with a mcnemar test . a hierarchical logistic regression analysis was performed to ascertain effects of baseline characteristics on the likelihood to change from frequent exacerbation type 1 year before pr to infrequent exacerbation type 1 year after pr ( 1 = change from frequent to infrequent ; 0 = unchanged or change from infrequent to frequent ) . four blocks of baseline parameters were tested : age , sex , body mass index ( block one ) ; forced expiratory volume in 1 second ( percent predicted [ % pred ] ) , quadriceps force ( % pred ) , wmax ( % pred ) ( block two ) ; sgrq total score ( block three ) ; and exacerbation frequency in the year preceding pr ( block four ) . these parameters were chosen because they are commonly tested before pr and because they represent different domains of the disease . another similar hierarchical logistic regression analysis was done , with the same baseline characteristic but with reduction in exacerbation frequency ( at least one exacerbation less in the following pr compared to the year preceding pr ) ( = 1 ) or no reduction in exacerbation frequency as dependent variable ( = 0 ) . finally , a logistic regression analysis was performed with change from frequent to infrequent exacerbation pattern as dependent variable and change in sgrq total score , constant work rate test , and 6mwd after pr as independent variables . a hierarchical logistic regression analysis was performed to ascertain effects of baseline characteristics on the likelihood to change from frequent exacerbation type 1 year before pr to infrequent exacerbation type 1 year after pr ( 1 = change from frequent to infrequent ; 0 = unchanged or change from infrequent to frequent ) . four blocks of baseline parameters were tested : age , sex , body mass index ( block one ) ; forced expiratory volume in 1 second ( percent predicted [ % pred ] ) , quadriceps force ( % pred ) , wmax ( % pred ) ( block two ) ; sgrq total score ( block three ) ; and exacerbation frequency in the year preceding pr ( block four ) . these parameters were chosen because they are commonly tested before pr and because they represent different domains of the disease . another similar hierarchical logistic regression analysis was done , with the same baseline characteristic but with reduction in exacerbation frequency ( at least one exacerbation less in the following pr compared to the year preceding pr ) ( = 1 ) or no reduction in exacerbation frequency as dependent variable ( = 0 ) . finally , a logistic regression analysis was performed with change from frequent to infrequent exacerbation pattern as dependent variable and change in sgrq total score , constant work rate test , and 6mwd after pr as independent variables . in total , 537 patients completed the initial program of 12 weeks and were eligible for follow - up . during the 1-year follow - up period after pr , 38 patients died ( 7% ) ( deaths ) . in addition , 156 patients ( 29% ) did not answer the questionnaire regarding exacerbation and hospitalization frequency sufficiently and were considered as lost to follow - up ( dropouts ) . for 343 patients , complete data on exacerbation and hospitalization frequency was available during the 1-year follow - up and were eligible for analysis ( completers ) . patient characteristics for completers , dropouts , and deaths are shown in table 1 . compared with patients that completed the follow - up , patients that were lost to follow - up were more often female , were younger , and had a lower score on the mental component scale the sf-36 . when comparing participants that died during follow - up with completers and dropouts , they had a higher medical research council scale and bode index , more hyperinflation , lower wmax ( % pred ) , and lower 6mwd than the survivors . in addition , patients who died during follow - up had a lower diffusion capacity and higher sgrq total score compared with completers . the mean number of exacerbations decreased significantly after participating in a prp by 1.37 exacerbations / year ( 95% confidence interval 1.029 to 1.717 ) from 4.563.26 exacerbations in the year preceding pr to 3.182.53 in the year following pr ( t=7.847 , p<0.0005 , d=0.42 ) . the number of hospitalizations due to exacerbations decreased significantly by 0.68 hospitalizations / year ( 95% confidence interval 0.467 to 0.903 ) from 1.481.84 in the year preceding pr to 0.801.31 hospitalizations / year in the year following pr ( t=6.184 , p<0.0005 , d=0.33 ) . the mean number of exacerbations , hospitalizations , and exacerbations out of hospital 1 year before and 1 year after pr are presented in table 2 and in figure 1 . one hundred and ninety - seven of 343 participants ( 57.4% ) experienced a reduction in total exacerbation frequency of at least 1 exacerbation , and 163 participants experienced a reduction of at least 1 hospitalization ( 47.5% ) . for 56 ( 16.3% ) participants , total exacerbation frequency was unchanged , and for 118 ( 34.4% ) participants , hospitalization frequency was unchanged . exacerbation and hospitalization frequency was increased for 90 ( 26.2% ) and 62 ( 18.1% ) participants , respectively . of the 343 participants , 236 ( 69% ) had frequent exacerbations and 107 ( 31% ) had infrequent exacerbations 1 year before pr . during the year after pr , 185 ( 54% ) this change was a consequence of 85 frequent exacerbators becoming infrequent exacerbators and 34 infrequent exacerbators becoming frequent exacerbators . a mcnemar test determined that the difference in the proportion of frequent exacerbators pre- and post - pr was statistically significant ( x=21.008 , p<0.0005 ) . the proportion of frequent and infrequent exacerbation type before and after pr is graphically presented in figure 2 . a hierarchical logistic regression analysis was performed to ascertain effects of baseline characteristics on the likelihood to change from frequent exacerbator 1 year before pr to infrequent exacerbator 1 year after pr , testing four blocks of baseline parameters ( age , sex , body mass index [ block one ] ; forced expiratory volume in 1 second [ % pred ] , quadriceps force [ % pred ] , wmax [ % pred ] [ block two ] ; sgrq total score [ block three ] ; and exacerbation frequency in the year preceding pr [ block four ] ) . although this model was statistically significant ( x=15.576 , p=0.049 ) , only a very small percentage of 6.6% of the variance could be explained . two variables ( sgrq total score and exacerbation frequency preceding pr ) contributed statistically significantly to the effect , with exacerbations in the year preceding pr accounting for the majority of the effect , with an increase of explained variance from 2.8% to 6.6% after adding the variable to the model with the previous blocks of variables . when a similar procedure was performed to ascertain the effect of the same baseline characteristics on the likelihood that participants would have at least one exacerbation less in the year following pr compared to the year preceding pr , the model was statistically significant ( x=99.419 , p<0.005 ) , and a larger proportion of the variance was explained ( 34.1% ) . in this model , only exacerbation frequency in the year preceding pr contributed statistically significantly , showing an increase of explained variance from 5.3% to 34.1% after adding the variable to the previous blocks of variables . a logistic regression model with change from frequent to infrequent exacerbator as dependent variable and variables that represent change after pr as independent variables ( change in 6mwd , cycle endurance time , sgrq total score ) did not reveal any statistically significant variables that might predict a change from frequent to infrequent exacerbation type . in total , 537 patients completed the initial program of 12 weeks and were eligible for follow - up . during the 1-year follow - up period after pr , 38 patients died ( 7% ) ( deaths ) . in addition , 156 patients ( 29% ) did not answer the questionnaire regarding exacerbation and hospitalization frequency sufficiently and were considered as lost to follow - up ( dropouts ) . for 343 patients , complete data on exacerbation and hospitalization frequency was available during the 1-year follow - up and were eligible for analysis ( completers ) . patient characteristics for completers , dropouts , and deaths are shown in table 1 . compared with patients that completed the follow - up , patients that were lost to follow - up were more often female , were younger , and had a lower score on the mental component scale the sf-36 . when comparing participants that died during follow - up with completers and dropouts , they had a higher medical research council scale and bode index , more hyperinflation , lower wmax ( % pred ) , and lower 6mwd than the survivors . in addition , patients who died during follow - up had a lower diffusion capacity and higher sgrq total score compared with completers . the mean number of exacerbations decreased significantly after participating in a prp by 1.37 exacerbations / year ( 95% confidence interval 1.029 to 1.717 ) from 4.563.26 exacerbations in the year preceding pr to 3.182.53 in the year following pr ( t=7.847 , p<0.0005 , d=0.42 ) . the number of hospitalizations due to exacerbations decreased significantly by 0.68 hospitalizations / year ( 95% confidence interval 0.467 to 0.903 ) from 1.481.84 in the year preceding pr to 0.801.31 hospitalizations / year in the year following pr ( t=6.184 , p<0.0005 , d=0.33 ) . the mean number of exacerbations , hospitalizations , and exacerbations out of hospital 1 year before and 1 year after pr are presented in table 2 and in figure 1 . one hundred and ninety - seven of 343 participants ( 57.4% ) experienced a reduction in total exacerbation frequency of at least 1 exacerbation , and 163 participants experienced a reduction of at least 1 hospitalization ( 47.5% ) . for 56 ( 16.3% ) participants , total exacerbation frequency was unchanged , and for 118 ( 34.4% ) participants , hospitalization frequency was unchanged . exacerbation and hospitalization frequency was increased for 90 ( 26.2% ) and 62 ( 18.1% ) participants , respectively . of the 343 participants , 236 ( 69% ) had frequent exacerbations and 107 ( 31% ) had infrequent exacerbations 1 year before pr . during the year after pr , 185 ( 54% ) this change was a consequence of 85 frequent exacerbators becoming infrequent exacerbators and 34 infrequent exacerbators becoming frequent exacerbators . a mcnemar test determined that the difference in the proportion of frequent exacerbators pre- and post - pr was statistically significant ( x=21.008 , p<0.0005 ) . the proportion of frequent and infrequent exacerbation type before and after pr is graphically presented in figure 2 . a hierarchical logistic regression analysis was performed to ascertain effects of baseline characteristics on the likelihood to change from frequent exacerbator 1 year before pr to infrequent exacerbator 1 year after pr , testing four blocks of baseline parameters ( age , sex , body mass index [ block one ] ; forced expiratory volume in 1 second [ % pred ] , quadriceps force [ % pred ] , wmax [ % pred ] [ block two ] ; sgrq total score [ block three ] ; and exacerbation frequency in the year preceding pr [ block four ] ) . although this model was statistically significant ( x=15.576 , p=0.049 ) , only a very small percentage of 6.6% of the variance could be explained . two variables ( sgrq total score and exacerbation frequency preceding pr ) contributed statistically significantly to the effect , with exacerbations in the year preceding pr accounting for the majority of the effect , with an increase of explained variance from 2.8% to 6.6% after adding the variable to the model with the previous blocks of variables . when a similar procedure was performed to ascertain the effect of the same baseline characteristics on the likelihood that participants would have at least one exacerbation less in the year following pr compared to the year preceding pr , the model was statistically significant ( x=99.419 , p<0.005 ) , and a larger proportion of the variance was explained ( 34.1% ) . in this model , only exacerbation frequency in the year preceding pr contributed statistically significantly , showing an increase of explained variance from 5.3% to 34.1% after adding the variable to the previous blocks of variables . a logistic regression model with change from frequent to infrequent exacerbator as dependent variable and variables that represent change after pr as independent variables ( change in 6mwd , cycle endurance time , sgrq total score ) did not reveal any statistically significant variables that might predict a change from frequent to infrequent exacerbation type . this reduction largely exceeds the suggested minimal clinically important difference.30 not only was the mean number of exacerbations reduced but there was also a significant reduction in the proportion of patients that were classified as frequent exacerbator . baseline characteristics and change during the program are not very useful to predict a change in exacerbation frequency . however , at baseline , higher exacerbation frequency in the year preceding pr predicts a larger reduction in exacerbation frequency in the year after pr . patients with a frequent exacerbation phenotype seem to have the largest potential for reducing the exacerbation frequency and may have greater benefits from participation in the prp on that long - term outcome . change in outcome measurements after completing the prp did not significantly predict effects on exacerbation rate . one of the main advantages of this study is the large number of severely disabled patients with copd , with high impact on physical performance and hrqol . in a publication in 2012 , ochmann et al3 reviewed 20 studies performed between 2000 and 2010 that described exacerbation rate and/or copd - related health care utilization after pr , with the number of participants ranging between 24 and 522 . these reviewed studies varied considerably in patient characteristics , components of the prp , and outcome measurements . about half of the 20 studies showed a reduction in exacerbation rate and/or health care utilization . the number of hospitalizations preceding pr in these studies is lower than in our group , ranging from 0.3 to 2 per year whereas , in our study , the exacerbation rate before pr is higher , with a mean of 4.56 exacerbations per year . this finding , combined with low hrqol , indicates that our population is a severely disabled group . long - term improvement in this population will only be possible by offering an intensive prp , where integrated care is the cornerstone of the program . the importance of reduction of exacerbation frequency can not be underestimated ; it might contribute to preservation of quality of life and even survival . by influencing exacerbation frequency , we might influence the natural course of the disease since frequent exacerbations are associated with more rapid lung function decline.9 we believe that the reduction in exacerbation frequency is influenced by the improvement of self - management and alterations in health behavior.31,32 because individualized action plans without a prp do not always improve exacerbation rates and health care utilization , the integration of self - management improvement in the prp seems to be a key factor to improve health care utilization.33,34 increased awareness of body symptoms and knowledge of symptoms related to exacerbations was an important part of the program . earlier recognition and subsequent treatment of exacerbations can lead to earlier recovery and probably less recurrence of exacerbation.35 although it could also be expected that an increased awareness of symptoms related to exacerbations might lead to an increase in exacerbation treatment , this was not observed . this might indicate that the prp has caused a sustained improvement in health - related behavior , possibly by improving self - management skills and physical activity in daily life.36 in the present study , we do not have data on pulmonary function or physical performance at 1 year following pr , so we do not know whether these parameters are associated with a reduction of exacerbation frequency . the identification of factors that might be associated with reduction in exacerbation frequency is an interesting area for future research . the reduction seen in exacerbation frequency subsequently seems to lead to a very significant reduction in exacerbation - related hospitalization frequency , which has the potential to reduce health care costs dramatically . we know that the majority of the costs incurred in chronic respiratory patients are associated with the costs of hospitalization.3739 in our study , the total number of hospitalizations for the whole group decreased from 535 hospitalizations to 344 , or a reduction of 191 hospitalizations . baseline characteristics and their change after participation in the prp are not very useful predictors for change in exacerbation frequency . a recent study by donaldson et al40 also found that a change in exacerbation frequency seems difficult to predict . this lack of predictors could be explained because of the heterogeneity of copd and because the program components are individually tailored according to the observations during the assessment . because , in the present study , we did not record the content and duration of individual components , however , because in a cochrane review , pr has been proven to be effective , at least on the short term , this eliminates the need for randomized studies.1 our population is very severely impaired and experiences a major impact on quality of life , so we believe it is unethical not to offer a proven intervention to these patients . this also points to another possible limitation : our population is a selected group for which the high rate of exacerbation and hospitalization frequency is one of the main reasons for referral to our center . the increase in exacerbation and hospitalization rate in the year before pr might be incidental , and we do not know what the natural course of the disease in these patients might be . however , there are indications that patients with frequent exacerbations have a more rapid deterioration in health status , so it seems unlikely that , without intervention , the exacerbation rate would be spontaneously reversed.31 the number of dropouts during the follow - up period is significant but can be expected in a long - term observational study , with the majority of the participants experiencing a severely impaired health status . significant differences in patient characteristics at baseline between completers and dropouts that might predict dropout are limited . specifically , there are no significant differences in comorbidity or in exacerbation or hospitalization frequency in the year preceding pr . differences in change in outcome during the prp between completers and dropouts are limited , which means that the absence of improvement is unlikely to predict dropout . the recorded exacerbations and hospitalizations are self - reported , which might lead to under or overreporting . data regarding the year preceding pr were retrospectively recorded by asking the participants about the frequency of these events at initial assessment , after informing the participants of the definition of exacerbation . this information was verified again by a respiratory nurse at the start of the prp . because the definition of exacerbations was treatment based , and information was verified twice , there would be a bias , we would rather expect an undercount of events pre - pr since participants have to recall information over a year . despite this the data in the year following pr were collected prospectively by asking the participants to record the exacerbation and exacerbation - related hospitalization frequency on a questionnaire sent to the participants at 3 , 6 , and 12 months post - pr . because participants were informed that they would be asked about the occurrence of these events , the potential for underreporting seems to be limited . in conclusion , participation in a comprehensive prp is associated with a significant reduction in self - reported exacerbation rate and exacerbation - related hospitalization rate in the year following pr . this might positively influence the progression of the disease , despite severely impaired health status of the participants at inclusion . the observed reduction in exacerbation rate might lead to a substantial reduction in health care utilization , with positive effects on health care costs . future research should be directed to an evaluation of factors that might be associated with reduction in exacerbation and hospitalization frequency . this could lead to development of more specific pr components and maintenance strategies , in order to further improve long - term outcomes in this group of patients .

rna has the dual capability of serving as a genetic polymer that mediates the inheritance of information and as a catalyst for chemical transformations and is therefore a promising candidate as an ancestral biopolymer . in early protocells , prior to the emergence of an rna replicase , nonenzymatic template - directed polymerization has been hypothesized to be responsible for rna copying . in this context , a template is a single - stranded rna oligomer that , by watson crick base - pairing , directs the sequence - specific polymerization of ribonucleotides to form its complementary strand . this process is frequently modeled as a repeated series of single - nucleotide primer extension events . each primer extension step is a supramolecular event that takes place at the 3-end of a primer annealed to a template and is orchestrated by noncovalent binding interactions , particularly hydrogen bonding and nucleobase stacking . a high rate of primer extension is critical for efficient rna copying ; this rate is affected initially by how tightly nucleotides bind to the complex formed by the template and the growing primer strand . furthermore , the fidelity of primer extension is influenced by the affinity of nucleotides that form wobble base pairs as well as other mismatches , and the overall rate of polymerization decreases significantly as a result of mismatches that lead to stalling of primer extension . understanding the thermodynamics of the binding interactions between a ribonucleotide and an rna primer template complex is of fundamental importance toward elucidating the mechanism as well as optimizing the rates of prebiotic replication of rna . however , quantitative physical measurements of mononucleotide binding thermodynamics , as opposed to values derived from reaction kinetics , are limited . early work by kanavarioti et al . examined the binding of multiple guanosine monomers on a polycytidylate template using differences in uv vis absorption . more recently , richert and co - workers have reported thermodynamic investigations of the binding of nucleotides to modified oligonucleotide duplexes , wherein they employed nmr spectroscopy to study the affinity of rgmp for a model rna primer template complex comprised of a hexaethylene glycol ( heg)-linked hairpin , 5-ccag(heg)cug-3. ( a ) selected model rna and dna sequences used in this study . ( b ) energy - minimized models of the a - form 5-ucaauauug-3 rna duplex in both free and ramp - bound states . all components were solvated in water boxes , neutralized by na , and minimized using the program namd 2.9 with charmm 36 parameter set ( see methods for further details on the modeling and si for other minimized systems ) . while these pioneering studies provided the physical measurements of rgmp binding to rna templates , studies of the other ribonucleotides , of context effects , and of non - watson crick binding interactions would be extremely useful in efforts to improve nonenzymatic rna copying . here we present the results of the noncovalent binding of ribonucleotide monophosphates ( rnmps ) to both rna and dna primer template complexes in neutral aqueous media using h nmr spectroscopy supplemented by molecular dynamics ( md ) structural calculations . we measure the thermodynamic association constants , kas , of nucleotide monomers for oligonucleotide duplexes by monitoring the resonances of the imino protons of all base - pairs in the duplexes , as the free monomer concentrations are increased incrementally . our approach provides insight into nonenzymatic template - directed rna polymerization by allowing the binding event to be separated from the subsequent chemical steps of primer extension . all of the rna and dna sequences that we studied ( figure 1a ) are self - complementary and form stable palindromic duplexes in aqueous media at ph 7 and 12 c , as verified by circular dichroism ( cd ) spectroscopy ( see figures s1s2 , si ) , uv - melting experiments ( figures s3s4 , si ) , and h nmr investigations ( figure s5 , si ) . the 5-single - nucleotide overhangs on either side of the duplexes , where x represents g , c , a , or u / t , serve as the template to which nucleotides can bind through hydrogen bonding , while the 3-ends serve as the primer providing a stacking surface that enhances nucleotide interactions . the c2 axis of symmetry in these palindromic duplexes not only allows a straightforward interpretation of the nmr spectra of the imino protons but also enables the use of a simple isotherm model to measure the binding constants of the titrated monomers ( see si for mathematical derivations ) we began by carrying out a detailed structural characterization of all oligonucleotide duplexes that were used for nmr titration experiments . as a representative example , here we focus on the rna duplex obtained from the 5-ucaauauug-3 sequence ( figures 1b and 2 ) . a qualitative analysis to confirm its self - hybridization and to determine the type of helical geometry was performed by variable - temperature cd spectroscopy . the cd spectra ( figure 2a ) acquired over a temperature range of 480 c revealed the characteristic features of a global a - form helical geometry , namely a small negative peak at ca . likewise , the cd spectra for all other rna duplexes were consistent with global a - form conformations ( figure s1 , si ) . as expected , the cd spectra for all dna duplexes ( figure s2 , si ) were consistent with the b - form conformation . we then performed md simulations to derive energy - minimized models of the primer all cd spectra and structural md models , see figures s1s2 and s27s34 in the si , respectively . in order to evaluate the thermodynamic parameters associated with duplex formation under identical conditions used for the nmr spectroscopic titrations , we obtained a melting profile ( figure 2b , left panel ) by carrying out variable - temperature uv absorption experiments at 260 nm , for a range of total strand concentrations between 1 and 100 m . a vant hoff analysis ( figure 2b , right panel ) of the melting temperature as a function of the total concentration of rna revealed enthalpic and entropic contributions to duplex formation of h = 71.4 kcal these parameters allowed us to calculate the concentration of free and duplexed rna at a total strand concentration of 3 mm at 12 c , the conditions under which all nmr monomer titrations were performed . under these conditions , the concentration of single - stranded rna is on the order of 1 m ( i.e. , < 0.04 mol % ) , a fraction small enough to be neglected when considering the mechanism of ribonucleotide binding . for similar calculations carried out on other sequences , ( a ) cd spectral overlay of the native 5-ucaauauug-3 rna duplex solution ( 0.2 mm ) acquired at variable - temperature [ 4 c intervals from 4 ( blue ) to 80 c ( red ) ] , revealing that the sequence forms an a - form helix at room temperature and below . ( b ) ( left ) uv - melting experiment for this rna sequence at 0.1 mm concentration , confirming that it forms a stable duplex at ph 7.0 . ( right ) vant hoff analysis of the melting temperature ( tm ) measured in the uv - melting experiment as a function of the total concentration of rna . in this particular analysis , double helix formation is driven by an enthalpic contribution ( h ) of 71.4 kcal mol with an entropic cost ( s ) of 213 cal mol k. ( c ) ( left ) h nmr ( 400 mhz ) spectrum of the same rna duplex ( 1.5 mm ) dissolved in h2o / d2o ( 9:1 ) in the presence of nacl ( 500 mm ) at 12 c and ph 7.0 ( 0.1 ) . the spectrum was acquired using the watergate flip - back sequence to suppress the bulk water peak . chemical shift values are externally referenced to pentafluorobenzaldehyde ( ext = 10.285 ppm ) , which is dissolved in cdcl3 ( 0.05% v / v tetramethylsilane ) and applied within a thin coaxial nmr tube . ( right ) expanded imino proton region ( 1214 ppm ) , wherein the three u(h3 ) and one g(h1 ) protons resonate . ( d ) variable - temperature nmr experiment performed for the same solution to assign the imino protons based on water exchange rates . faster exchange rates , which lead to greater line broadening , are indicative of closer proximity to the duplex termini . ( e ) expanded imino proton region of the h h 2d noesy nmr spectrum ( 200 ms mixing time ) , where the noe cross - peaks represent the interaction between neighboring imino protons . crick g : c and a : u / t base - pairs have distinct chemical shift values ranging between 12 and 13 ppm for g : c pairs and 13 and 15 ppm for a : u / t pairs . in the duplex formed from 5-ucaauauug-3 , the magnetically nonequivalent imino protons u5(h3 ) , u7(h3 ) , u8(h3 ) , and g9(h1 ) resonate as expected in the region from 12 to 15 ppm ( figure 2c ) . in order to assign these resonances , we relied on a combination of variable - temperature ( figure 2d ) and h we corroborated the assignment of the most upfield signal ( red ) to g9(h1 ) based on its greater line width compared to the other resonances , as expected from the fact that the terminal base pair allows faster g9(h1 ) exchange with h2o on the time scale of the nmr experiment . we analyzed the change in the chemical shift ( ) and the line width of the duplex imino proton resonances , as we gradually increased the concentration of the free nucleotide monomers . duplex solutions were titrated with ribonucleotide monomer solutions ( up to 250 mm ) , which contained the same concentration of the duplex ( 1.5 mm ) . the total cation ( na ) concentration ( 500 mm ) was also maintained at a constant value in a similar manner ( methods , si ) . all titration experiments were carried out using 90% h2o/10% d2o solutions of the rnmp and rna or dna duplex . spectra were recorded using a watergate flip - back suppression pulse sequence to obtain the signals of the imino protons while effectively suppressing the bulk water peak ( figure 2c ) . ( a ) single titration experiment carried out with ramp for the 5-ucaauauug-3 rna duplex . errors obtained from the best fit are shown as the deviation in the least significant digit placed in parentheses ( see the si for the best fit curves ) . the statistical model assumes that two binding sites are identical and noninteracting , reducing to a single binding site isotherm . the interacting model assumes that the two binding sites are identical but can also interact ( see the si for details ) . we monitored the h nmr spectra of the 5-u overhang rna duplex ( figure 3a ) as we titrated ramp from 0 to 250 mm . no new signals in the 1215 ppm region emerged throughout the titration , while the g9(h1 ) resonance ( red ) moved upfield from 12.4 ppm ( no monomer present ) to 12.0 ppm ( near saturation of the duplex ) . this upfield shift is presumably due to the fact that g9(h1 ) becomes magnetically shielded by the ring current of adenine upon ramp binding ( see md predicted structures in figure s29 , si ) . this aromatic ring - current effect decreases , as expected , upon moving away from the binding site , such that the second strongest change in the chemical shift is exhibited by u8(h3 ) ( green ) , and the two most internal protons , u5(h3 ) ( yellow ) and u7(h3 ) ( blue ) , show the smallest changes , which are likely due to subtle alterations in conformation of the duplex upon binding . significantly , g9(h1 ) changes hyperbolically as a function of concentration of ramp . together , these observations are consistent with the specific binding of the ramp monomers to both of the 5-u overhangs of the duplex , a process that is in fast equilibrium on the nmr time scale . in order to confirm the 2:1 monomer - duplex binding stoichiometry , we performed an ramp titration by continuous variation on the analogous dna sequence , 5-d(tcaatattg)-3 , which shows similar hyperbolic behavior in terms of the g9(h1 ) . we used a dna duplex for this experiment because of the large amounts of material required . we maintained a constant total concentration of the dna duplex and ramp and monitored the chemical shift of g9(h1 ) while varying the ratio of these two species . we constructed a job plot from these data using g9(h1 ) and the mole fraction of the duplex ( xdupl ) . we observed a maximum in the job plot at a value of x = 0.35 . this value of x , which is related to the stoichiometric ratio n by the formula x = 1/(1 + n ) , is consistent with a 2:1 binding stoichiometry of ramp to the duplex ( figure s23 ) . we globally fit the chemical shift values of all four imino proton resonances to two binding isotherm models , which we have called statistical and interacting , respectively ( figure 3b ) . the statistical model assumes that the two binding sites are identical and noninteracting . in this mechanism , the two stepwise macroscopic binding constants k1 and k2 ( also see figure 1b ) are related to each other statistically through an intrinsic binding constant ka ( /2k1 = 2k2 = ka ; k1k2= ka ) . this statistical model has the same mathematical form as a single binding site isotherm . the value of ka using this model was determined to be 16(1 ) m for the single titration experiment shown in figure 3a . on the other hand , the interacting model places no constraints on the macroscopic binding constants k1 and k2 in order to account for the possibility that the binding of the first monomer has an effect on the binding of the second . the fit to the statistical isotherm gave identical results within error to the fit with the interacting model , which yielded values of k1 and k2 as 32(1 ) and 8(1 ) m , respectively . these results are likely explained by the supposition that the binding of the first ramp does not significantly alter the local conformation of the second binding site on the other end of the duplex . nearly all of the fits performed on the data obtained from the other rna and dna duplexes yielded either indistinguishable results with respect to the values determined from these two binding models ( figure 3b ) or only slightly deviated from statistical behavior . the most significant exception to statistical binding was observed in the case of the 3-c/5-g rna primer template complex ( figure 5a ) . for a single titration experiment , k1 and k2 were measured to be 42(4 ) and 30(5 ) m , respectively , which suggests that a slight amount of positive cooperativity is at play . this positive cooperativity may be the result of changes in conformation of the duplex that occur after the first rcmp binds , which enhances the binding of the second . nevertheless , the value of k1k2 , 35(3 ) m from the interacting model , is almost the same as value of ka , 29(1 ) m , lying just outside of the errors from the fits . ( a d ) selected single nmr titrations carried out for all - rna systems ( see the si for best fit curves to the data in panels a and b ) . errors obtained from the best fits are shown as the deviations in the least significant digit(s ) placed in parentheses . h2o / d2o ( 9:1 ) , nacl ( 500 mm ) at ph 7.0 ( 0.1 ) and 12 c . ka represents the observed association constant assuming that all ratios of activity coefficients are unity . figure 4 shows a compilation of the results of the single titration experiments carried out on other all - rna systems , and table 1 displays a summary of the thermodynamic binding properties measured for monomers including the binding constants ka ( statistical model ) , the change in gibbs free energy g , maximum change in the chemical shift of the g - imino proton maxg9(h1 ) , and the r values obtained from the fits . all values reported in table 1 represent the averages obtained over three titration experiments , and the associated errors are those calculated from the sample standard deviation , unless otherwise noted . the rna duplexes with 5-u , -c and -g overhangs showed hyperbolic g9(h1 ) when the complementary monomer was titrated . we obtained a ka of 52(5 ) m ( average of three experiments : table 1 , entry 1 ) for rgmp ( in 500 mm nacl , ph 7 , 12 c ) binding to the 5-c overhang of the duplex formed by 5-ccaauauug-3 ( single experiment : figure 4a ) . when we measured the binding of 2-deoxyguanosine monophosphate ( dgmp ) to the duplex formed by 5-ccaauauug-3 , we obtained a ka of 41(5 ) m ( table 1 , entry 5 ) , slightly weaker than that of rgmp for the same template . lastly , we found that rcmp binding to the 5-g overhang ( figure 4b ) exhibited a ka of 66(15 ) m ( table 1 , entry 2 ) . an exception to those studied above was of the titration performed with ump ( figure 4c ) , in which the binding constant was too small to be reliably quantified . in order to examine possible effects of unbound monomer on the chemical environment of a duplex , we carried out a titration of rcmp into the blunt - ended duplex formed from 5-caauauug-3 rna , which does not possess any monomer binding site ( figure 4d ) . only modest changes in the chemical shifts of all four imino proton resonances were observed , ranging from 0.01 to 0.02 ppm , indicating that little to no interaction takes place between rcmp and the fully paired double helix . notably , the g8(h1 ) resonance undergoes significant line broadening as the concentration of rcmp increases . figure 4b ) observed when rcmp is titrated into a solution of the 5-gcaauauug-3 duplex . this control experiment suggests that excess phosphate monoester likely acts as a proton acceptor and facilitates imino proton exchange , whereas monomer binding to the overhang slows this process down . the binding constants of rgmp , rcmp and ramp to the analogous dna primer template complexes are all less than their rna counterparts [ ka of 12(2 ) , 13(3 ) and 7(2 ) m , respectively , table 1 , entries 68 ] . in addition to the titration of free ribonucleotides , we also studied the binding affinity of dgmp toward the 5-c overhang dna duplex ( table 1 , entry 10 ) . under the same conditions ( ph 7 , 12 c ) , the ka of dgmp was measured as 13(2 ) m , which is the same within error as that obtained for rgmp , 12(2 ) m. in addition to the titration studies described above , we carried out experiments to ascertain the influence of sequence context on binding affinity . such context effects would presumably be mediated largely by differences in stacking interactions but could also be affected by sequence dependent changes in helical geometry . as a preliminary effort to measure the significance of such potential effects , we measured the ka of rcmp for 5-g overhang rna and dna duplexes with a primer terminated by a 3-c instead of a 3-g ( figure 5a , b ) . the binding constant for rcmp to 5-ggaauauuc-3 rna was measured to be 22(7 ) m ( average of three experiments : table 1 , entry 11 ) , a decrease of about 3-fold compared to that [ 66(15 ) m ] for 5-gcaauauug-3. in the case of the 5-d(ggaatattc)-3 dna duplex , the ka value of rcmp ( table 1 , entry 2 ) was too small to measure , as no hyperbolic change was observed in g2(h1 ) . finally , we tested the strength of the g : u wobble base pair , the major source of nonenzymatic replication error , by performing a titration of rgmp into a solution of 5-ucaauauug-3 rna duplex ( figure 5c ) . the binding constant for rgmp was estimated to be 6 m ( table 1 , entry 13 ) , a value which is approaching the lower limit of our ability to quantify reliably . nevertheless , this value is only about two times less than the binding constant for ramp to the same template ( table 1 , entry 3 ) . this difference in ka corresponds to a difference in g of binding of approximately 0.5 kcal mol . our titration experiments show that the noncovalent binding affinities between rnmps and rna primer template complexes follow the order of c > g > a > u. our initial expectation , based upon primer extension experiments , was that rgmp would be the tightest binder instead of rcmp . the rather surprising result that rcmp actually binds more tightly motivated us to carry - out an analysis relying on well - established nearest - neighbor ( nn ) models in order to gain additional insight into the parameters governing the binding event . in our analysis , we first calculated the duplex energies of the full - length double - stranded 10-mers , which serve as models for the noncovalently bound rnmp rna complexes . we next calculated the duplex energies of the rna complexes with rnmp - free overhangs that were used during the titration experiments . the differences in duplex energies between the full - length and dangling - end duplexes are plotted ( figure 6 ) along with the corresponding experimentally determined rnmp binding energies . the relative order of the experimental binding energies is in good agreement with those predicted from the nn analysis , i.e. , c > g > a > u. the nn analysis also accurately predicts that the energy of binding for rcmp with the 5-ggaauauuc-3 duplex ( cmp * in figure 6 ) lies in between those for ramp and rgmp . the values predicted by the nn analysis are independent of the core sequence and only depend on the identities of the dangling - end and terminal base pairs ( for more details , see s24 in si ) . the most significant difference between the experimental and predicted nn binding energies is that the predicted nn energies are all approximately 1.5 kcal mol more negative than the experimental values . as a consequence of the fact that the nn analysis does not take into consideration that the rnmp monomers are only noncovalently bound , the predicted energies should be expected to be more negative than what is observed . we hypothesize that this roughly constant difference of 1.5 kcal mol is due to the negative change in entropy related to losses in translational and rotational degrees of freedom that occur upon rnmp monomer binding , and which are approximately the same for each monomer . extrapolating this 1.5 kcal mol difference in the case of ump allows us to estimate the binding energy to be on the order of 0.5 kcal mol , corresponding to a ka of 2 m at 12 c , a value which is below our ability to quantify reliably under our experimental setup . ( a , b ) titrations carried out to investigate the effect of primer base stacking surface on the binding strength of free monomer using ( a ) the 5-ggaauauuc-3 rna duplex and ( b ) the dna analog of the same sequence . ( c ) titration experiment performed to quantify the affinity of rgmp toward a 3-g/5-u primer / template in order to determine the strength of the g : u wobble base - pair formation . errors obtained from the best fits are shown as the deviations in the least significant digit placed in parentheses ( see the si for best fit curves to the data in panels a and c ) . a comparison of experimental to predicted binding energies of the rnmps to their complementary rna primer template complexes . the red bars are for the predicted values obtained from a nn analysis ( 12 c ) , the blue bars are the experimental values obtained from titration data , while the green bars are the differences between experimental and predicted energies . the dashed bars for ump are assuming a 1.5 kcal mol difference between predicted and experimental energies . the excellent agreement between the measured binding energies of the rnmps to the rna primer template complexes with the values predicted from the nn analyses suggests that rnmp binding to rna is subject to similar structural constraints to those that determine duplex stability . duplex stability is dominated by hydrogen bonding and base - stacking , and these short - range interactions depend mostly on the identities of adjacent nucleotides . the differences in binding energy between rcmp and rgmp , for example , are primarily the result of the greater stability of the gc / cg ( g = 4.25 kcal mol , 12 c ) propagation sequence in comparison to the gg / cc counterpart ( g = 3.74 kcal mol , 12 c ) . the contribution arising from the differences in energies between the gc / g ( g = 0.17 kcal mol , 12 c ) and cc / g ( g = 0.06 kcal mol , 12 c ) dangling ends is much less significant . the larger binding constant of rcmp contrasts with the observations reported by wu and orgel using hairpin templates that the 5-phosphor-2-methylimidazolide of rgmp ( 2-meimpg ) has better primer extension reactivity than that of rcmp ( 2-meimpc ) . one possible explanation may be that 2-meimpg is more tightly bound than 2-meimpc in the presence of mg , conditions under which the majority of template - directed primer extensions take place . our data on rgmp and rcmp binding may differ from the binding of the corresponding 2-methylimidazolides . affinity , however , is not the only factor affecting the rate of template - directed polymerization . template complex , 2-meimpg is sterically better positioned to react than is 2-meimpc , giving rise to a larger rate constant . under conditions in which the primer template complex is fully occupied with activated monomers , a larger rate constant for 2-meimpg would lead to a faster primer extension regardless of the value of their respective noncovalent binding constants , ka . the direct experimental measurement of the binding of 2-methylimidazole - activated monomers is difficult since these compounds hydrolyze on the time scale of the nmr titration experiments . we are therefore currently exploring the possibility of measuring the binding of nonhydrolyzable analogs of the activated nucleotides . when comparing the rnmp binding energies for the rna versus the dna primer template complexes , the contribution of stacking energy at the primer template binding site is likely to be affected by the backbone preorganization and helical parameters of the duplex , which are significantly different for a - form rna and b - form dna helices . we hypothesize that the local geometry of the 5-overhangs , which depend primarily on whether the primer template complexes are in the a- or b - form conformations , is the dominant factor that leads to the differences in binding energies observed between rna and dna . the structural models obtained from md calculations ( see figures s27s34 , si ) lend support to this hypothesis in that these calculations do indeed yield significant differences in the local geometry of the rnmp monomers depending on whether they are bound to a - form rna or b - form dna . one of the key differences may be the greater degree of interstrand base - stacking present in a - form rna in comparison to b - form dna , a structural feature which is often considered important in providing at least a partial explanation for the greater duplex stability of rna . the differences in binding energy between rgmp and dgmp when binding to either the rna or dna primer template complexes are likely accounted for by small differences in the intrinsic conformational preferences of these monomers . the ka values of rgmp [ 12(2 ) m ] and dgmp [ 13(2 ) m ] for dna duplexes are most likely the same within error , while the ka of dgmp [ 41(5 ) m ] is slightly less than that of rgmp [ 52(5 ) m ] for the rna analogs . we have shown previously that both dgmp and rgmp bind to dna primer template complexes in the 2-endo sugar pucker conformation , which likely explains why their binding constants are essentially the same within error . in the case of binding to an rna primer template complex , rgmp was observed to make a switch to the 3-endo conformation , while dgmp stayed in the 2-endo conformation . we hypothesize that the preferred 2-endo sugar puckering of the bound dgmp gives rise to the slightly smaller binding energy compared to that of rgmp observed herein . the result for the g : u mismatch experiment shows that the binding affinity of rgmp to the 5-u rna duplex is comparable to that of ramp to the same duplex . this finding is in agreement with the duplex stability experiments carried out for oligoribonucleotides with g : u mismatches and points to the future challenge of devising a way to improve the selectivity of ramp over rgmp for a u template in order to avoid an inevitable error catastrophe . the noncovalent binding constant of rgmp that we measured for 5-ccaauauug-3 [ 52(5 ) m ] appears to be slightly lower than that ( 71 m ) reported by richert et al . employing the 5-ccag(heg)cug-3 rna hairpin in the presence of mg ( 2 mm hairpin , 200 mm na - phosphate , ph 7.0 , 400 mm nacl , 80 mm mgcl2 , 20 c ) . this difference might not be surprising given that no divalent cations ( e.g. , mg ) are present in our titration conditions . similarly , the ka of 6 m that we measured for a rg : u wobble base - pair is slightly weaker than that ( 10 m ) reported by richert et al . for a dg : t wobble pair . finally , our observation that both ump and tmp monomers bind very poorly to 5-a rna and dna duplexes , respectively , is in agreement with the results of richert et al . in their dna system . on the other hand , there are some differences in the relative binding affinities of monomers to the rna and dna duplex systems that we studied , compared to those reported by richert et al . for their heg - linked dna hairpins ( conditions as above except ph 8.9 ) . for example , their measured ka values for the deoxy - monomers to the complementary templates are in the order dgmp ( 100 m ) > damp ( 26 m ) dcmp ( 25 m ) > tmp ( 4 m ) , where the titrations of dgmp , dcmp , and tmp were carried out with dna hairpins terminated by a 3-a instead of a 3-g . in contrast , our measured binding strength of ramp is always lower than that of rcmp for both the rna ( 14 vs 66 m ) and dna ( 7 vs 13 m ) duplexes we employed , in which the incoming monomer is always stacked against a 3-g residue . in addition , our ka values of rgmp for the 5-d(ccaatattg)-3 dna duplex [ 12(2 ) m ] and of dgmp for the same duplex [ 13(2 ) m ] are both about an order of magnitude lower than that of dgmp reported by richert et al . for the 5-d(ctgc(heg)gca)-3 dna hairpin ( 100 m ) and five times lower than that of dgmp for the 5-d(ctctgc(heg)gca)-3 dna hairpin ( 63 m ) . these differences could in part be due to the presence of magnesium in the experiments performed by richert et al . , or alternatively could reflect structural differences between our symmetrical native duplexes and their short heg - linked hairpin duplexes . further work will be required to fully assess the context dependence of monomer binding to rna and dna templates . in summary , we have presented results from an nmr - based quantitative approach to the study of the noncovalent interactions between ribonucleotide monophosphates and primer template complexes , through which we obtained the thermodynamic binding constants , kas , of the ribonucleotide monomers . application of solution - phase techniques common for supramolecular and host guest chemistry , in particular nmr spectroscopy , played an important role in developing the template - directed syntheses that led to a variety of complex macromolecular structures . accordingly , a thorough understanding of monomer duplex binding interactions will provide a strong foundation on which to optimize the template - directed polymerization of rna . these same techniques can also be applied to experiments with structurally alternative polymers that may have played a role in the early evolution of life . the higher affinity of monomers for rna primer template complexes than for the corresponding dna complexes may explain in part the advantage of rna over dna templates in nonenzymatic replication . in fact , similar arguments have been made through qualitative studies showing how the ribonucleotide monomers adapt more productive conformations upon binding to rna templates . we are currently extending our h nmr experiments to the measurement of binding kinetics , binding affinity of higher order nucleotides ( dimers and trimers ) , and , in the case of the templates with multiple binding sites , the effect of cooperativity on monomer binding . rna duplexes were synthesized by standard solid - phase phosphoramidite chemistry ( for detailed procedures see the si ) . each oligonucleotide duplex was titrated with the selected ribonucleotide monophosphate ( up to ca . monomer solutions contained the same concentration of the oligonucleotide duplex ( 1.5 mm ) in order to maintain a constant duplex concentration throughout the titration experiments . this was also the case for the total cation ( na ) concentration ( 500 mm ) . the ph of both duplex and monomer solutions was adjusted to 7.0 ( 0.1 ) with either naoh or hcl , and the nmr spectra were acquired at 12 c , unless otherwise noted . monomer titration , ph , and temperature gradient experiments were performed on a varian inova 400 mhz nmr spectrometer . initial concentrations of the duplex and monomer were determined by uv ( nanodrop ) measurements and confirmed by ( 31)p nmr ( 161 mhz ) spectroscopy using a potassium sodium phosphate buffer concentrate ( supelco ) , which was applied using a coaxial nmr tube . for each sequence , an ideal a - form 10-mer rna and b - form 10-mer dna duplex generated by coot was used to model both the free and monomer - bound states of the 9-mer duplex with single - nucleotide 5-overhangs at both ends . two systems were modeled for the monomer - bound states , each containing two dianionic nucleoside monophosphates . 48 64 48 tip3p water boxes and neutralized by na using vmd . these systems were minimized using the program namd 2.9 with a charmm 36 parameter set . the bonded , nonbonded , and electrostatic interactions were calculated at time steps of 1 , 2 , and 4 fs , respectively . the switching ( cutoff ) distance for nonbonded interaction was set at 10 ( 12 ) . to compute long - range electrostatic interactions , the particle mesh ewald method with a grid density of at least 1 was used . a four - step minimization protocol similar to that used by eargle et al . was applied . in the first 10,000 steps of minimization , all heavy atoms were fixed . in the next 20,000 steps , ions , nucleobases , and part of the sugar were then freed while keeping the nucleic acid backbone fixed for the next 20,000 steps .

the development of an artificial endocrine pancreas ( aep ) , composed of a system of continuous blood glucose monitoring and automated insulin infusion , has been a long sought - after solution in diabetic care . the intravascular approach to both glucose sensing and insulin delivery was abandoned in the late 80s , so current research is focused on subcutaneous glucose measurements and subcutaneous insulin administration . however , the performance of these systems are still less than satisfactory , even though there have been some recent advances [ 35 ] . we think it is time to reexamine the intravascular approach as this calls for substantial benefits despite its invasiveness . a convincing body of evidence indicates that insulin is secreted in synchronized bursts from the entire pancreas into the portal blood stream [ 68 ] . likewise , multiple studies in humans and animals have described the oscillatory nature of systemic levels of blood glucose and insulin [ 912 ] . pulsed intravenous insulin delivery has been shown to be more effective in lowering bgl compared to equal doses of continuously infused insulin [ 14 , 15 ] , and the pulsatile nature of endogenous insulin secretion has been mimicked for therapeutic reasons as pulsatile intravenous insulin therapy . compared to standard therapy , pulsed therapy has shown better metabolic control , less end - organ damage , and restoration of normal pulsatile pancreatic function in type 2 diabetes [ 1618 ] . in this study , we used a novel intravascular continuous glucose sensor , the ivs-1 ( invivosense , trondheim , norway ) ( figure 1(a ) ) . our group has tested the ivs-1 in preclinical in vivo studies in pigs and found that it demonstrated high accuracy and a rapid response time . the sensor was originally developed to meet the need for better control of blood glucose levels ( bgl ) in patients in intensive care units . however , when we discovered the potential of our technology , we started to work towards the development of an aep . we realized that the sensor was able to instantaneously detect very small changes in bgl , and we found small oscillations in bgl with a period of approximately 10 minutes ( figure 1(b ) ) . when we first started to infuse insulin intravenously to regulate bgl , we found that the time from start of the infusion until a new steady state bgl was reached to be two hours or longer , which is very long . this means that it would take several hours to adjust any insulin infusion to the correct rate to achieve an appropriate and stable bgl . the insulin resistance in a single individual is constantly changing , and some authors even suggest that the regulatory system includes deterministic chaotic components that would render it impossible to foresee the effect of insulin on bgl during a given time using ordinary linear methods . a control system based on continuous infusions will therefore always be running to catch up and will have great difficulties in lowering a patient 's bgl to a sufficient degree without risking hypoglycemia . the use of subcutaneous sensors and infusions would increase the time delay and complicate the situation even more . given this background , we conducted a series of experiments to characterize the effects of intravenous bolus injections of insulin ( ib ) in a previous study . here , we found the time lag from an ib until a first observed decrease in bgl becomes four to six minutes . the maximum rate of decrease in bgl occurred shortly thereafter , and a nadir was reached 1520 minutes after the ib . these time intervals seemed to be rather dose independent as long as the ib dose was sufficient to yield any change in bgl at all . based on these observations , we hypothesized that it should be possible to construct an aep using the biological pancreas as a model . the ivs-1 would detect early changes in bgl , and the system should respond instantaneously with adjusted repetitive ibs . we had to construct a novel algorithm with the target of establishing and maintaining a normal fasting bgl , defined as 4.56.0 bgl regulation had to be a two - stage process , with the first step to establish glycemic control in a hyperglycemic subject by rapidly bringing bgl to the desired level and thereafter to maintain the desired glucose level over time . we identified the following key elements that had to be incorporated in the algorithm.the overall goal was to bring bgl to within a predefined range of 4.56.0 mmol / l and to keep it in this range , including small oscillations around the middle value in this range.ib was administered whenever needed in accordance with the continuous bgl readings ; adjusting the ib doses was kept simple with only three alternatives according to the previously administered ib : the same dose as the previous one , half the previous dose , or twice the previous dose.any decrease in bgl should be observable within five minutes after an ib ; otherwise , a new ib should be administered.blood glucose control should be established by a rapid decrease in bgl from hyperglycemic levels with a series of consecutive ibs . the bgl should be lowered 1 mmol / l for each ib injected.as soon as the bgl dropped below 6.0 mmol / l , blood glucose control should be maintained by meticulously timed ibs to allow small fluctuations in the blood glucose curve . the overall goal was to bring bgl to within a predefined range of 4.56.0 mmol / l and to keep it in this range , including small oscillations around the middle value in this range . ib was administered whenever needed in accordance with the continuous bgl readings ; adjusting the ib doses was kept simple with only three alternatives according to the previously administered ib : the same dose as the previous one , half the previous dose , or twice the previous dose . any decrease in bgl should be observable within five minutes after an ib ; otherwise , a new ib should be administered . blood glucose control should be established by a rapid decrease in bgl from hyperglycemic levels with a series of consecutive ibs . as soon as the bgl dropped below 6.0 mmol / l , blood glucose control should be maintained by meticulously timed ibs to allow small fluctuations in the blood glucose curve . the insulin algorithm was constructed as a flowchart based on simple if - then decisions , and the details of the algorithm evolved as the study went on . the current version of the algorithm is shown in figure 2 . human recombinant insulin 0.1 iu / ml ( actrapid , novonordisk , bagsvrd , denmark ) was used in the study . the drug was manually administered from a syringe pump ( using the bolus function ) ( alaris cc plus , carefusion , san diego , ca , usa ) in accordance with the insulin algorithm . forty - eight hours prior to the main experiments , we induced diabetes in healthy pigs by destroying their pancreatic beta cells using the cytotoxic agent streptozotocin 200 mg / kg i.v . ( zanosar , teva parenteral medicines , irvine , ca , usa ) [ 24 , 25 ] . on the day of the experiment , the animals were put under general anesthesia , an arterial line was established for monitoring and blood samples , central venous access was obtained , and the animals were instrumented with two ivs-1 sensors , one in each femoral artery . animal handling , anesthesia , and surgical intervention have been described in previous studies [ 20 , 23 ] . in order to achieve real - time continuous bgl output , the ivs-1 software had to be updated and a preinsertion in vitro calibration procedure had to be determined . we applied a calibration procedure by exposing the sensor to buffer solutions with glucose concentrations of 0.0 , 2.0 , and 10.0 mmol / l . a nonlinear least square algorithm was used to compute the two calibration parameters describing the nonlinear calibration function . the time series from the ivs-1 was calibrated using the computed calibration parameters and a one - point calibration method to set the off - set parameter by adjusting the ivs-1 level with the blood glucose level achieved from a simultaneously drawn blood - sample analyzed on a bed - side radiometer abl 720 blood - gas analyzer ( radiometer , brnshy , denmark ) . throughout the study , several in vivo calibration procedures to ensure we had redundancy , we instrumented each animal with two ivs-1 sensors hardwired to the same monitor . after the first in vivo calibration we chose whichever one had the most stable signal and used data from this sensor for the rest of the experiment . after the experiment , the ivs-1 data was retrieved by using data from the repetitive blood samples as calibration parameters . to transform the interferometric length measurement data into glucose concentration data we compensated for baseline drift by using a fixed baseline drift rate and compensated for ph interference by using the ph values from the blood - gas analyzer to compute ph corrected calibration parameters . the ph dependence of the calibration parameters was found from a set of in vitro experiments . this paper presents data from four animals , with the details shown in figure 3 and table 1 . the glucose values presented are those after the calibration procedures described in the previous paragraphs . the effect of the streptozotocin pretreatment varied between animals ; at the time of the first insulin bolus the starting bgl value ( bgl0 ) ranged from 7.46 to 14.06 mmol / l . we were able to establish glycemic control by bringing the bgl below 6.0 mmol / l ( test ) in 21 to 121 minutes , with the longest time in animals with a high bgl0 value . in animals 24 , the rate of decrease ( rd ) from the first insulin bolus until the 6.0 mmol / l limit was reached was relatively high , from 0.064 to 0.077 ( mmol / l)/min . ( as a comparison , we found the maximum rd to be approximately 0.1 ( mmol / l)/min after a single bolus dose of insulin in our earlier experiments . ) after reaching the target range , the animals were kept under glycemic control ( tctrl ) from 128 to 238 minutes . the total time with bgl values below the lower limit of the range , that is , 4.5 mmol / l ( tlow ) , varied from 3 to 51 min in the four animals . the lowest bgl measured in the four animals ( bgllow ) varied from 3.81 to 4.44 mmol / l . the intuitive interpretation of figure 3 is that the fundamental principles of this aep model work . bgl was rapidly and safely brought within the predefined range and kept within the range during the study time . we tried to administer ib as best we could according to the algorithm ; however , small details in the control algorithm had to be updated and changed as the experiments went on . these were mainly the timing of the boluses as the bgl tends to rise quite fast , and it is important not to delay the ib as it takes approximately 4 minutes from bolus given until a change in bgl starts to occur . however , the time lag between ib and effect was predictable and in accordance with our previous research . we believe that the amplitude can be substantially decreased by further improvement in blood glucose sensor stability and improvement in the insulin regulatory algorithm . as explained in the introduction , actual pancreatic beta cells oscillate with a fixed time interval of approximately five minutes , whereas the amount of insulin released with each pulse is constantly changing . as the effect of a single ib starts to lower bgl in four to six minutes and the nadir bgl values are reached after approximately 15 minutes , consecutive insulin bursts from the pancreas with an interval of five minutes will yield a carryover effect where several bursts interfere with bgl at a given time . we found this regulation system to be too complicated to model at this stage and therefore constructed a simplified system where the effect of each ib was followed throughout its effect period before a new ib was administered . in the future , a model able to correct for , and take advantage of , this carryover effect might be constructed , either through the use of fixed interval ibs and/or ibs combined with a small continuous infusion of insulin . another limitation of our aep is that insulin is infused into the systemic circulation , whereas the pancreas secretes the insulin into the portal vein . this means that in our model ( as in all other available insulin infusion regimes ) the liver receives relatively small amount of insulin compared to the rest of the body . the insulin infusion algorithms are based on what is called model predictive control , where the pharmacokinetic properties of insulin are modeled in a series of equations that are used to calculate the correct insulin dose at a given time [ 26 , 27 ] . several studies have shown the feasibility of such systems in controlling bgl overnight in diabetic subjects . however , it is very challenging to calculate the correct insulin dose when the subject is eating , exercising , or ill , and a fully automatic aep thus has yet to be constructed [ 28 , 29 ] . we believe a more empirical system like ours could tackle such obstacles , as it allows for the correct insulin dose to be calculated and timed continuously based on the effect of the previous ib . in this system , it is very unlikely that bgl would drift into hypoglycemic levels or that it would rise into gross hyperglycemia . the main challenge during the study was that the ivs-1 was not optimally calibrated at all times , which led to small errors in the real - time bgl display . we therefore to some extent had to rely on a combination of the ivs-1 output and the blood samples to estimate the correct real - time bgl in order to use the insulin algorithm properly . the observed periods of bgl below the range 's lower limit were caused by a discrepancy between the observed real - time bgl and the correct bgl calculated after the experiment ( the reported bgl ) . as such , the major limitation of the current technology is a background swelling of the glucose - sensing hydrogel , which resulted in a drift in the ivs-1 output . there will always be some doubt as to whether results from animal studies are valid in a human population ; on the other hand , the model makes it possible to manipulate bgl and to instrument study subjects without fear of any iatrogenic damage to healthy volunteers or patients . the ivs-1 probes used are all handcrafted and the setup of the animal experiment is complicated , which is why the number of animals used in the study is low . however , all of the animals studied ( both the final four as well as animals in our earlier studies ) displayed the same behavior . the main point of this paper has been to illustrate the physiological principle of using nature 's own regulatory system in an artificial control system , without fine tuning the details . we conclude that the use of real - time accurate intravasal glucose monitoring in combination with repetitive bolus injections of insulin , administered to mimic the natural pulsatility of endogenous insulin , is a promising method for a future artificial pancreas .

in recent years , the demand for herbal remedies in nigeria has been on the rise . yet there are little or no scientific data to support medicinal claims of these herbal formulations . herbals are usually sought after for their health benefits and these have become common medicines in many nigerian homes . it is recognized that herbal remedies prepared from medicinal plants may have several traditional applications especially in developing nations like nigeria where access to formal healthcare is limited . however , the increasing commercial promotion of herbals necessitate the need for assessment of safety and validity of medicinal claims . in recent past , several studies have demonstrated the toxic effects of indiscriminate use of packaged herbals [ 1 , 4 , 5 ] . furthermore , the general belief that herbal medicines are natural , effective , and without adverse effects has immensely contributed to the upsurge in the patronage of herbal formulations [ 68 ] . the popular belief that herbal remedies are without toxic or undesirable effects these and other factors form part of several concerns associated with the use of herbal remedies in developing nations . in nigeria , studies have revealed that the number of nigerian medicinal plants screened for the validation of biological activity far outweighs the assessment for potential toxic compounds and contaminants [ 2 , 10 , 11 ] . also , many herbal formulations being promoted and marketed in nigeria lack scientific data to support acclaimed medicinal benefits and may pose great health risk to the numerous unsuspecting consumers . according to the fijk leaflet , the herbal is prepared from a mixture of 13 medicinal plants including cassia alata , citrus medica var acida , aloe vera , cassia augustifolia , cassia siamea , kyaha sengalensis , xylopia aethiopica , gongronema latifolium , khaya grandifoliola , morinda lucida , anthocelesta djalonensis , citrullus lanatus , and aloe barbaris . this must have contributed to the wide patronage and consumption among the nigerian populace . according to the fijk label , the herbal remedy is indicated for treatments of several ailments which include obesity , diabetes , sexual improvement , menstruation pains , infertility , high blood pressure , crawling sensation , and malaria , amongst others . there are scientific reports on the medicinal relevance of the individual plant constituents of the fijk mixture . however , there are no empirical data to support claims of medicinal benefits or otherwise of fijk . a search through online research libraries including the cochrane , pubmed , and google - scholar revealed the absence of scientific data on fijk herbal remedy . the scenario makes likely toxic potential from repeated exposure to the fijk mixture largely unknown . for the safety of public health , it is essential that herbal remedies have available scientific data on safety / toxicity profiles in order to aid informed choices by consumers . this study aimed to determine the effect of oral and repeated exposure to fijk on rat plasma biochemical indices and liver morphology . commercial reagent kits for the assay of alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferases ( alt ) , total cholesterol ( tc ) , total protein ( tp ) , triglyceride ( tag ) , and high density lipoprotein cholesterol ( hdl - c ) were as supplied by randox diagnostics , crumlin , uk . twenty - four male wistar rats with weight between 190 and 200 g were obtained from the experimental animal farm at the university of ilorin , ilorin , nigeria . the wistar rats were housed in animal cages in a well - ventilated experimental room . the rats were allowed to acclimatize for a period of 14 days before the commencement of treatments . handling of animals was in accordance with relevant institutional and ethical guidelines as approved for scientific study . mg / kg : received fijk at a dosage of 15 mg / kg body weight . fijk 30 mg / kg : received fijk at a dosage of 30 mg / kg body weight . fijk 45 mg / kg : received fijk at a dosage of 45 mg / kg body weight . control : received distilled water . 15 mg / kg : received fijk at a dosage of 15 mg / kg body weight . fijk 30 mg / kg : received fijk at a dosage of 30 mg / kg body weight . fijk 45 mg / kg : received fijk at a dosage of 45 mg / kg body weight . the dosages were selected based on corresponding therapeutic doses for human consumption as recommended by the herbal producer . the treatment was daily and lasted for 21 days . at the end of treatments , the blood samples were collected into edta bottles , spun at 5000 g for 10 minutes using a refrigerated centrifuge ( anke tdl-5000b , shanghai , china ) to obtain the plasma , which was subsequently used for the biochemical analysis . the liver tissues were excised , weighed , and fixed in buffered neutral formalin and used for histopathological examinations . the biochemical indices were determined in rat plasma using a uv / vis spectrophotometer ( jenway , staffordshire , united kingdom ) where applicable . the levels of rat plasma total protein ( tp ) , aspartate aminotransferase ( ast - ec : 2.6.1.1 ) , alanine aminotransferase ( alt - ec : 2.6.1.2 ) , alkaline phosphatase ( alp - ec : 3.1.3.1 ) , and lipid profile including total cholesterol ( tc ) , triglyceride ( tag ) , high - density lipoprotein - cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) were determined using randox assay kits . reduced glutathione level ( gsh ) was determined by the procedure described by ellman with slight modification . lipid peroxidation was ascertained by monitoring the degrees of lipid peroxides in the supernatant fraction of plasma using the method described by niehaus jr . and samuelsson . the rat liver was fixed in 10% buffered neutral formalin ( bnf ) immediately following excision from animals . capture and scoring for morphological changes were done by a pathologist blind to the treatments , at the pathology unit , university of ilorin teaching hospital , ilorin , nigeria . data were subjected to statistical analysis using the one - way anova ( graphpad software inc . , san diego , ca ) . the oral administration of fijk to rats did not produce significant change to average body weights of animals , even though gradual decreases in rat body weights were observed for groups that received fijk herbal mixture ( figure 1 ) . likewise , the relative organ to body weight ratio showed no significant difference among the various treatment groups ( figure 2 ) . however , the oral administration of fijk herbal mixture at double and triple doses caused significant reduction in rat plasma protein level compared to the control group . the determination of lipid profile showed that the oral exposure of rats to fijk at the various doses did not produce significant change in the plasma level of total cholesterol when compared to the control . however , the exposure to fijk at the highest dosage caused significant elevation in rat plasma level of tryglyceride . further assessment of lipid profile revealed that the daily administration of fijk mixture depleted rat plasma level of hdl - c ( figure 3 ) . meanwhile , the level of rat plasma ldl - c rose with increasing doses of fijk herbal . furthermore , the atherogenic index was elevated significantly in the groups exposed to fijk mixture ( figure 4 ) . on the other hand , rat plasma alp level was lowered ( p < 0.05 ) following oral exposure to fijk herbal mixture but the reduction was only significant in the group that received the highest dose of fijk . in contrast , treatment of rats with fijk herbal mixture at all doses significantly increased the levels of plasma alt and ast relative to the control group ( figure 5 ) . to assess whether oral exposure of fijk in rats would predispose to lipid peroxidation , the plasma level of mda was determined . there was significant elevation in rat plasma level of mda in the fijk treated groups relative to the control ( figure 6 ) . conversely , oral administration of fijk to rats significantly depleted the plasma levels of gsh compared to the control . the light microscopic examination of rat liver sections for morphological changes revealed lesions caused by fijk herbal mixture ( figure 7 ) . the fijk herbal mixture caused hepatic alterations including hypochromic , haemorrahage , ruptured vascular channels , and hyperemia . however , the rat liver in the control group had no incidence of this cellular lession . unfortunately the use of herbal products is not strictly regulated in nigeria thus making them freely available , a scenario which predisposes to possible abuse by consumers . although herbal mixtures enjoy wide patronage in nigeria , little is known about likely toxicity that may be associated with repeated consumption . herbal remedies may have recognizable therapeutic effects ; they also may have toxic side effect . more so , many herbal preparations lack scientific facts to back up acclaimed medicinal benefits . currently , there are no available empirical data on fijk herbal mixture . the present study sought to determine the influence of oral and repeated adminstration of fijk herbal mixture on rat biochemical and morphological parameters . this is coming against the background that previous studies have demonstrated toxic potentials of packaged herbals [ 4 , 10 , 11 ] . in the present study , although rat liver weights were reduced in the treatment groups , the relative organ weights were not significantly affected by exposure fijk mixture . however , the evaluation of rat lipid profile revealed that oral administration of fijk herbal mixture did not significantly alter the total cholesterol in all the treatment groups . however , there were dose - dependent increases ( p < 0.05 ) to the plasma levels of tag and ldl - c in rats orally exposed to fijk herbal mixture . conversely , levels of plasma hdl - c were significantly lowered by repeated administration of fijk mixture and the effect was dose dependent . the lipid profile indices are useful in monitoring health status of the cardiovascular system . although the rat total cholesterol level was not affected by fijk herbal mixture administration , elevated levels of tag and ldl - c may predispose to cardiovascular related disorders . increased level of ldl - c has been associated with higher risk of atherosclerosis while elevated level of hdl - c is linked to reduced occurences of cardiovascular disorder [ 19 , 20 ] . the level of rat plasma hdl - c decreased with increasing doses of fijk leading to elevated atherogenic index . the atherogenic index can be used to predict the risk for development of cardiovascular disorders . therefore , the low ratio of hdl - c to ldl - c , caused by repeated oral exposure to fijk , may implicate increased tendency for the development of atherosclerosis . the rat plasma levels of alt and ast were elevated by exposure to repeated administration of fijk mixture relative to control . the alt and ast are normally found in the red blood cells , liver , heart , and kidney tissues . the levels of alt and ast have long been used to assess the functions of liver . increased plasma levels of both alt and ast have been linked to tissue toxicity [ 21 , 22 ] . normally , a basal level of the enzymes is found in the plasma ; however , when there is cellular damage , the enzymes extrude into the extracellular fluid thus raising the concentrations in the plasma . in the present study , the significant alterations to levels of rat plasma alt and ast this is further supported by the fact that the ratio of ast to alt in plasma was less than one ( < 1 ) , which may indicate ensuing acute or chronic liver injury . on the other hand , the rat plasma alp levels were significantly reduced in the group which received the highest dosage of fijk herbal mixture . the decreased level may be as a result of inactivation or decreased protein synthesis . previous studies have shown that herbal mixtures have capability to alter the levels of alt , ast , and alp in rats [ 1 , 9 ] . furthermore , in order to evaluate whether the oral and repeated exposure to fijk mixture was capable of predisposing to oxidative stress , we determined the levels of rat plasma mda and gsh . the mda is a by - product of lipid peroxidation while the gsh level could be used to assess the antioxidant status of a cellular system . the increased level of rat plasma mda suggested the presence of lipid peroxidation caused by oral exposure to fijk herbal mixture . the increased mda level in rats may be due to the generation of free radical species potentiated by exposure to fijk mixture . on the other hand , ensuing oxidative stress caused by the oral administration of fijk was further demonstrated by the depleted level of rat plasma gsh . the gsh molecule is a nonenzymatic antioxidant capable of scavenging free radicals and it could also serve as substrate to other antioxidant enzymes like the glutathione peroxidase . the low level of gsh may be attributed to increased usage in order to scavenge free radical species or consumption as substrate by antioxidant enzymes which function to protect against oxidative damage . recent studies have associated oral and repeated exposure to herbal mixtures with lipid peroxidation and oxidative stress [ 1 , 5 ] . the light microscopic examinations of rat liver sections showed intact and normal cellular architecture in the control group . however , the examination of rat liver sections in groups exposed to various dosages of fijk mixture revealed incidences of morphological lesions . the rat hepatic alterations caused by exposure to fijk herbal mixture included haemorrahage , ruptured vascular channels , hyperemia , and inflammation . the histopathological findings support the biochemical alterations caused by oral and repeated exposure to fijk herbal mixture and are reminiscent of rat hepatic injury . this is consistent with a previous report which demonstrated organ toxicity caused by herbal formulations . the study revealed elevated atherogenic index caused by oral and repeated exposure to fijk mixture in rats . furthermore , oral and repeated exposure to fijk herbal mixture in rats caused lipid peroxidation and potentiated hepatic lesion .

hallucinations , predominantly visual in nature , can occur in parkinson disease ( pd ) and are often related to the antiparkinsonian drugs . visual hallucinations are a well - recognized problem . besides visual hallucinations , patients with pd uncommonly experience different forms of hallucinations , such as auditory hallucinations . auditory hallucinations occur in 8% to 13% of patients with pd and are generally accompanied by visual hallucinations . auditory musical hallucinations ( amhs ) are rare complex auditory hallucinations in pd that have been reported in only 8 patients . four patients for whom detailed information is available have been documented , and 2 of these patients were deaf ( table 1 ) . in patients with amhs caused by conditions other than pd , amhs are characterized by repetitive and usually uncontrollable musical patterns , unrelated to external stimulation . in december 2005 , a 72-year - old right - handed woman with no clinically significant history of disease , including epilepsy , migraines , or mental disease such as schizophrenia , noticed a tremor in her right hand . in december 2007 , she noted slowness of movement , difficulty in walking in narrow spaces , and the onset of left hand tremor in addition to right hand tremor and presented at our hospital . she showed features of moderate parkinsonism , including masked face , retropulsion , bradykinesia , and right - side - dominant rigidity and resting tremor . the scores on parts i , ii , iii , and iv of the unified parkinson disease rating scale ( updrs ) were 0 , 14 , 24 , and 0 , respectively . the scores on the mini - mental status examination ( mmse ) and frontal assessment battery were normal ( 30/30 ) and decreased ( 16/18 ) , respectively . she was given levodopa ( 250 mg / day ) and immediate - release pramipexole ( 1.5 mg / day ) , and the severity of these symptoms had decreased in may 2008 . in august 2008 , she showed wearing - off phenomenon and had freezing of gait . subsequently , she was given entacapone ( 200 mg / day ) in june 2010 and zonisamide ( 25 mg / day ) in july 2011 . the scores on the mmse and zung depression scale were 29 and 45 , respectively . in july 2012 , selegiline ( 5 mg / day ) was started since the severity of freezing gait had intensified , and the patient frequently fell . she was then given stable doses of levodopa ( 500 mg / day ) , immediate - release pramipexole ( 1.5 mg / day , 3 times per day ) , entacapone ( 200 mg / day ) , zonisamide ( 25 mg / day ) , and selegiline ( 5 mg / day ) . neuroleptic drugs were not used . in december 2012 , we switched from immediate- to once - daily extended - release pramipexole in stable dosage because off - periods had increased . after that , she often had visual hallucinations such as animals or human hands and auditory hallucinations such as rustling sounds . in april 2013 , the amhs were a quiet piano or often songs on a loud radio or background music over other sounds . amhs did not occur every day , but when they did the music persisted all day while the patient was awake . the amhs were unrelated to visual hallucinations as well as other auditory hallucinations of children talking together . the music was unpleasant , but not objectionable , threatening or ego - syntonic , and it did not interrupt her daily activities . although the hallucinations persisted , she requested no further measures to suppress the hallucinations , such as the reducing the doses of anti - parkinsonian drugs or using neuroleptic agents , because the hallucinations were non - threatening and tolerable . in october 2013 , zonisamide was increased to 50 mg / day because off - periods had increased . after receiving the patient 's informed consent , we switched back from extended- to immediate - release pramipexole in a stable dosage in january 2014 to determine whether extended - release pramipexole was causing the amhs . when the patient visited to our hospital 5 day after switching back to immediate - release pramipexole , she reported that recognizable subjects such as animals or persons that she previously saw clearly as visual hallucinations had disappeared , whereas some unrecognizable shadows were often evident . amhs sometimes occurred for several hours , particularly in the early morning or evening , and other auditory hallucinations became faded . twelve days after retuning to immediate - release pramipexole , visual hallucinations rarely occurred , and amhs and other auditory hallucinations had disappeared . among the 5 patients including ours for whom detailed information are available , amhs in pd were non - specific incomprehensible tunes or different familiar or popular songs such as folk or lyrical songs , which were unrelated to an external stimulus . the inability of patients to interrupt amhs might have been uncomfortable , but was not threatening . female sex , hearing impairment , advanced age , psychiatric conditions , cognitive disorders , alcoholism , obsessive compulsive disorders , and neurological conditions ( eg , seizure disorders , stroke , tumors , central nervous systems infections , and brain lesions in the dorsal pons , temporal lobes , and frontal lobes ) were reported as risk factors for amhs . our patient had none of these risk factors except for female sex and advanced age . in the 5 patients including ours , neither cognitive decline nor depression was evident during amhs , and disease severity and off - periods were apparently unrelated to amhs . the intervals between the time of altering treatment with anti - parkinsonian drugs and the onset of amhs were long in 2 previous patients , and the authors did not mention any relation between amhs and anti - parkinsonian drugs . in another patient , however , short - term amhs were elicited by amantadine . in our patient , although other preceding dopaminergic treatments such as levodopa , entacapone , and selegiline might have been prerequisite to the development of the hallucinations , the auditory and visual hallucinations occurred after immediate - release pramipexole was switched to extended - release pramipexole , and similar findings have not been reported previously . several months after that , amhs began . in studies of patients with advanced or early pd , switching from immediate- to extended - release pramipexole was safe , and the most frequently reported side effects were dyskinesia or somnolence . in healthy male volunteers , the pharmacokinetics and tolerability of a once - daily extended - release formulation of pramipexole were similar to those of an immediate - release formulation of pramipexole given 3 times daily . however , 2 patients with pd in whom dopamine dysregulation syndrome developed after switching from immediate - release to extended - release pramipexole have been reported . amhs in pd were non - threatening , and dopaminergic treatment may predispose patients to amhs or be a unique possible cause of amhs .

recently , the importance of quality of life research has been accepted . regardless of the disease process , improving a person 's quality of life has become an important goal of treatment programs . in the pediatric literature , quality of life studies have been largely confined to single disease states . usually absent from such studies is an understanding of the quality of life of children with heterogeneous diagnoses often participating in treatment programs with unpredictable prognoses including a deterioration in physical and cognitive function . have recently reported detailed findings on the quality of life ( physical and psychosocial ) of children and youth both healthy and with varying chronic conditions such as asthma , cancer , cerebral palsy , and psychiatric disorders using the pediatric quality of life inventory ( pedsql ) . the quality of life of a complex multidiagnosis group of special needs children and youth has not been reported . research is emerging that investigates associations between child / family variables and physical and psychosocial quality of life in special needs children in an attempt to gain a better understanding of the factors influencing child well - being . it is becoming clear that factors affecting a child 's physical quality of life differ from those affecting psychosocial quality of life . aged 612 with cerebral palsy ( cp ) , majnemar showed that the strongest predictor of physical functioning ( using the pedsql ) was the gmfm ( gross motor function measure , r = 0.79 ) . the child 's general competency in performing tasks ( motivation ) also correlated with better physical functioning ( r = 0.45 ) . psychosocial well - being was associated strongly with few behavior difficulties ( r = 0.62 ) and low parent distress ( r = 0.43 ) . in another cp group ( n = 39 , aged 618 ) autonomous / allowing parenting style was associated with physical ( r = 0.40 ) and psychosocial ( r = 0.40 ) well - being measured using the child health questionnaire . used the kidscreen quality of life measure in children with cp aged 812 ( n = 818 ) . they showed that children scoring in the lowest quartile for physical function had low gross motor functioning , high pain levels , and high parent stress . kids in the lowest psychosocial quartile had high pain levels and high parent distress . in children with attention deficit hyperactivity disorder ( adhd ) psychosocial well - being ( child health questionnaire ) was associated with fewer symptoms of adhd and less comorbid psychiatric diagnosis . no association was found between reported symptoms of adhd and co - morbid psychiatric diagnosis and the physical quality of life of these children . an understanding of the associations of child and family variables with child 's quality of life ( physical and psychosocial ) in multidiagnosis special needs population would provide a clearer picture of the issues faced by these individuals and their families . little is known about the relationship between health care expenditures and quality of life of children with special needs . seid et al . measured the direct costs ( excluding pharmacy and mental health ) of an inclusive ( healthy and special needs ) group of children ( ages 218 ) that were members of federally supported managed health care plan in san diego . an inverse relationship between health - related quality of life and direct pediatric health care costs was reported . multiple regression analysis showed that the presence of a chronic health condition and lower physical functioning scale scores consistently accounted for the greatest amount of variance ( 21.2% ) in predicting healthcare costs at 24 months . no research study could be found that related quality of life to direct and indirect health care costs in a group of special needs children from a societal perspective . the objectives of this paper were threefold : first , to report the quality of life ( physical and psychosocial ) in a multi - diagnosis , 219-year - old group of special needs children / youth ; next , to investigate associations between family / child variables with the child 's physical and psychosocial quality of life ; it was hypothesized that psychosocial quality of life would have stronger associations with child / family variables ( parent distress , child behaviors , parenting styles , social support , family functioning , and overall impact on family ) than physical quality of life ; and finally , to explore trends in the direct and indirect costs associated with quality of life in these families . this descriptive study is part of a cohort study examining the effects and expense of more and less integration of services that provide treatment and rehabilitation for children with complex needs . the cohort is enrolled in the newly modeled children 's treatment network ( ctn ) of simcoe / york counties in ontario . the ctn approach is unique in that it is based on the collaboration of numerous existing autonomous , local service agencies utilizing the service coordination , and electronic record functions of the ctn . ethics approval was obtained for the study by the research ethics board of mcmaster university . this was a cross - sectional survey of families with a special needs child enrolled in the ctn from may to december 2007 . families were deemed eligible if the child was age 219 years , they were residents of simcoe / york , and there were multiple needs within the family ( child 's complex needs and/or families needs for example , a parent 's medical or mental health problem ) . the consenting parent / guardian most knowledgeable ( pmk ) returned a signed consent form to mcmaster university indicating their willingness to participate . the pmk then completed a telephone interview ( 1 hour ) by one of three trained interviewers from mcmaster university . child quality of lifethe pedsql is a generic measurement system developed by varni et al . for use in children ages 218 years . the shortened version consists of 15-items comprising three core scales and addresses the physical ( 5 items ) , emotional ( 4 items ) , social ( 3 items ) , and school functioning ( 3 items ) . item wording differs for children ages 24 , 57 , and 818 . each item asks how much of a problem it has been during the past month on a five - point scale ( 0-never a problem to 4-almost always a problem ) . the questionnaire appropriate for 818 years old was also used for 19 year old children in this study . parent proxy report formats were used for all eligible children due to the inclusion of children with limited cognitive or communicative abilities . items are reverse - scored and linearly transformed to a 0100 scale so that higher scores indicate better quality of life . psychosocial quality of life ( 10 items ) is computed as the sum of the emotional , social and school scale scores ( range 0100 ) . the minimally clinically important difference has been reported for the parent proxy total score ( standard error of the mean ( sem ) = 4.50 ) , psychosocial health score ( sem = 5.49 ) and physical health score ( sem = 6.92 ) in a population health survey in the state of california . the pedsql is a generic measurement system developed by varni et al . for use in children ages 218 years . the shortened version consists of 15-items comprising three core scales and addresses the physical ( 5 items ) , emotional ( 4 items ) , social ( 3 items ) , and school functioning ( 3 items ) . item wording differs for children ages 24 , 57 , and 818 . each item asks how much of a problem it has been during the past month on a five - point scale ( 0-never a problem to 4-almost always a problem ) . the questionnaire appropriate for 818 years old was also used for 19 year old children in this study . parent proxy report formats were used for all eligible children due to the inclusion of children with limited cognitive or communicative abilities . items are reverse - scored and linearly transformed to a 0100 scale so that higher scores indicate better quality of life . psychosocial quality of life ( 10 items ) is computed as the sum of the emotional , social and school scale scores ( range 0100 ) . the minimally clinically important difference has been reported for the parent proxy total score ( standard error of the mean ( sem ) = 4.50 ) , psychosocial health score ( sem = 5.49 ) and physical health score ( sem = 6.92 ) in a population health survey in the state of california . child behaviorbehavior is measured using thecanadiannational longitudinal survey of children and youth ( nlscy ) behavior questionnaire for children ages 219 . the questionnaire asks about how the child seems to feel or act regarding age specific behaviors such as getting into fights , inability to sit still , and worrying . the parent is asked to rate the specific behavior from 1-never to 3-often . behavior subscales include hyperactivity / inattentive , prosocial , anxiety / emotional disorder , conduct disorder/ physical aggression , indirect aggression , and property offence . behavior is measured using thecanadiannational longitudinal survey of children and youth ( nlscy ) behavior questionnaire for children ages 219 . the questionnaire asks about how the child seems to feel or act regarding age specific behaviors such as getting into fights , inability to sit still , and worrying . the parent is asked to rate the specific behavior from 1-never to 3-often . behavior subscales include hyperactivity / inattentive , prosocial , anxiety / emotional disorder , conduct disorder/ physical aggression , indirect aggression , and property offence . demographics of the childit includes child age , grade , and pmk report of the main medical and other important diagnosis . it includes child age , grade , and pmk report of the main medical and other important diagnosis . health of pmkthekessler scale ( k10 ) measures pmk symptoms of depression and anxiety , a frequent accompaniment of depression . ten questions measure the frequency of feeling : sad , nervous , restless , hopeless , worthless , everything was an effort , tired for no good reason , so nervous that nothing could calm down , fidgety , so restless could not sit still , or depressed during the past month . chronic aspects of distress in the past month are examined on a five - point scale ( 1-all of the time to 5-none of the time ) . scores range for 1050 where 19 indicates no clinically important level of distress , 2024 indicates mild distress , 2529 moderate distress , and 3050 severe distress . thekessler scale ( k10 ) measures pmk symptoms of depression and anxiety , a frequent accompaniment of depression . ten questions measure the frequency of feeling : sad , nervous , restless , hopeless , worthless , everything was an effort , tired for no good reason , so nervous that nothing could calm down , fidgety , so restless could not sit still , or depressed during the past month . chronic aspects of distress in the past month are examined on a five - point scale ( 1-all of the time to 5-none of the time ) . scores range for 1050 where 19 indicates no clinically important level of distress , 2024 indicates mild distress , 2529 moderate distress , and 3050 severe distress . parent wellbeingparents were asked to rate their mental , physical health , and life satisfaction on a five - point scale ( 1-very satisfied or these questions were taken from the canadian community health survey ( cchs 2.2 ) . parents were asked to rate their mental , physical health , and life satisfaction on a five - point scale ( 1-very satisfied or these questions were taken from the canadian community health survey ( cchs 2.2 ) . parenting practicesthe nlscy parenting scale was used and consists of twenty - five questions adapted from the parent practices scale . the following four parenting behaviors were measured : positive interaction ( praise , play ) , hostility ( anger , discipline ) , consistency ( follow through ) , and punitive ( yelling , physical punishment ) . pmk rated each item ( e.g. , do something special with your child that he / she enjoys ) in terms of frequency from 0-never to 4-many times each day . internal consistency is reported by subscale and age group ( cronbachs alpha 0.390.75 ) . the nlscy parenting scale was used and consists of twenty - five questions adapted from the parent practices scale . the following four parenting behaviors were measured : positive interaction ( praise , play ) , hostility ( anger , discipline ) , consistency ( follow through ) , and punitive ( yelling , physical punishment ) . pmk rated each item ( e.g. , do something special with your child that he / she enjoys ) in terms of frequency from 0-never to 4-many times each day . internal consistency is reported by subscale and age group ( cronbachs alpha 0.390.75 ) . family functioningthirteen items taken from the nlscy population survey , based on a subscale of the mcmaster family assessment device , were used to gather information on various aspects of family functioning , namely , problem solving , communication , roles , affective responsiveness , affective involvement , and behavior control . pmk rated each item ( e.g. , we avoid discussing our fears or concerns ) along a four - point scale from 0-strongly agree to 3-strongly disagree . thirteen items taken from the nlscy population survey , based on a subscale of the mcmaster family assessment device , were used to gather information on various aspects of family functioning , namely , problem solving , communication , roles , affective responsiveness , affective involvement , and behavior control . pmk rated each item ( e.g. , we avoid discussing our fears or concerns ) along a four - point scale from 0-strongly agree to 3-strongly disagree . . social supportthe level of social support of the pmk was assessed using an eight item shortened version of the social provisions scale . different social support constructs were measured : guidance , reliable alliance ( i.e. , feeling assured that others would be available to offer practical help ) , and attachment . pmk rated each item along a four - point scale from 0-strongly disagree to 3-strongly agree . the level of social support of the pmk was assessed using an eight item shortened version of the social provisions scale . different social support constructs were measured : guidance , reliable alliance ( i.e. , feeling assured that others would be available to offer practical help ) , and attachment . pmk rated each item along a four - point scale from 0-strongly disagree to 3-strongly agree . caregiver burdenthe impact on family ( iof ) scale determines the effects of a chronic illness on parents and families . parents respond on a four - point scale to the degree that statements apply to their family ( 1-strongly agree to 4-strongly disagree ) . the revised iof scale ( 15 items ) has been validated [ 19 , 20 ] . statements cover four dimensions : financial burden , family / social impact , personal strain , and mastery ( e.g. , fatigue is a problem , see family and friends less , need to change plans at last minute , little desire to go out ) . the impact on family ( iof ) scale determines the effects of a chronic illness on parents and families . parents respond on a four - point scale to the degree that statements apply to their family ( 1-strongly agree to 4-strongly disagree ) . the revised iof scale ( 15 items ) has been validated [ 19 , 20 ] . statements cover four dimensions : financial burden , family / social impact , personal strain , and mastery ( e.g. , fatigue is a problem , see family and friends less , need to change plans at last minute , little desire to go out ) . demographics of the familya standard form including spiritual or faith orientation , ethnicity , and languages was selected from the canadian national longitudinal survey on children and youth ( nlscy ) that also includes community dwelling disabled children . sociodemographic data were gathered on the pmk gender , age , and educational level as well as on household income and family status . a standard form including spiritual or faith orientation , ethnicity , and languages was selected from the canadian national longitudinal survey on children and youth ( nlscy ) that also includes community dwelling disabled children . sociodemographic data were gathered on the pmk gender , age , and educational level as well as on household income and family status . costs for health and social serviceshealth and social service utilization is measured by an inventory developed initially by browne et al . and this measure has consistently distinguished expenditures for use of services by youth with and without behavior problems , people with and without mental illness , with and without a range of chronic diseases , with and without poverty [ 23 , 24 ] . it consists of questions about the respondent 's use of eight categories of direct health services : primary care , emergency room , specialists , hospital episodes , hospital days ( irrespective of episode ) , emergency room specialists , seven types of other community health professionals , and laboratory services . recall that data are used in order to assess the patient 's use of all health services . inquiries are restricted to the reliable duration of recall span : 6-months for remembering a hospitalization , 2 weeks for a visit to a physician , and 2 days for the consumption of a prescription medication . to calculate 6-month utilization , the various spans of time are extended to yield a 6-month rate of utilization per category of health service , as proposed by spitzer and petrou et al . . the 6 month rate per category of service is multiplied by the 2006 unit cost ( canadian $ ) for that service . [ 27 , 28 ] as one of the few measures of ambulatory utilization published and empirically validated . health and social service utilization is measured by an inventory developed initially by browne et al . and this measure has consistently distinguished expenditures for use of services by youth with and without behavior problems , people with and without mental illness , with and without a range of chronic diseases , with and without poverty [ 23 , 24 ] . it consists of questions about the respondent 's use of eight categories of direct health services : primary care , emergency room , specialists , hospital episodes , hospital days ( irrespective of episode ) , emergency room specialists , seven types of other community health professionals , and laboratory services . recall that data are used in order to assess the patient 's use of all health services . inquiries are restricted to the reliable duration of recall span : 6-months for remembering a hospitalization , 2 weeks for a visit to a physician , and 2 days for the consumption of a prescription medication . to calculate 6-month utilization , the various spans of time are extended to yield a 6-month rate of utilization per category of health service , as proposed by spitzer and petrou et al . . the 6 month rate per category of service is multiplied by the 2006 unit cost ( canadian $ ) for that service . [ 27 , 28 ] as one of the few measures of ambulatory utilization published and empirically validated . descriptive statistics ( numbers , percentages , means , and standard deviations ) were calculated for demographic data , child / family variables , and expenditures . the behavior subscale measures have different numbers of items applicable to different age groups : children 2 to 5 years , and children and youth 6 to 19 years . the behavior scales for different age groups were transformed using the interpolation technique where the mean of the behavior scale scores for children 2 to 5 years with fewer items was multiplied times the number of items for older children . there were reports on two or three children with complex needs in the same family and only one report of parent variables . in these instances , the pmk was counted 2 or 3 times as appropriate to ensure a matched number of children and parents in the analysis . pearson correlation coefficients were calculated for quality of life variables and other child / family variables . differences between dichotomized psychosocial ( 3 or <3 often or almost always responses ) and physical ( 2 or < 2 often or almost always responses ) quality of life and family / child variables were compared using chi - square and t - tests . these cut - offs were chosen as it was thought to represent a child with clinically important psychosocial issues , requiring professional follow - up and clinically important physical restrictions . table 1 shows the demographic characteristics of participating families . the majority of pmk were mothers of the children ( 85% ) , born in canada ( 75% ) , and spoke english ( 90% ) . the average pmk was 41 years , 90% were female , 85% were married / common - law , 69% were employed , and the median household income was $ 60$69,000 . there was an even split between families residing in simcoe ( 52% ) and york ( 49% ) county . the average child age at interview was 8 years with 66% of the sample being male . thirty - eight percent of the children were in preschool ( up to and including kindergarten ) , 36% in grade 15 ( elementary ) , and 26% in grade 6 and up ( junior ) . sixty percent of children were receiving service from community care access centres and school boards at time of entry into the ctn . the top pmk reported diagnoses for the children were mental and behavioral disorders ( 85% ) , one of which was autism ( 25% ) ; diseases of the nervous system ( 34% ) ; and congenital malformations , deformations , and chromosomal abnormalities ( 19% ) ( table 2 ) . fifty one percent of children had more than one reported medical problem . child quality of life scores is presented in table 3 ( n = 429 ) . these differences exceeded minimally clinically important differences ( mcids ) reported by varni et al . between the three age groups . physical well - being was the lowest in the oldest age group ( grades 6&up ) and psychosocial well - being was the lowest in school aged children ( grades 15 and grades 6&up ) . these differences also exceeded the mcid . within this population of children with complex needs there was no correlation between the child 's physical functioning and their psychosocial functioning ( table 4 ) . physical well - being showed moderate associations with less adverse impact on the family and the presence of prosocial behavior ( r = 0.30.5 ) . the child 's physical functioning had weak associations ( r = 0.1 < 0.3 ) with social support , parental distress and life satisfaction , hostile and consistency parenting styles , family functioning , and negative child behaviors . psychosocial well - being in the children / youth was strongly associated ( r > 0.5 ) with reports of fewer anxiety / emotional behaviors . psychosocial well - being was moderately associated with lower levels of adverse family impact , less hostile parenting , less child hyperactivity , conduct disorder , indirect aggression , and property offence behaviors . parental distress and life satisfaction , positive parenting styles , and family functioning showed weak associations with child psychosocial functioning ( r = 0.1 < 0.3 ) . in this sample , the child 's psychosocial functioning was unrelated to pmk punitive ( emotionally charged ) parenting or prosocial behavior . 67% of punitive parenting levels were none to mild , 33% moderate , and 0% severe in this sample . table 5 presents family / child variables dichotomized by responding often or almost always to 2 or < 2 items ( 5 total ) in the physical function domains . in children with 2 or more identified physical challenges there were lower levels of pmk social support , greater overall adverse impact on the family , less consistent parenting , less hostile parenting , and less prosocial and hyperactivity behaviors in the children . the number of physical challenges in the children was not related to family function , child anxiety , conduct disorder , property offense , parent symptoms of depression / anxiety , positive , nor punitive parenting . table 6 presents family / child variables dichotomized by responding often or almost always to 3 or <3 items ( 10 total ) about the child 's emotional , social , and school function domains . in children with 3 or more identified psychosocial issues there was less pmk social support , greater report of adverse impact on the family , and poorer overall family functioning . parent distress was higher , parenting practices ( positive interaction , consistency , hostile ) were worse , and child behavior scores were all higher ( poorer ) in the children and youth with poor psychosocial function . prosocial behavior was also higher in children and youth with fewer symptoms of psychosocial problems . table 7 shows per 6 month expenditures for the child 's use of human services by their physical function . although overall primary care use was not statistically significant , in the group with 2 challenges there was greater use of ambulance and 911 calls . overall physician specialist costs were higher in this group . economically important higher use of neurologists and pediatricians was observed . economically and statistically significant higher use of physiotherapists , nutritionists , nurses , personal support workers and special ed . the overall costs of other health and social support services were not different between the groups largely due to the greater increased use of social and recreations programs in children less physically challenged ( < 2 items ) group . community supports , outpatient lab test costs , medication and supply costs , and hospital and respite costs were all higher in the group with 2 physical challenges . table 8 shows per 6 month expenditures for the child 's use of human services by their psychosocial well - being . total primary and secondary care costs were similar between children exhibiting 3 or <3 psychosocial behaviors . total other health and other service provider costs were similar between the two groups ; however , economically important increased use of psychologists , chiropractors , and mental health counselors was noted in the group exhibiting more psychosocial distress . also , greater use of physiotherapists , nutritionists , nurses , personal support workers , daycare , and device costs was seen in the group of children with less psychosocial distress . this is the first study to present quality of life scores in a heterogeneous group of special needs children ages 219 . quality of life scores were much lower than reported mean scores for healthy children ( physical score 87.84 , psychosocial 81.87 ) . they were also lower than scores reported by 10 disease clusters ( physical score range 64.4085.89 , psychosocial range 67.4677.34 ) . the scores obtained in this multidiagnosis sample were comparable to reported scores for children with cerebral palsy attending a cp clinic in san diego ( physical 43.19 , psychosocial 55.91 ) . children in varni 's cp sample , however , were excluded if they were not able to self - report pedsql scores . generally the physical score in the cp group is lower likely because of the inclusion of children with quadriplegia while the psychosocial score is higher likely due to our inclusion of kids with mental and behavioral diagnosis . the lower scores illustrate the multifaceted needs and issues faced by this heterogeneous group of children and youth with complex needs usually excluded from other studies . the finding of declining physical and psychosocial well - being in these children / youth with advancing age is troubling . perhaps this finding demonstrates the value of the recent early intervention services in ontario aged 06 years and captures the population of adolescents that did not receive this . perhaps , fewer formal services / programs are available for older youth thus possibly explaining their declining physical function or perhaps older children simply decide not to pursue further efforts that maintain / improve the physical aspects of their lives . nevertheless , the ctn decision to include children up to age 19 is supported by this finding . the decline in psychosocial function could be the results of accumulating parent distress , less favorable parenting styles , and/or the child exclusion from peers and activities when in school . the psychosocial well - being of complex needs children was more strongly related to child and family variables than physical functioning in this sample . there was a weak association with punitive parenting , however , which is likely explained by the absence of severe punition in this sample . these findings are similar to previous reports in special needs populations of parent distress , negative child behavior , and negative parenting practicing being associated with lower child psychosocial quality of life [ 25 ] . there may be complex interactions between physical and psychosocial factors within the child and parent that could lead to parent distress , ineffective parenting , child behavior , and family functioning . it is the associations and interactions of these variables overtime that help explain the child 's quality of life . the increased use of specialist costs in children with greater physical need is not surprising nor is the increased use of lab tests , medications , community support , special ed . the overall costs to the health care system in children with greater physical disability , however , are largely offset by the increased use of social and recreation programs by those children less affected . the more physically affected children are not using the social and recreational sector to the same extent than those less affected . the presence of psychosocial problems was not associated with a greater use of any services . economic trends of increased use of mental health professionals , however , are seen in children with more psychosocial problems . the use of special education services was similar between the two psychosocial groups perhaps indicating under recognition of psychosocial issues in school aged kids . this is the first comprehensive presentation of health service utilization data in relation to the quality of life of children with complex needs . results and findings are difficult to generalize outside of this study population as contexts may differ . this study may be missing important information from working , lower educated , single parents and their children , likely those with greater need and harder to reach . also , quality of life data were parent - reported . generally , parents underestimate their child 's quality of life compared to child self reports . therefore , associations may differ when child self report data are used , particularly for older kids . it was not feasible to obtain self - report data from this complex needs group due to the wide range of limitations present and budget constraints of the study . quality of life scores decreased with advancing age in these complex , multidiagnosis children with special needs aged 219 years . psychosocial quality of life showed stronger univariate associations with child and family variables measured compared with physical quality of life . health and social service utilization costs were higher in children with greater physical challenges mainly due to increased use of medications , treatment , supplies , and aids .

cardiotoxicity and associated drug failures in clinical development or even withdrawals from the market contribute significantly to the rising cost of drug discovery and development in the pharmaceutical industry . it has been reported that 28% of drug withdrawals in the usa are associated with negative cardiovascular events , and because of amortized drug failures , the cost of developing a new drug has been estimated to be as high as $ 5 billion . traditionally used biomarkers of cardiovascular diseases ( cvd ) , however , have been proven unreliable in predicting cadiotoxicity in a number of settings . for example , although highly active antiretroviral therapy ( haart ) has reduced aids - related deaths , cvd and cvd - related deaths have increased in the hiv - infected population by 1.52 times in comparison with hiv - negative individuals . yet , there is a lack of safety biomarkers addressing both drug - induced and immune - associated cvd in therapeutics for hiv . one of the strategies to address this problem is to use improved safety biomarkers to predict drug cardiotoxicity before advancing to human clinical trials . in particular , biomarkers that can reliably predict drug - induced cardiac injury in animals will decrease costs by excluding nonviable drug candidates and identifying those that have a greater probability of success in clinical trials . numerous articles have reported the use of myocardium - specific proteins as biomarkers for congestive heart failure , myocardial infarction and other heart diseases . these cardiac biomarkers include the cardiac troponins i and t , cardiac natriuretic peptides , creatine kinase isoenzyme mb ( ck - mb ) , and lactate dehydrogenase ( ldh ) . among the biomarkers , cardiac troponins are perhaps the gold standard for cardiac injury , considering their sensitivity and specificity ; however , the troponins have a half - life of 2 h , which makes them difficult to use for routine evaluation in toxicology studies . one major problem associated with these current biomarkers is that they typically show significant changes only in the first 1224 h postdose , returning to normal levels after 24 h. therefore , there is an urgent need for reliable cardiac biomarkers that can predict drug cardiotoxicity over longer periods of time and be incorporated in routine toxicology studies . acute myocardial infarction , the most common cause for cvd - related death , was found showing increased risk among hiv - positive people . in our study , isoproterenol ( iso ) , a beta - adrenergic agonist used to treat bradycardia or heart block , has been used as a model compound to induce myocardial infarct - like lesions . various studies have been conducted to evaluate myocyte damage following exposure to this drug in rat and monkey species . we previously reported a panel of nine serum proteins showing significant change at 48 and 72 h after iso treatment in african green monkeys ( afg ) ; these proteins can potentially be used for predicting long - term cardiac injury . to corroborate our studies , we conducted similar protein profiling of serum proteins at different times after iso injection in cynomolgus macaques ( cm ) and rhesus macaques ( rm ) . the serum proteins showing long - term alterations were compared among all three nonhuman primate ( nhp ) species . moreover , proteins from four different heart compartments ( left ventricle , right ventricle , left atrium , right atrium ) were systematically analyzed to elucidate the cellular mechanism of the drug - induced cardiac injury . energy imbalance , altered calcium flux , formation of reactive oxygen species , and structural degradation have been implicated in the literature as mechanisms of iso - induced cardiotoxicity in mouse and rat species using immunoassays and histopathological and immunohistochemical studies ; however , few studies have been conducted on the protein profile of injured heart tissue . ms / ms quantitative proteomics analysis on serum proteins and heart tissue proteins from cm and rm . a filter - aided sample preparation ( fasp ) strategy was used for protein extraction and digestion for individual animals ; tandem mass tag ( tmt ) was used for isotopic labeling of the digested peptides from different animals for quantitation purposes . the yield and reproducibility of this protocol has been evaluated for technical and biological replicates in previous studies , and the results showed it is highly reproducible and reliable for protein identification and quantitation . in this study , we applied a similar approach to analyze serum proteins from two other nhp models to continue the cardiac biomarker discovery and expanded the analysis to include tissue proteins from four different compartments to understand the cellular mechanism of drug - induced heart injury . to our knowledge , this is the first systematic quantitative proteomic analysis on four different heart compartments from nhp models . a total of six non - nave cynomolgus macaques ( macaca fascicularis , mauritius origin , 710 years of age , nave to isoproterenol ) and six non - nave rhesus macaques ( macaca mulatta , unknown origin , 1013 years of age , nave to isoproterenol ) were assigned to the study . for each species , two male and two female non - nave animals were administered isoproterenol hydrochloride ( iso , sigma - aldrich , st . louis , mo ) as a single subcutaneous ( s.c . ) dose of 4 mg / kg ( concentration 2 mg / ml ) on day 1 . an additional one male and one female of each species were administered saline and served as vehicle control animals . blood was collected predose , and at 1 , 4 , 24 , 48 , and 72 h postdose from each animal , processed to serum , and flash - frozen with liquid nitrogen . on day 4 , animals were sedated with ketamine ( buttler schein , ca ) and euthanized with an overdose of sodium pentobarbital ( buttler schein , ca ) . slices 4 mm thick from the left and right ventricular free walls and the left and right atria of each animal were placed into screw - cap vials and flash - frozen with liquid nitrogen . heart tissue samples were thawed on ice , weighed , and washed with phosphate buffered saline ( pbs ) . for tissue protein extraction , 100 mg of heart tissue was minced into 2 mm pieces in 1 ml of lysis buffer ( 50 mm tris , ph 7.4 , 0.5% sds , edta - free protease inhibitor cocktail ) . about 0.2 ml of glass beads ( 0.5 mm , sigma - aldrich , st . subsequently , samples were homogenized five times for 1 min each by a bullet - blender ( next advance , averill park , ny ) at 4 c and then left on ice for 1 h. debris was removed by centrifugation at 12 000 g for 15 min at 4 c , and supernatants were stored at 80 c for proteomics sample preparation . total protein of the samples was measured using a nanodrop 2000 ( thermo scientific , san jose , ca ) . both serum samples and tissue protein lysates were purified and digested using the fasp protocol developed by mann s group . briefly , 200 l of diluted ( 1:20 dilution ratio ) serum or tissue lysate was transferred into a 1.5 ml microcon ym-10 centrifugal unit ( millipore , billerica , ma ) . protein reduction , alkylation , and tryptic digestion were conducted in the centrifugal unit . after overnight tryptic digestion at 37 c , the peptides were eluted twice with 150 l of 50 mm ammonium bicarbonate . the total protein or peptide concentration in each step the eluted peptides were dried by vacuum centrifugation and then resuspended in 50 mm tetraethylammonium bromide for tmt labeling . for each sample , 50 g of digested peptides was incubated with an amine - reactive 6-plex tmt tag ( thermo scientific , san jose , ca ) for 2 h at room temperature ( rt ) . reactions were quenched by adding 8 l 5% hydroxylamine and incubated at rt for 15 min . the labeled peptides were then combined , and excess tmt tags were removed by a 3 8 mm strong cation exchange ( scx ) trap column , then desalted by a 3 8 mm c18 reverse phase ( rp ) trap column ( bruker - michrom , ca ) . purified labeled peptides were dried by vacuum centrifugation and then resuspended in 0.1% formic acid ( fa ) for multidimensional protein identification technology ( mudpit ) analysis . each tmt labeled sample was separated using a nano - lc system ( agilent 1200 , palo alto , ca ) . briefly , 15 g peptide mixtures were separated first by an scx column ( self - packed , 100 m 100 mm , 5 m , 300 polysulfethyl ) . fractions were eluted stepwise with a nine - step ammonium formate concentration gradient ( 5 , 15 , 25 , 50 , 75 , 100 , 200 , 500 , and 1500 mm ) . each salt step eluent was further separated on a capillary c18 rp column ( 75 m 150 mm , 3 m particle size ) with a 146 min gradient from 2 to 97% acetonitrile in 0.1% fa at a constant flow rate of 300 nl / min . the peptides eluted from the c18 column were directly analyzed with an ltq orbitrap xl ( thermo fisher scientific , san jose , ca ) equipped with a nanoelectrospray ionization source . full ms spectra were acquired in positive mode over a 3501800 m / z range , followed by four cid ( collision induced dissociation ) and four hcd ( higher - energy collisional dissociation ) events on the four most intense ions selected from the full ms spectrum using a dynamic exclusion time of 30 s. four cid scans ( maximum inject time 100 ms , minimum signal threshold 500 counts , collision energy 35% , activation time 30 ms , isolation width 1.0 m / z ) were used for peptide identification , and four corresponding hcd scans ( maximum inject time 300 ms , minimum signal threshold 500 counts , collision energy 45% , activation time 30 ms , isolation width 1.0 m / z ) were used for quantitation . acquired tandem mass spectra were searched against the national center for biotechnology information macaca protein database . a decoy database containing the reverse sequence of all the proteins was appended to estimate false discovery rate ( fdr ) . the search was performed using the sequest algorithm incorporated in proteome discoverer 1.3.0.339 ( thermo finnigan , san jose , ca ) , and a cutoff of 5% fdr was applied . the precursor mass accuracy was limited to 13 ppm , and fragment ion mass tolerance was set at 1.1 da . fixed modifications included carbamidomethylation on cysteines , and variable modifications included oxidation on methionines and tmt adduction to peptide n - termini , lysines , and tyrosines . the fdr , calculated through a decoy database , was set as 0.05 using percolator scoring with posterior error probability validation . for protein identification the intensity of each peptide was normalized to protein median intensity before calculating the ratio of different tags from the same peptides . peptide quantitation data from both unique and nonunique peptides were averaged to determine the protein relative abundance at different time points versus predose . a student s t - test was applied on the proteins showing average abundance change > 1.5-fold upon iso treatment , and a cutoff of p - value < 0.05 was applied . pathway and network analysis on changed heart tissue proteins was performed using ingenuity pathway analysis software ( redwood city , ca ) . human c - reactive protein and pancreatic -amylase elisa kits were purchased from abcam ( cambridge , ma ) . monkey serum amyloid a , and -1-acid glycoprotein elisa kits were purchased from kamiya biomedical ( seattle , wa ) . each serum sample was tested in duplicate , and the average value was calculated and plotted graphically . for western blot assays , cardiac troponin i antibody and -2-microglobulin antibody were purchased from abcam ( cambridge , ma ) . briefly , 50 g of proteins was run on 420% precast polyacrylamide gel ( bio - rad , hercules , ca ) and transferred to nitrocellulose membranes . membranes were blocked with odyssey blocking buffer ( li - cor , lincoln , ne ) , followed by incubation with primary antibodies at 1:1000 dilutions . membranes were washed three times with 1x pbs containing 0.05% tween20 ( v / v ) , incubated with irdye secondary antibodies ( li - cor , lincoln , ne ) for 1 h , and washed again to remove unbound antibody . odyssey clx ( li - cor , lincoln , ne ) was used to detect bound antibody complexes . monkeys treated with iso demonstrated a variety of adverse cardiac effects . the cardiac effect of iso in afg has been previously reported . heart rate for cm , as measured by electrocardiograms ( ecg ) , decreased within 1 h and in general remained below control and prestudy values for 72 h. at 24 h postdose , heart rate was 34.7% and 22% lower than the control group for males and females , respectively ( figure 1 ) . cardiac abnormalities observed in cm during the first 24 h postdose included hypotension ; occasional ventricular premature complexes ; ventricular bigeminy ; atrial premature complexes , with or without aberrant conduction ; and st segment elevation . a majority of the treatment - related findings were noted within 1560 min or 4565 min . these increased within 1 h post - treatment and remained elevated for up to 24 h , then dropped and remained below the control and prestudy values for the remainder of the study ( figure s1 , supporting information ) . heart rates of ( a ) male and ( b ) female cynomolgus macaques following a single s.c . heart rates were measured by ecg at time 0 ( immediately prior to dose administration ) , 15 min , 1 , 4 , 24 , 48 , and 72 h postdose . a total of 375 proteins were identified and quantified in cm serum samples . the 0 h time point was used as control , and the ratios of the intensity of the reporter ions associated with other time points ( 1 , 4 , 24 , 48 , 72 h ) versus the reporter ion of 0 h time point were acquired as relative peptide abundance . table 1 shows that after iso injection , seven proteins were significantly up - regulated , and three proteins were down - regulated in cm serum ( table s2 , supporting information ) . these effects lasted through 72 h , except pancreatic -amylase isoform 2 , which still maintained a high level of concentration at 48 h post dose but dropped to baseline at 72 h. data on protein relative abundance was acquired via the ratio of intensity of reporter ions from hcd spectra ( figure s3 , supporting information ) . all the cid spectra of selected proteins were manually inspected ( figure s4 , supporting information ) to ensure accurate identification . of the 10 proteins showing significant alteration in cm serum , 5 they are -1-acid glycoprotein 1-like isoform 2 ( a1ag ) , c - reactive protein isoform 2 ( crp ) , fructose - bisphosphate aldolase a isoform 1 ( fbaa ) , serum amyloid a protein - like isoform 1 ( saa ) and apolipoprotein a-1 isoform 1 ( apo a1 ) . to further assess the potential of these proteins to serve as biomarkers for evaluating heart injury , we tested the serum samples from four rms after iso injection . serum samples were taken at 4 and 72 h post dose , and the protein levels were compared with the sample from the same monkey taken predose . three proteins a1ag , saa and apo a1were detected as significantly changed after iso injection in all three species ( figure 2 ) . the level of saa reached the peak at 24 h post dose and still maintained a high level ( 5-fold increase ) at 48 h in cm . in afg , saa showed its highest level at 48 h post dose , which was about a 7-fold increase compared with predose . because the 24 and 48 h time points were not analyzed in rm , we can not tell at which point saa showed maximum concentration ; however , at 72 h post dose , saa still showed as much as a 10-fold increase . the other two proteins , a1ag and apo a1 , also showed consistent increases or decreases in all three species after iso injection . proteins showed significant change after iso injection in serum in all three species ( african green monkey , rhesus macaque , and cynomolgus macaque ) . the protein relative abundances over 0 h ( predose ) were determined by lc ms / ms . values shown represent protein relative abundance over 0 h values averaged from four iso - treated monkeys ( males and females combined ) . crp , saa , a1ag , and pancreatic -amylase ( pa)were selected for elisa validation in cm ( figure 3 and table s5 , supporting information ) . saa and a1ag were confirmed to be significantly changed after iso injection in all three species and were also validated in rm sera by elisa ( table s6 , supporting information ) . crp was on the list of changed proteins in both afg and cm studies . crp and saa have been previously reported as acute - phase proteins , and their levels are often correlated . previous studies showed that elevated levels of crp and saa can be correlated with higher mortality rates in patients with acute coronary syndromes , even in the patients with negative rapid cardiac troponin t ( ctnt ) assay . quantitative crp and saa can thus provide additional prognostic information for predicting cardiac necrosis beyond the information provided by ctnt alone . serum concentration of crp increased from 173 64 ng / ml at 0 h to the maximum concentration of 3846 240 ng / ml at 24 h post dose . the concentration of saa was also elevated significantly after iso injection and reached its peak concentration at 48 h post dose ( 45 32 g / ml , which is about a 50-fold increase compared with the concentration at 0 h of 0.9 0.09 g / ml ) . elisa validation of ( a ) c - reactive protein ; ( b ) pancreatic -amylase ; ( c ) serum amyloid a ; ( d ) -1-acid glycoprotein in serum from four iso - treated cynomolgus macaques . the serum concentration of a1ag , also an acute - phase protein , has been reported to increase in response to tissue injury , inflammation , infection , and cancer . the elisa results showed that a1ag increased from 262 95 g / ml at 0 h to 650 147 g / ml at 24 h post dose and stayed as high as 502 81 g / ml at 72 h post dose . the elevation of pa in serum after iso treatment was also validated by elisa . the concentration of pa increased significantly and stayed at the peak concentration ( 2500 u / ml ) from 4 to 24 h post dose , which is 25-fold higher than predose . pa can regulate the blood glucose level , and it has been used as a drug target for treating diabetes and obesity . there are , as yet , no studies showing a direct correlation between pa elevation and cardiac injury , making this the first report of increased pa level in serum associated with heart injury . all the validated proteins did not show any changes in the control animals , except for crp , which showed elevation in one control animal but still lower than the protein level in the treated animals . to understand the cellular mechanism of drug - induced cardiac injury , heart tissue proteins from each cm sacrificed at 72 h post dose each heart tissue was divided into four compartments : left ventricle , right ventricle , left atrium , and right atrium . six tmt tags were used to label six different macaques ( four iso - treated and two controls ) . a total of 1320 and 1335 proteins were identified and quantified with high confidence ( fdr < 0.05 ) in the left ventricle and right ventricle compartments , respectively . 3 , 4 , 5 , and 6 ( iso - treated ) were compared with the vehicle - treated monkeys nos . 1 and 2 . a student s t - test was applied to screen the proteins showing an average abundance change > 1.5-fold upon iso treatment . previous studies on iso - induced cardiac toxicity in a rat model reported severe hypertrophy and myocardial necrosis in the left ventricle . in our study , moderate myofiber degeneration was observed in the left ventricle of iso - treated cm by histopathology ( data not shown ) . the results of left ventricle protein profiling showed that the expression level of 12 proteins increased and 7 proteins decreased more than 1.5-fold in iso - treated animals compared with the control with a p - value < 0.05 ( table 2 and table s7 , supporting information ) . network analysis of the changed proteins in the left ventricle showed that their functions are associated with cardiac necrosis , cardiovascular system development and function , and cell death ( figure 4 ) . compared with the left ventricle , protein profiling showed fewer and less severe changes in the right ventricle , suggesting that iso might have less deleterious effects on the right ventricle ( table s8 , supporting information ) . network analysis of the changed proteins in cynomolgus macaque left ventricles after iso treatment : cardiac necrosis / cell death , cardiovascular system development and function , cell death , and survival . proteins labeled red are up - regulated ; proteins labeled green are down - regulated ; proteins shown in gray are not identified in our data . interestingly , we observed the most iso treatment - induced protein expression level changes in the right atrium compartment . because of the limited amount of right atrium tissue sample from monkey no . 2 , the right atrium protein profiling was performed only for monkeys 1 , 3 , 4 , 5 , and 6 . of the total 1140 identified proteins in the right atrium , 19 proteins were shown to be down - regulated , and 2 proteins were up - regulated in iso - treated monkeys compared with the controls using a p - value < 0.05 ( table 3 and table s9 , supporting information ) . levels of atrial natriuretic factor ( anf ) , a polypeptide hormone secreted by the heart muscle cells in response to atrial stretch , were significantly increased in both left and right atria upon iso injection . an enhanced plasma concentration of anf has been reported in the literature as associated with cardiac dysfunction and has been suggested for diagnosis and risk stratification in heart failure . we observed less and moderate protein changes in the left atrium than in the right atrium after iso injection ( table s10 , supporting information ) . to verify the changed proteins identified in heart tissue upon iso treatment , western blot assays were conducted on three tissue proteins : cardiac troponin i ( tnni3 ) , cytochrome c1 ( cyc1 ) , and -2-microglubulin ( b2 m ) , from both the left ventricle and the right atrium compartments ( figure 5 ) . dissociation and release of troponin from the myofibril has been reported in the literature after myocardial infarction , which result in the observed elevation of troponin serum level . in our proteomic analysis , cardiac troponin i and t and troponin c were all identified as decreased in the right atrium tissue lysate , indicating dissociation of troponin from the myofibril . western blot verification showed that the level of tnni3 significantly decreased in the right atrium tissue lysate of iso - treated monkeys ( animals 3 , 4 , 5 , 6 ) compared with the control monkey ( animal 1 ) . alteration of cyc1 could cause the disruption of mitochondrial function and contribute to cell apoptosis . the decrease in cyc1 identified by proteomic analysis was also verified by western blot in both the left ventricle and right atrium tissue lysate . in addition , b2 m was indicated as a biomarker to evaluate cardiovascular diseases , and an elevated b2 m level in plasma was reported to be associated with cardiovascular mortality and cardiovascular events in patients at different stages of chronic kidney disease . in our current study , western blot analysis of ( a ) cyc1 and b2 m in left ventricle tissue lysate ; ( b ) tnni3 and cyc1 in right atrium tissue lysate . animals 1 and 2 were vehicle - treated ; animals 36 were iso - treated . data show the cyc1 level decreased in both the left ventricle and right atrium of iso - treated animal tissue lysate compared with vehicle - treated ones . the level of b2 m elevated upon iso administration in the left ventricle tissue lysate , whereas tnni3 decreased after iso treatment in the right atrium tissue lysate . cardiac toxicity is now the leading cause for drug discontinuation in clinical trials and even postmarket surveillance in the u.s . with antiretroviral therapy , patients infected with hiv are living longer ; however , studies showed increased rates of cvd and cvd - related mortality in this population . these cardiac events may be due to a combination of factors from antiretroviral therapy and the hiv virus . preclinical evaluations provide an invaluable opportunity to identify drug toxicity before administration of potential new antiretroviral therapeutics or other drugs to human subjects . although cardiac troponins have gained much attention and interest for their potential to provide sensitive early warning of heart injury , significant uncertainty surrounding the interpretation , narrow kinetic range , and variability in troponin response level among species make routine application of troponin in preclinical trials controversial . thus , there is an urgent need for safety biomarkers that can be used in routine evaluation of short- and long - term cardiac injury in preclinical trials . we previously reported nine serum proteins that showed significant changes at 48 and 72 h after iso injection in afg monkeys . to expand on this study , similar proteomic screening has been conducted on the serum proteins from two other nhp models , cm and rm . a panel of 10 proteins showed significant alteration at 48 and 72 h after iso injection in cm . among these , three proteins ( saa , a1ag , and apo a1 ) were confirmed to be changed in all three species . multiple proteins such as saa , a1ag , and crp are involved in inflammation , whereas several other proteins are associated with cardiac hypertrophy and necrosis , such as carbonic anhydrase , tropomyosin , and -1-antichymotrypsin . our results suggest that these proteins have the potential to be used in preclinical evaluation of cardiac toxicity for both short- and long - term studies . understanding the cellular mechanism of drug - induced heart injury is essential for physicians to be aware of potential interactions with other drugs and dose limitation for prevention of adverse effects . in our study , interrogation of proteins from four different heart compartments identified the left ventricle and right atrium as the compartments showing the greatest alterations after iso administration . pathway analysis of the altered proteins in heart tissue suggested two major pathways that are responsible for cardiotoxicity : calcium signaling and mitochondrial dysfunction . since sydney ringer showed over 100 years ago that the heart could not beat in the absence of ca , numerous studies have been conducted to understand the role of calcium in cardiac function and the progression of heart disease , such as cardiac hypertrophy and heart failure . calcium plays a crucial role in regulating contraction and intracellular signaling , which are vital for healthy heart function . in our study , two of the up - regulated proteins identified in left ventricle were involved in the calcium signaling pathway : calreticulin and glutamate [ nmda ] receptor subunit -3-like ( grin2c ) . it is essential for cardiac development , and its gene is tightly regulated during cardiogenesis . calreticulin is involved in regulation of intracellular ca homeostasis , and overexpression of calreticulin can disrupt ca storage and release . seidman s group reported increased expression levels of calreticulin in calsequestrin 2 mutant mice with ventricular tachycardia . grin2c is a subunit of glutamate receptors , which are found in the central nervous system and also play a role in the calcium signaling pathway . although glutamate receptors are mostly implicated in neurological disorders such as parkinson s and huntington s diseases , these receptors have also been found to be overactivated or abnormal in diabetes and ischemia . there are currently no reports of altered grin2c levels associated with any cardiac diseases in the literature . at the end of the calcium signaling pathway , ca directly activates the myofilaments for heart muscle contraction by binding to the troponin complex . when ca concentration declines , it dissociates from troponin , and muscle fiber relaxation occurs . the troponin complex has three units : troponin i , t , and c. although troponin c has no cardiac specificity , both troponin i and t have cardiac isoforms . thus , cardiac troponin i ( ctni ) and troponin t ( ctnt ) are used as biomarkers for the detection of cardiac injury . interestingly , we observed decreased levels of tnc , ctni , and ctnt in right atria upon iso treatment . this could be due to the degradation of the structural pool of the troponin complex and release of troponin into the circulatory system , which is consistent with the elevated blood levels of troponin observed in myocardial infarction and other cardiac injuries in clinical studies . other myofilament proteins , such as myosin-6 , myosin light chain 4 , tropomyosin -chain isoform 11 , and tropomyosin -4 chain isoform 17 , are also detected as altered upon iso treatment . the decrease in troponins and other interacting proteins suggests that myofilaments , the contractile elements of the heart , could be one of the drug targets . mitochondria generate most of the cell s supply of adenosine triphosphate ( atp ) . in addition to supplying cellular energy , mitochondria play an important role in many other tasks , such as cellular proliferation , cell death , regulation of the cellular redox state , and fatty acid oxidation . mitochondrial mutation and dysfunction have been implicated in many diseases , including heart and neurological diseases , and may also play a role in the aging process . oxidative phosphorylation is the metabolic pathway in which the mitochondria can generate atp by the oxidation of nutrients . in our study of cm heart tissue proteins , we identified several proteins involved in the oxidative phosphorylation pathway that are significantly down - regulated upon iso treatment ( figure 6 ) . among these proteins , atp5b , atp5c1 , atp5d , atp5f1 , and atp5j2 belong to complex v , which is the final enzyme in the oxidative phosphorylation pathway to generate atp . uqcrc1 and cyc1 , and cox5a are present in complex iii and iv , respectively , and are responsible for transferring electrons to proteins and oxygen in the electron transfer chain . down - regulation of these proteins could lower the rate of oxidative phosphorylation and impair energy production . damage to mitochondrial function could also cause the release of cytochrome c , which is a key step leading to programmed cell death . we observed decreased levels of cyc1 , which is a subunit responsible of transferring electrons to cytochrome c , in the heart tissue . previous studies on cardiac diseases reported damage to mitochondria by decreased energy production , accumulation of reactive oxygen species , and release of cytochrome c in ischemia , reperfusion , cardiomyopathy , and heart failure . thus , a drug - induced decrease in energy production and loss of mitochondrial function could very likely contribute to myocyte apoptosis and heart tissue injury . proteins showing significant down - regulation upon iso treatment are involved in the electron transport and oxidative phosphorylation pathway . in summary , we applied a tmt labeling - based quantitative proteomics approach to three nhp species for safety biomarker discovery to evaluate drug cardiotoxicity . saa , a1ag , and apo a1in all three species upon iso injection , indicating the potential of including these proteins in preclinical evaluation of drug - induced cardiac injury . in addition , proteomic analysis of injured heart tissue proteins demonstrated detrimental effects on energy generation and mitochondrial function of cardiac myocytes , which can provide insights for reducing cardiotoxicity in future drug discovery and development . it is worth noting that cardiac troponins were not identified in serum , but were identified as decreased in heart tissue lysate along with other cardiac - specific proteins . in serum samples , the top 10 most abundant proteins account for 90% of the total protein content , making it difficult to detect these medium- or low - abundance cardiac - specific proteins with nontargeted profiling mass spectrometry . immunoaffinity enrichment and targeted mass spectrometry were used for detection of cardiac troponins in serum samples . our results suggest that proteomic screening of heart tissue proteins , followed by verification in serum samples using targeted mass spectrometry or other sensitive measures , could be a new strategy to discover improved cardiac biomarkers with high specificity in future studies .

blunt traumatic rupture of the diaphragm is a serious injury that is often difficult to diagnose . ( 1 ) it is an uncommon injury whose incidence is rising because of the increasing number of road traffic accidents in the last few years , together with better pre - hospital and hospital resuscitation of severely injured patients and improved diagnostic facilities . ( 2 ) in developing countries where initial care of severely injured patients and diagnostic facilities are less than optimal , blunt traumatic diaphragmatic rupture ( btdr ) may go undiagnosed . high index of clinical suspicion is required because of the missed diagnosis and potential for delayed presentation . we report a case of delayed presentation of post traumatic diaphragmatic hernia and reviewed the literature to highlight challenges in diagnosis and management of such cases . a 32 year old male was admitted to the emergency department with a history of nausea , vomiting , pain in the chest and upper abdomen as well as breathlessness of 3 days duration . he had a history of blunt trauma to the abdomen following a fall from a tree 4 years previously . he was then treated conservatively and no diaphragmatic hernia was detected . on physical examination the patient appeared pale , dehydrated and had dyspnea . examination of the left chest showed decreased movement on respiration , absent breath sounds , bowel sounds were present and dullness on percussion . apart from a mild anaemia ( 9gm / dl ) , all blood tests were within normal limits . the chest x - ray showed a large air - fluid level in the left thoracic cavity with a nasogastric tube within it , collapse of left lung field , obliteration of cardio - phrenic angle and shifting of the mediastinum toward the right side . ( figure 1 ) the presence of the nasogastric tube was confirmed with the presence of gurgling sound on auscultation after administration of air through it . barium meal was carried out to localize the stomach and showed the presence of the stomach in the thoracic cavity confirming diagnosis of post - traumatic diaphragmatic hernia . ( figure 2 ) the patient was counseled and prepared for surgery through a left sub costal incision . the findings at surgery were that two - thirds of the stomach , omentum and spleen had herniated through a 10 cm x 7 cm tear in left hemidiaphragm into the chest . riolfi performed the first successful repair in 1886 . not until 1951 , when carter et al . ( 3 ) approximately 0.8%-1.6% of patients with blunt trauma shows a rupture in the diaphragm . ( 4 ) blunt trauma accounts for 75% of ruptures , and penetrating trauma accounts for the rest . approximately 69% of hernias are left - sided , 24% are right - sided , and 15% are bilateral . ( 5,6 ) patients with delayed diaphragmatic herniation frequently present months to years after the initial injury with manifestations of visceral herniation , incarceration , obstruction , ischemia from strangulation , or perforation . in our case symbas et al . , observed a delay in diagnosis in 8% of cases of diaphragmatic injury from 18 h to 15 years after injury . ( 7 ) chest radiography is the standard in the advanced trauma life support ( atls ) protocol for a trauma workup . approximately 23 - 73% of traumatic diaphragmatic ruptures will be detected by initial chest radiograph , with an additional 25% found with subsequent films . chest radiographic findings that indicate traumatic rupture include the following ( 8) : abdominal contents in the thorax , with or without signs of focal constriction ( collar sign)nasogastric tube seen in the thorax , elevated hemidiaphragm ( > 4 cm higher on left vs right ) anddistortion of diaphragmatic margin . abdominal contents in the thorax , with or without signs of focal constriction ( collar sign ) nasogastric tube seen in the thorax , elevated hemidiaphragm ( > 4 cm higher on left vs right ) and distortion of diaphragmatic margin . conventional ct scan has been reported to have a sensitivity of 14%-82% , with a specificity of 87% . helical ct has increased sensitivity 71 - 100% , with higher sensitivity for left vs right . ct findings indicating rupture include the following ( 8) : direct visualization of injurysegmental diaphragm non - visualizationintrathoracic herniation of visceracollar signperidiaphragmatic active contrast extravasation direct visualization of injury segmental diaphragm non - visualization intrathoracic herniation of viscera peridiaphragmatic active contrast extravasation ultrasonography ( focused assessment with sonography for trauma [ fast ] scan ) has been reported to detect diaphragmatic hernias . ( 9 ) our patient was diagnosed by abdomino - thoracic x - ray and barium meal study . the difference is based on the degree of adhesion present in the thoracic cavity and the state of the herniated organs . after reduction of the abdominal contents , the diaphragm is usually repaired simply with monofilament nonabsorbable suture as in our case . delay in presentation of a diaphragmatic hernia in this case could be explained by various hypotheses . delayed rupture of a devitalized diaphragmatic muscle may occur several days after the initial injury . however the more likely explanation is a possible delayed detection assuming that the diaphragmatic defect occurring with injury manifests only when herniation occurs . in conclusion , a history of trauma must be investigated to diagnose patients with a delayed post traumatic diaphragmatic hernia . a high index of suspicion and the use of relevant radiological investigation is essential for early diagnosis . all cases whether diagnosed pre - operatively or intraoperatively must be repaired surgically either by laparotomy , thoracotomy , a thoraco - abdominal approach or by minimal access surgery .

the jun activation - domain binding protein 1 ( jab1 ) is initially identified as a coactivator of activator protein ( ap-1 ) transcription factor and found a component of the cop9 signalosome ( csn ) complex , contained modulating signal transduction , gene transcription , and protein stability.12 jab1 interacted with p27 and translocate it 's from nuclear to cytoplasm which resulted in acceleration of p27 degradation through the ubiquitin - dependent / proteasome pathway and promotes cell - cycle progression.3 p27 is an inhibitor of cyclin - dependent kinases ( cdks ) , performing potential tumor suppressor gene in diversity of human 's cancer . cdk is a family of protein kinases , the activity of the serine / threonine kinases known as cdk that are under the strict control of positive and negative regulators . until now studies have found that p27 promotes apoptosis , growth inhibition and cell cycle arrest.45 reduction of p27 protein means high resistance and poor prognosis to anticancer drug . down regulation of p27 protein is often discovered by human cancers including breast and ovarian cancer and is normally correlated with poor clinical outcome . as a result , the concentration of the p27 protein can be expressed as an important marker in the development of human cancer . accordingly , this study examines a correlation of p27 and jab1 , will reveal the relevant disease in women . jab1 has been involved to the tumorigenic process , therefore , jab1 have the potential to be an effective beneficial target that affect the interaction of many tumor stage . size of jab1 is an -40 kda soluble protein , also jab1 is located on chromosome 8 . jab1 interacted with p27 and improved its cytoplasmic translocation which resulted in acceleration of p27 degradation through the ubiquitin and proteasome pathway . it is well known that p27 protein levels are mainly regulated through degradation by ubiquitin - dependent proteolysis . control of p27 and jab1 is important process in cancer progression , which is controlled by both the positive and negative regulators . p27 is a kind of cdk inhibitors , suppresses the g1-to - s cell cycle progression , functions as a main negative regulator of apoptosis and is , thus , considered a tumor suppressor.6 the main function of this p27 is to inhibit the cdk2-cyclin e complex by controlling at the g1 to s tarnsition in normal cells . jab1 is an important protein in breast cancer progression comes from the recent study that it is a downstream target for her2 - 2/neu.7 amplification and overexpression of her-2/neu was established in many cancers and upregulating of this oncogene was associated with increased metastasis and prognosis . especially , her-2/neu oncogene ( known for erbb2 ) is a critical biomarker , target of therapy for roughly 30% of breast cancer patients and expression of her-2/neu is upregulated in the existence of tamoxifen , leading to tamoxifen resistance in breast cancer . the role of jab1 interacts with c - myc gene in breast cancer cells.8 c - myc is regulator gene , including apoptosis , transformation , metabolism and cell cycle progression . on the other hand , estrogen receptor - negative and progesterone receptor - positive p27 showed a positive correlation between low levels in breast cancer . endometriosis is the endometrial tissue in the uterus that should be present in the abdominal cavity outside the uterus,9 a common disease that occurs in approximately 10 to 15% woman of childbearing age . the cause of endometriosis , though not known as yet , regurgitation of menstrual blood , immunological factors , genetic factors and family history factors have been established occurrence of endometriosis . according to previous research compared to the healthy women were reported for endometriosis and endometriosis in infertile women , following studies have been reported to demonstrate the changes in the endometrium of women with disease.1011 moreover the before - mentioned transformation demanded the establishment of the endometrium in the peritoneal , recent evidence indicated that uterine mucosal cells of patients with endometriosis do not follow the normal growth pattern and apoptosis.121314 in the midst of cdk inhibitors , we should be focused on p27 . the critical function of p27 is to inhibit cdk - cyclin e complex by controlling a checkpoint in the g1 in normal cells . when p27 is not present in the cells , cells are not follow a cell cycle control signal and proliferation.15 because endometrial cell cycle changes may be involved in cell cycle regulation of endometriosis in women with diseases significance of p27 protein level it can be seen that a change in the lining of a particular cell cycle is important . ovarian cancer is malignant tumor , the most widely occurring between 50 to 70 years old woman.1617 the second most common cause of gynecologic cancer in succession cervical cancer has been identified as a family history , a lot of ovulation number . no proven method for ovarian cancer is often rapidly progressive and fatal disease early symptoms rarely.5 as a negative regulator of the cell cycle , p27 is tumor suppressor,6 which inhibits cyclin - cdk in a dosage dependent manner to control cell cycle progression.78 recently , decreased expression of p27 has been frequently detected in human cancers,5101112131415 including ovarian carcinoma . recently , the expression of jab1 and p27 in a malignant epithelial ovarian cancer , was found to be correlated with the reverse p27 expression levels as a result jab1 negative regulator.18 that is a negative regulator of p27 jab1 has been associated with the development of the ovarian tumor , progression and prognosis . jab1 is a protein that enhances the activity of the c - jun gene and induce cell proliferation , p27 is a strong reduction in the tumor suppressor gene p27 has a high resistance and poor prognosis in cancer . recently , jab1 and p27 has been reported that many women involved in diseases such as breast cancer , endometriosis and ovarian cancer . as well as jab1 and p27 started to reveal that the association became known widely in hepatocellular carcinoma , laryngeal carcinoma and nerves . in conclusion , a wide range of research on jab1 and p27 is expected to play a major role in reproductive system , kidney , blood vessel tissues and nerves system further physiological pathophysiological .

the retinal pigment epithelium ( rpe ) performs highly specialized metabolic and transport functions essential for homeostasis of the neural retina ( bok 1993 ) . these include phagocytosis of photoreceptor - shed outer segments , transport of nutrients into and removal of waste products from photoreceptor cells and retinoid transport and regeneration . the rpe is a low cuboidal epithelium containing very long thin and sheet - like microvilli on its apical surface that project into the interphotoreceptor matrix where they interact with the tips of the rod and cone photoreceptor outer segments ( bok 1993 ) . the apical surface of rpe cells supports and carries out the diurnal phagocytic removal of spent photoreceptor tips . one rpe cell supports 3050 photoreceptors , which shed daily ~5% of their outer segment mass ( zinn and benjamin - henkind 1979 ) . the basal surface of rpe cells displays highly convoluted basal infoldings that attach to a specialized bruch s basement membrane , an acellular layer separating the rpe from the choriocapillaris . the rpe s basal surface participates in extensive metabolic exchanges with the blood vessels in the underlying choriocapillaris . an accumulation of discrete but pronounced structural changes occurs in aging eyes . in the aged retina , an overall thinning is apparent , due to loss of neurons from all the neuronal cells and also shortening of photoreceptor cells . the rpe specifically is known to undergo several structural changes , including loss of melanin granules , increase in the number of residual bodies , accumulation of the age pigment lipofuscin , accumulation of basal deposits on or within bruch s membrane ( bm ) , formation of drusen ( between the basal lamina of the rpe and the inner collagenous layer of bm ) , thickening of bm , rpe microvilli atrophy and disorganization of basal infoldings ( boulton and dayhaw - barker 2001 ) . some of these changes are shown in figure 1 ( b , d , f , h , j ) and they will be discussed in detail in the following text . the rpe aging changes progress slowly and are of varying severity in different eyes . melanin is an insoluble high molecular weight polymer derived from the enzymatic oxidation of tyrosine and dihydroxyphenylalanine , linked to proteins and contained in membrane - limited granules in the rpe melanosomes . recently a comprehensive determination of the protein composition of melanosomes isolated from human melanoma cells was reported using proteomics ( chi et al 2006 ) . the identified proteins included 16 homologs to mouse coat color genes , many associated with human pigmentary diseases , pigment epithelium - derived factor ( pedf ) and slc24a5 ( sodium / potassium / calcium exchanger 5 , nckx5 ) . rpe melanin originates from neural ectoderm , whereas the one in melanocytes originates from neural crest ( feeney 1978 ) . in aged rpe cells melanin granules are frequently seen in association with lysosomes ( melanolysosomes , mll ) and lipofuscin granules ( melanolipofuscin , mlf ; figure 1h ) , which suggests that any protein bound to melanin may be degraded . in addition , melanosomes may undergo photobleaching with aging , which can diminish the antioxidant efficiency of melanin ( sarna et al 2003 ) . altogether , these observations suggest that changes in melanin granules possibly contribute to some of the senile changes evident in the rpe . a recent manuscript observed the accumulation of mlf in human rpe from different decades of life and assessed their phototoxicity to rpe cultures in vitro . the analysis of the composition of mlf granules suggested that , in contrast to lipofuscin , they do not contain photoreceptor - specific proteins . the authors suggest that mlf may not originate from photoreceptor outer segments phagocytosis but that mlf accumulates as a result of the melanosomal autophagocytosis of rpe cells ( warburton et al 2006 ) . accumulation of secondary lysosomes and residual bodies containing lipofuscin , known as dense bodies , has been observed in post - mitotic and intermitotic cells during aging ( schmucker and sachs 2002 ; morales et al 2004 ; kubasik - juraniec et al 2004 ) . the general consensus is that the accumulation of these dense bodies represents lysosomal aging and is a universal index of cellular senescence ( schmucker and sachs 2002 ; terman et al 2007 ) . it has been well established that the rpe has an extremely active lysosomal system capable of degrading thousands of phagocytosed outer segment disks per day ( young 1971 ; zinn and benjamin - henkind 1979 ) . the aged rpe accumulates indigested residues of this phagocytic process as residual bodies ( feeney - burns et al 1987 ) . lipofuscin pigment has been described as intracellular yellow - brown autofluorescent granules exhibiting sudanophilic , osmiophilic , argyrophilic and periodic acid - schiff - positive and acid - fast staining characteristics ( feeney 1978 ) . lipofuscin is a heterogeneous material composed of a mixture of lipids and different fluorescent compounds , the main fluorophore of which has been identified as the pyridinium bis - retinoid , n -retinylidene - n -retinylethanolamine ( a2e ) , a derivative of vitamin a. rpe lipofuscin is unique because it originates mainly from the phagocytosed photoreceptor outer segments as was demonstrated in early studies . for instance , analysis of the chemical composition of rpe cells revealed that it is different from the photoreceptor outer segments ( berman et al 1974 ) . in addition , investigations undertaken on the royal college of surgeons ( rcs ) rats showed that in this strain , which fails to phagocytose shed outer segments , lipofuscin is significantly diminished ( katz et al 1986 ; eldred and lasky 1993 ) . moreover , the accumulation of autofluorescent debris was observed in a transgenic mouse line expressing a mutated form of cathepsin d that is enzymatically inactive , thereby impairing the processing of phagocytosed photoreceptor outer segments by the rpe cells ( rakoczy et al 2002 ) . a recent study established the presence of extragranular material present in preparations of lipofuscin routinely isolated by sucrose density gradient centrifugation . in this study , the lipofuscin granules were isolated and further purified by digestion of the extragranular material with proteinase k or by wash with sds detergent . the results demonstrated that : debris - free granules contain little or no protein ; the protein associated with lipofuscin granules is essentially all extra - granular and appears to be significantly modified by posttranslational modifications ( renganathan et al 2007 ) . lipofuscin granules first appear in the basal portions of rpe cells of young eyes ( 1st decade , figure 1c ) , whereas in older eyes ( 9th decade , figure 1d ) , lipofuscin granules form into clumps and fill the entire rpe cell cytoplasm ( wing et al 1978 ) . it is suggested that the accumulation of lipofuscin in aged rpe is connected to rpe functional degeneration either by clogging of the cytoplasm or by increased oxidative stress in the cell . support for the first mechanism ( clogging of the cytoplasm ) comes from a recent study , which implanted glycoxidized microspheres ( glycox - ms ) as imitation for lipofuscin into the subretinal space of 1012 week - old rabbits . glycox - ms stagnated for a prolonged period in the cytoplasm of rpe cells and eyes implanted with glycox - ms produced drusen - like deposits at a significantly higher frequency ( yasukawa et al 2007 ) . support for the second mechanism ( increased oxidative stress in the rpe cells ) comes from observations that lipofuscin is a photoinducible generator of superoxide anion , singlet oxygen and hydrogen peroxide ( boulton et al 1993 ; gaillard et al 1995 ; rozanowska et al 1995 , 1998 ) . thus , visible - light irradiation ( 4001100 nm ) of lipofuscin granules results in extra - granular oxidation of lipids and inactivation of lysosomal and antioxidant enzymes ( wassel et al 1999 ) . in addition , it was shown that a2e has phototoxic and detergent properties and is capable of inducing disintegration of membrane - bound organelles in rpe cultures . finally , lipofuscin can also interfere with the antioxidant properties of melanin ( boulton et al 1993 ; rozanowska et al 1995 ; schutt et al 2001 , 2002 ; wang et al 2006 ) . the aged rpe displays increased intracellular accumulation of the blue - shifted autofluorescence lipofuscin granules , which coincides with the depletion of melanin pigments ( feeney - burns 1984 ; han et al 2007 ) . rpe lipofuscin granules exhibit a broad band emission spectrum with a peak at 600 nm and subsidiary shoulders located at 470 and 550 nm when excited at 364 nm ; a 680 peak appears with increasing age ( boulton et al 1990 ) . bruch s membrane ( bm ) is a pentalaminar structure composed of the rpe basement membrane , inner collagenous layer , middle elastic layer , outer collagenous layer , and the choroidal endothelial cell basement membrane ( hogan and alvarado 1967 ) . this acellular extracellular meshwork found between the rpe and the choroid , which is 24 m thick is known to undergo increased thickening ( figure 1j ) , chemical reconfiguration of both proteins and lipids , and debris accumulation during aging ( pauleikhoff et al 1990 ; okubo et al 1999 ; zarbin 2004 ) . the aged bm displays an exponential increase in phospholipids , triglycerides , fatty acids , and free cholesterol content ( sheraidah et al 1993 ) . protein reconfiguration in the form of post - translational modifications has been reported in the bm . immunore - activity to some of the advanced glycation end product ( age ) adducts increases in the aged bm ( farboud et al 1999 ; handa et al 1999 ) . although the precise contribution of ages to the retinal pathology remains to be elucidated , ages are recognized as important initiators of age - related dysfunction , inasmuch as they are known to cause protein cross - linking , reduced solubility , enzymatic dysfunction , and loss of receptor recognition ( baynes 2001 ) . a recent study combined both raman microscopy and specific chemical quantification to assess defined age adducts and quantify age - related spectral alterations in aged bm of postmortem eyes ( glenn et al 2007 ) . the analysis showed that the ages pentosidine , carboxymethyllysine ( cml ) , and carboxyethyl - lysine ( cel ) occurred at significantly higher levels in bm - ch with age ( glenn et al 2007 ) . in addition , several recent studies demonstrated that tissue metalloproteinase inhibitor 3 ( timp-3 ) , vitronectin , annexins , crystallins , clathrin and adaptin proteins were crosslinked as evidenced from western blots that showed the presence of these proteins at several regions of the gel ( nakata et al 2005 ; rayborn et al 2006 ; bando et al 2007 ) . in addition , bm is under constant cycles of pressure - induced stress as a result of the choroidal flow oscillating with the cardiac rhythm . specifically , the elastic properties of bm will determine its ability to sustain potentially damaging stress and strain perturbations . this study demonstrated that the elasticity of human bm - ch complex decreased linearly with aging after the age of 21 with an approximate reduction of 1% per year . on the other hand , the recoil capacity of bruch s membrane - choroid was not affected by aging ( ugarte et al 2006 ) . drusen are debris - like deposits that accumulate below the rpe along bm ( figure 1f ) . clinically , they are characterized by the terms hard and soft according to their size and their appearance in fluorescein angiography . hard drusen are small , hard , round and have well defined borders ( marshall et al 1998 ) . hard drusen occur in 80% of postmortem eyes , and are usually small , they are hyperfluorescent on fluorescein angiography , a characteristic that may be related to the fact that they are enriched in phospholipids ( bird and marshall 1986 ; pauleikhoff et al 1992 ; arnold et al 1997 ) . on the other hand , soft drusen are extensive , diffuse , large deposits , which have borders not sharply defined , and rarely occur before the age of 55 ( garner et al 1994 ; marshall et al 1998 ) . soft drusen are hypofluorescent in fluorescein angiography and display a high content of neutral fats ( pauleikhoff et al 1992 ; arnold et al 1997 ) , vesicles , membranous debris , and wide - spaced collagen . generally , hard drusen do not lead to loss of vision , but soft drusen are considered contributors to the pathology of age - related macular degeneration ( amd ) . soft drusen deposition in the macula precedes visual loss ; it defines the early stages of amd together with pigmentary changes of the rpe . deposition of soft drusen in the macula is considered the precursor lesion that leads to the development of geographic atrophy ( dry amd ) and choroidal neovascularization ( wet amd ) , which are the late forms of amd . a recent proteomic study carried out on isolated drusen from both amd and normal donors found up to 65% of the proteins identified common to both donor types . timp-3 , clusterin , vitronectin , and serum albumin were the most common proteins observed in normal donor drusen , whereas crystallin was detected more frequently in amd donor drusen . in addition , protein from both normal and amd donors such as vitronectin , timp-3 , clusterin , complement c9 , lysosyme c , serum amyloid p , and apolipoprotein e migrated in multiple mass ranges from the top to the bottom of the gel , suggesting the presence of covalent crosslinks ( crabb et al 2002 ) . recent work from several groups also suggests that local inflammation plays a role in drusen formation in a process analogous to that which occurs in other age - related diseases such as alzheimer s disease and atherosclerosis , in which there is an accumulation of extracellular plaques and deposits causing a local chronic inflammatory response which in turn exacerbates the effects of the primary stimuli ( hageman et al 2001 ; johnson et al 2001 ; anderson et al 2002 ) . this hypothesis is supported by evidence revealing the localization of several proteins involved in the immune system such as immunoglobulins ; components of complement cascade ( such as c5b-9 complex , complement factor f ) ; mhc class ii antigens ; cell - associated molecules , including hla - dr and specific cd antigens ( mullins et al 2000 ; hageman et al 2001 ; johnson et al 2001 ; anderson et al 2002 ) . a finding common to all of them was the shortening of the rpe microvilli ( katz and robison 1984 ; lai and rana 1986 ; weisse 1995 ) . previously , we have been able to isolate intact rpe microvilli from mice ( bonilha et al 2004 ) and characterize its content using proteomics . several of the identified proteins in the microvilli fraction are antioxidant enzymes and have been shown to undergo specific modulation during aging . these include lactate dehydrogenase , glutathione s - transferase , peroxiredoxin , ceruloplasmin , and superoxide dismutase . our data are supported by several reports , which identified the presence of antioxidative enzymes in the microvilli of kidney ( davies et al 1993 ; muse et al 1994 ) , respiratory tract epithelium ( coursin et al 1992 ) , and intestine ( davis et al 1989 ) , among others . oxidation is a very important mechanism in aging ( kohen and nyska 2002 ; balazy and nigam 2003 ; van remmen et al 2003 ; kregel and zhang 2007 ) . rpe apical microvilli shortening is also expected to affect several of the key functions carried out by the apical surface . examples of these include phagocytosis of shed photoreceptor outer segments through the receptors v5 vitronectin receptor and the mer tyrosine kinase ( mertk ) receptor protein ; apical transport involving transporters such as na , k - atpase , the glucose transporter ( glut-1 ) , monocarboxylate transporter 1 ( mct1 ) , basigin , the kir7.1 k+ channel , chloride intracellular channel 6 , carbonic anhydrase xiv , among others ; and visual cycle function through the involvement of cralbp , rpe65 , irbp , and crbp ( rayborn et al 2005 ) . these changes could alter the retinal metabolic equilibrium and accelerate degenerative processes in the aging retina . our ongoing research aims to identify a protein profile that is uniquely present in aged rpe cells . our findings will lead to future studies on the functional consequence of these proteins and to a more complete understanding of the pathogenesis of amd . a good animal model should reduplicate biochemical , morphological , and molecular changes shown in humans during aging . much of our understanding of the biological changes that occur with aging has come from studies using rodents . similarities in the physiology and cell biology of aging in humans and rodents make rodents a valuable model with which to test therapeutic interventions for aging , and they are small enough to allow for the use of statistically robust sample sizes . the national institute on aging ( nia ) supports many resources to facilitate the use of animal models for biogerontological research , including aged rodent colonies , the aged rodent tissue bank , and tissue arrays from aged rodents ( nadon 2006 ) . one of the rodent models available for aging studies is the rat f1 f344/bn hybrid . rat displays several of the rpe age - related changes described above ; specifically , bm thickening , lipofuscin accumulation , accumulation of residual bodies , decrease in rpe density and microvilli atrophy when compared to young ( 34 month - old ) rats ( figure 2 ) . however , previous studies yielded contradictory results : some found that rpe density increased with age ( tso and friedman 1968 ; harman et al 1997 ; leung et al 2004 ) . others found that it decreased ( streeten 1969 ; gao and hollyfield 1992 ; watzke et al 1993 ) ; while yet another study found that it did not change with age at all ( dorey et al 1989 ) . the discrepancies among these studies can be explained by the number of eyes analyzed and by the nature of the analyses which were carried out . use of cross - sections allows analysis of a restricted number of cells , whereas whole - mount preparations allows analysis of the whole population of cells in the tissue . one study ( gao and hollyfield 1992 ) investigated this issue analyzing eyes from donors from the 2nd to the 9th decade . tissue fragments were obtained and analyzed as whole - mounts from the fovea and the retinal equator . observations suggested that foveal rpe is denser , with cells smaller and more homogeneous , independent of the age of the donor , as shown in figure 3a and c. linear regression of the obtained data yielded a significant negative slope of rpe density in the retinal equator , suggesting a uniform rate of equatorial rpe loss during aging . on the other hand , foveal rpe density was relatively stable from the 2nd through the 9th decades , with no significant decrease in cell density ( gao and hollyfield 1992 ) . another study investigated the age - related changes in rpe in an even larger number of eyes from donors from the 2nd to the 9th decade ( panda - jones et al 1996 ) . using a 3 mm trephine the authors collected rpe / retina / choroid in the fovea and in the superior , inferior , temporal , and nasal meridians in 6 rings that were arranged concentrically around the fovea . as in the previous study , the authors concluded that rpe density at the foveal center was the highest and that it decreased significantly from the fovea to the mid - retinal periphery . in the periphery , the age - related loss was most marked in the fovea and the mid periphery . the authors determined that rpe cell density in the fovea decreased significantly by about 0.3% per year with increasing age . the elderly suffer from loss of visual acuity ( weale 1975 ; del viva and agostini 2007 ) , color perception ( ohta and kato 1975 ; page and crognale 2005 ) , and dark - adaptation sensitivity ( mcfarland et al 1960 ; werner 2005 ) . one study specifically addressed this issue by analyzing age - related rpe apoptosis through terminal deoxynucleotidyl transferase - mediated dutp nick end ( tunel)-labeling in whole - mount preparations of eyes divided into 4 concentric regions centered on the fovea . there was a significant positive correlation between the donor s age and the number of apoptotic cells . analysis of the individual regions revealed that within zone 1 ( 01.5 mm radius ) the proportion of apoptotic rpe cells started to increase in the 6th decade . there was also an age - dependent increase in apoptosis within zone 2 ( 1.53.0 mm radius , at a much lower number ) ( del priore et al 2002 ) . amd is the most common cause of irreversible blindness in the elderly population in industrialized countries ( leibowitz et al 1980 ; klein et al 1995 ; kelin et al 2004 ) . although aging is an important event that contributes to the pathogenesis of amd , it does not directly lead to the occurrence of amd ( sarks 1976 ; young 1987 ) . amd occurs in two forms : neovascular or exudative ( wet ) and atrophic ( dry ) amd . neovascular amd is characterized by abnormal growth of capillaries from the choroid into the bruch s membrane and rpe and by subsequent exudation of fluid , lipid , and blood . it results ultimately in a disciform scar in the macula and is responsible for severe , sudden visual loss ( holloway and verhoeff 1929 ; verhoeff and grossman 1937 ) . atrophic amd , also known as geographic atrophy ( ga ) is characterized by a progressing course leading to degeneration of rpe and photoreceptors . studies have shown that the atrophy initially tends to develop in the perifoveal area , while the fovea may be spared until later during the clinical course ( sarks et al 1988 ; sunness 1999 ) . ga is characterized by a loss of the outer neurosen - sory retina , the rpe , and the choriocapillaris ( figure 4 ) . the primary dysfunction and cell death of the rpe cells is thought to occur initially , followed by collateral loss of neighboring photoreceptor cells and choriocapillaris ( sarks et al 1988 ; roth et al 2004 ) . previous studies have shown that photoreceptor apoptosis is involved in amd pathology ( green and enger 1993 ; xu et al 1996 ) . recently , another study quantified the number of tunel - positive cells in each retinal layer in cryosections of amd and control eyes . the authors showed that maculas with amd had a statistically significant increase in tunel - positive rpe cells compared with the control ones . in the ga eyes , tunel - positive rods and rpe nuclei were present near the edges of rpe atrophy ( dunaief et al 2002 ) . additional data also suggest that a2e , a lipofuscin component , induces apoptosis in rpe cultures ( suter et al 2000 ) . finally , apoptosis was also observed in surgically excised choroidal neovascular ( cnv ) membranes from amd - affected eyes ( hinton et al 1998 ) . clinical features common in both types of amd include the presence of drusen and hypo- and hyperpigmentation of the rpe . histological features of rpe in amd include accumulation of lipofuscin , formation of drusen and of basal deposits in the bm , and alteration in the bm extracellular matrix ( hogan 1972 ; sarks 1976 ; young 1987 ; green and enger 1993 ; roth et al 2004 ; nowak 2006 ) . as mentioned above , these features amd is a multifactor disease with genetic components ( klaver and allikmets 2003 ; gold et al 2006 ; hageman et al 2006 ; scholl et al 2007 ) . however , exogenous factors such as light exposure , a high fat diet , high blood pressure , and smoking ( leibowitz et al 1980 ; cruickshanks et al 1993 ; cousins et al 2002 ) are known to modulate its pathogenesis . in addition , abnormal regulation of the complement system , likely caused by the y402h polymorphism in the complement factor h gene on 1q , is a recognized risk factor for amd , as is the a69s variant in the poorly characterized loc387715 gene and the serine protease htra1 in multiple populations ( edwards et al 2005 ; haines et al 2005 ; jakobsdottir et al 2005 ; klein et al 2005 ; rivera et al 2005 ; dewan et al 2006 ; yang et al 2006 ) . on the other hand , polymorphisms in the factor b and complement component 2 are associated with decreased susceptibility to amd ( gold et al 2006 ; spencer et al 2007 ) . previous studies had identified the genes abc4 , apoe , tlr4 , and fbln5 as being associated with susceptibility to amd . however , the fraction of amd patients carrying sequence changes in these genes was very small ( scholl et al 2007 ) . as mentioned above , complement activation has been implicated in susceptibility to amd , mainly through complement factor h. it is synthesized mainly in the liver and released into the blood where it is transported to other tissues . however , high levels of complement factor h are detected in the retina - choroid interface ( mandal and ayyagari 2006 ; chen et al 2007 ) . in addition , it was suggested that the rpe synthesizes complement factor h locally ( hageman et al 2005 ) . the rpe production of factor h would increase the local concentration of complement regulators and provide protection to inappropriate complement activation at sites of infection and inflammation ( rodriguez et al 2004 ) . this mechanism would be particularly important in the retina , as the complement factors supplied by the blood may be restricted through the blood - brain barrier ( mandal and ayyagari 2006 ) . several reports have shown that oxidative mechanisms constitute the initial stimulus that triggers apoptosis , thereby contributing to the progression of amd . the retina is highly susceptible to photo - oxidative damage due to its high oxygen demand , life - long exposure to light and the presence of poly - unsaturated fatty acids highly enriched in the photoreceptor outer segments ( beatty et al 2000 ; roth et al 2004 ) . this scenario is aggravated with age , for there is a reduction in the local antioxidative enzymes in the rpe and a decrease in macular pigment density , which serves not only as a filter for short - wavelength light , but also as an antioxidant , through its two constituents , lutein and zeaxanthin . the age - related increase in oxidative stress leads to cellular events which in turn induce the histopathological changes associated with amd , as described above . an animal model recently described the connection between oxidation , inflammation and pathology of amd . in this model , mice were injected with mouse serum albumin adducted with carboxyethylpyr - role , an oxidation fragment generated from the protoreceptor - enriched lipid docosahexaenoic acid . injected mice develop antibodies to this hapten , fix high amounts of complement component 3 in bruch s membrane , accumulate drusen below the rpe , and develop lesions in the rpe - photoreceptor interface that mimic amd ( hollyfield et al 2007 ) . the aged rpe is characterized by several structural changes , which are exacerbated in amd . these structural changes are known to be associated with an increase in oxidative stress and general decline of basic functions . recently , it became evident that rpe and choriocapillaris express many if not all of the components and regulators of the complement cascade . moreover , the rpe also plays an important role in the development of immune and inflammatory responses in the posterior part of the eye through production of cytokines . the connection between oxidation and immune system in aging will provide a powerful approach for the elucidation of the many senile degenerative macular and peripheral retinal diseases such as amd .

h. pylori eradication is strongly recommended in all patients with atrophic gastritis and peptic ulcer disease , but may also benefit subgroups of patients with dyspepsia , and patients who start with nsaid therapy [ 16 ] . h. pylori eradication therapy is an important component of guidelines concerning these patients [ 7 , 8 ] . currently , non - invasive management strategies and the widespread shortage in endoscopic capacity insure that many patients with h. pylori are managed without upper gastrointestinal endoscopy . the american college of gastroenterology recommends that when an endoscopy is not performed , a serological test , which is the least expensive means of evaluating for evidence of h. pylori infection , should be done . when endoscopy is indicated , biopsy specimens can be taken for microscopic demonstration of the organism , culture , histology or urease testing . nowadays , in the netherlands , biopsies are not routinely sent for culture and susceptibility testing of the infecting strain because of the high costs . apart from patient compliance , resistance of helicobacter pylori to antibiotics can decrease the success of h. pylori eradication therapy . regimens of choice for eradication of h. pylori should be guided by local antibiotic resistance rates . in the netherlands , the overall prevalence of resistance to clarithromycin and metronidazol was lower than in some surrounding countries possibly due to restrictive use of antimicrobials [ 1012 ] . the advised treatment in the netherlands consists of a proton pump inhibitor ( ppi)-triple therapy for 7 days without prior susceptibility testing . an increase of resistance rates to antimicrobial agents is however expected because increasing number of patients treated and increasing consumption of antibiotics , in particular macrolides , was observed in recent years . the aim of the present study was firstly , to determine the efficacy of 7-day ppi - triple therapy for h. pylori in a well - defined group of patients with a rheumatic disease and serologic evidence of h. pylori infection who were on long - term nsaid therapy and secondly , to get insight in the prevalence of antibiotic resistance of h. pylori in the studied population . this study was part of a placebo - controlled randomized clinical trial of which the clinical results have been described elsewhere , wherein we described that h. pylori eradication has no beneficial effect on the incidence of gastroduodenal ulcers or occurrence of dyspepsia in patients on long - term nsaid treatment . between may 2000 and june 2002 , patients with a rheumatic disease were eligible for inclusion if they were between 40 and 80 years of age , were positive for h. pylori on serological testing and were on long - term nsaid treatment . forty - eight percent used a gastroprotective drug ( 7% h2 receptor antagonists [ h2ra ] , 37% proton pump inhibitors [ ppi ] , 7% misoprostol , 3% used a combination of these ) . exclusion criteria were previous eradication therapy for h. pylori , known allergy for the study medication or presence of severe concomitant disease . serologic testing for h. pylori igg - antibodies was performed with a commercial enzyme - linked immunosorbent assay ( pyloriset new eia - g , orion diagnostica , espoo , finland ) according to the manufacturer s instructions . a serum sample was considered positive for igg antibodies to h. pylori if the test result was 250 international units ( iu ) . this assay has been assessed , in a population similar to the population in the presented trial , and has proven a sensitivity and specificity in the netherlands of 98100% and 7985% , even in patients on acid suppressive therapy [ 1517 ] . the study protocol was approved by research and medical ethics committees of all participating centers and all patients gave written informed consent . after stratification by concurrent use of gastroprotective agents ( proton pump inhibitors , h2 receptor antagonists or misoprostol , but not prokinetics , or antacids ) , patients were randomly assigned to receive either h. pylori eradication therapy with omeprazole 20 mg , amoxicillin 1000 mg , and clarithromycin 500 mg ( oac ) twice daily for 7 days or placebo . patients with an allergy for amoxicillin were treated with omeprazole 20 mg , metronidazole 500 mg and clarithromycin 250 mg ( omc ) or placebo therapy twice daily for one week in a distinct stratum . all study personnel and participants were blinded to treatment assignment for the duration of the study . the study protocol was approved by research and medical ethics committees of all participating centres and all patients gave written informed consent . at the 2-week follow - up visit , unused study medication was returned and remaining tablets were counted in order to check compliance . patients were considered to be noncompliant if 6 days ( 85% ) of study medication were used . three months after baseline , and additionally if clinically indicated , patients underwent endoscopy of the upper gastrointestinal tract . four samples , two from the antrum and two from the corpus were used for histology . the slides were scored independently by an experienced gastrointestinal pathologist and the investigator ( hdl ) , blinded to treatment assignment and clinical data , according to the updated sydney classification . in case of discrepant results , the remaining four biopsies were sent to a microbiological laboratory for culture and storage at 70c . a patient was considered h. pylori - negative when histology as well as culture was negative . all isolated strains were assessed for susceptibility to clarithromycin , metronidazole , tetracycline and amoxicillin at the central laboratory . both biopsy specimens of corpus and antrum were streaked on columbia agar ( ca ) ( becton dickinson , cockeysville , md , usa ) with 10% lysed horse blood ( bio trading , mijdrecht , the netherlands ) , referred to as columbia agar plates , and on ca with h. pylori selective supplement ( oxoid , basingstoke , uk ) . plates were incubated for 72 h at 37c in a micro - aerophilic atmosphere ( 5% o2 , 10% co2 , 85% n2 ) . identification was carried out by gram s stain morphology , catalase , oxidase , and urea hydrolysis measurements . mics of metronidazole , clarithromycin , tetracycline and amoxicillin were determined by e - test ( ab biodisk , solna , sweden ) on ca plates essentially as described by glupczynski et al . . ca plates were inoculated with a bacterial suspension with a turbidity of a 3 mcfarland standard ( 2 10 cfu / ml ) . clsi ( tentative ) breakpoints 2009 for susceptibility ( s ) and resistance ( r ) were applied ( metronidazole mic 8 mg / l ( s ) and 16 mg / l ( r ) , amoxicillin mic 0.5 mg / l ( s ) and 2 mg / l ( r ) ; tetracycline mic 2 mg / l ( s ) and 8 mg / l ( r ) , and clarithromycin mic 0.25 mg / l ( s ) and 1 mg / l ( r ) ) . measurements with a gaussian distribution were expressed at baseline as mean and sd , and measures with a non - gaussian distribution were expressed as the median and interquartile range ( iqr ; expressed as the net result of 75th percentile25th percentile ) . an additional analysis compared outcomes ( presence of h. pylori after h. pylori eradication therapy or placebo ) between stratum ( patients on gastroprotective drugs ( n = 165 ) and not on gastroprotective drugs ( n = 182 ) by computing the homogeneity of the common odds ratio . differences in the proportions of patients with susceptible and resistant h. pylori strains and for compliant and non - compliant patients were analyzed with 95% confidence interval using the confidence interval analysis ( cia ) software for windows ( version 2.2.0 ) . the level of significance was set at p < 0.05 , two sided . a total of 347 patients consented to be randomly assigned to eradication therapy ( 172 patients ) or placebo ( 175 patients ) . pylori igg antibodies were present in all patients ( median titre 1689 [ iqr 700 - 3732 ] ) . the treatment groups were similar in terms of demographic , rheumatic disease , nsaid and other drug use . our eligibility criteria resulted in a study group with mainly inflammatory rheumatic diseases ( rheumatoid arthritis 61% , spondylarthropathy 8% , psoriatic arthritis 7% , osteoarthritis 9% , other 15% ) . the most commonly used nsaids were diclofenac ( 29% ) , naproxen ( 18% ) , and ibuprofen ( 13% ) , most at full therapeutic doses ( median relative daily dose 1 [ iqr 0.51 ] ) . the mean age was 60 years ( sd 10 ) , 61% was female . twenty - two patients had a known allergy for amoxicillin and received metronidazole instead ( 10 patients ) or placebo ( 12 patients ) . of these 347 patients , data on culture and histology of 304 patients were available ( table 1 ) . in two cases only culture data were available and in one case only histology result was available ; all three cases met the criteria for h. pylori - positivity and were found in the placebo group , but for clarity purposes were left out of table 1 . a total of 32 patients ( with no significant differences between eradication and placebo groups ) refused the 3-month endoscopy , withdrew informed consent , or could not undergo endoscopy because of adverse events . seven patients used anticoagulant therapy ruling out biopsy sampling according the protocol , and in one patient no biopsy specimens could be obtained because of discomfort requiring early completion of the procedure . table 1results of culture and histology on h. pyloricultureeradication group ( n = 152)placebo group ( n = 152)histologytotal patients , n = 304h . pylori negative9 ( 6%)132 ( 87%)22 ( 15%)32 ( 21% ) results of culture and histology on h. pylori at follow - up after 3 months , 79% ( 120 /152 ; 95% ci 7285% ) of the patients in the placebo group were h. pylori - positive by histology or culture of biopsy specimen . in the eradication group , this number was 13% ( 20/152 ; 95% ci 920% ) ( table 1 ) . patients in the placebo group who were h. pylori negative at 3 months as assessed by culture and histology had significantly lower titers of h. pylori anti igg antibodies at baseline than those who were h. pylori culture- and or histology - positive ( mean difference 1582 , 95% ci 2637 to 527 , p = 0.004 ) . there were no differences between strata according to the use of gastroprotective drugs for the presence of h. pylori by culture and or histology ( p = 0.454 ) . compliance was 89% in patients in the eradication group and 98% in the placebo group with the assigned regimen ( p < 0.001 ) . in the eradication group , h. pylori could not be demonstrated in 91% of patients with full compliance ( n = 136 ) . in patients who did not take all 7 days of eradication therapy ( n = 16 ) , h. pylori was found in 50% ( difference of 41% ; 95% ci 1863% ) . a total of 105 clinical isolates of h. pylori were available for susceptibility testing ( one isolate per patient ; 95 isolates from the placebo group , ten from the eradication group ) from the six participating laboratories in the netherlands . table 2antibiotic resistance of h. pylori isolatesantibioticresistance ratesplacebo group , n = 95eradication group , n = 10clarithromycin4%20%metronidazole19%30%tetracycline2%0amoxicillin1%0all patients in the eradication group who were still h. pylori positive were assigned oac ( omeprazole 20 mg , amoxicillin 1000 mg , and clarithromycin 500 mg ) and not omc ( omeprazole 20 mg , metronidazole 500 mg and clarithromycin 250 mg)intermediate susceptible antibiotic resistance of h. pylori isolates all patients in the eradication group who were still h. pylori positive were assigned oac ( omeprazole 20 mg , amoxicillin 1000 mg , and clarithromycin 500 mg ) and not omc ( omeprazole 20 mg , metronidazole 500 mg and clarithromycin 250 mg ) intermediate susceptible in the placebo group ( n = 95 ) , resistance was found in 4% ( 4/95 ) to clarithromycin ( mic 1 mg / l ) , in 1% ( 1/95 ) and 2% ( 2/95 ) intermediate susceptibility to amoxicillin ( mic 1 mg / l ) and tetracycline ( mic 4 mg / l ) , respectively , and in 19% ( 18/95 ) resistance to metronidazole . amongst these 95 isolates two were resistant to metronidazole in combination with intermediate susceptible to tetracycline , and one strain was resistant to metronidazole and clarithromycin . the placebo group had an mic90 for clarithromycin of 0.085 mg / l , for metronidazole > 256 mg / l , for tetracycline 0.341 one h. pylori strain was resistant to clarithromycin and metronidazole , and intermediate susceptible to tetracycline and amoxicillin . no difference was found in h. pylori resistance rates between men and women ( p = 0.217 ) or between patients who used gastroprotective agents and who those did not ( p = 0.25 ) . in the eradication group , two strains were resistant to clarithromycin and three to metronidazole . a total of 105 clinical isolates of h. pylori were available for susceptibility testing ( one isolate per patient ; 95 isolates from the placebo group , ten from the eradication group ) from the six participating laboratories in the netherlands . table 2antibiotic resistance of h. pylori isolatesantibioticresistance ratesplacebo group , n = 95eradication group , n = 10clarithromycin4%20%metronidazole19%30%tetracycline2%0amoxicillin1%0all patients in the eradication group who were still h. pylori positive were assigned oac ( omeprazole 20 mg , amoxicillin 1000 mg , and clarithromycin 500 mg ) and not omc ( omeprazole 20 mg , metronidazole 500 mg and clarithromycin 250 mg)intermediate susceptible antibiotic resistance of h. pylori isolates all patients in the eradication group who were still h. pylori positive were assigned oac ( omeprazole 20 mg , amoxicillin 1000 mg , and clarithromycin 500 mg ) and not omc ( omeprazole 20 mg , metronidazole 500 mg and clarithromycin 250 mg ) intermediate susceptible in the placebo group ( n = 95 ) , resistance was found in 4% ( 4/95 ) to clarithromycin ( mic 1 mg / l ) , in 1% ( 1/95 ) and 2% ( 2/95 ) intermediate susceptibility to amoxicillin ( mic 1 mg / l ) and tetracycline ( mic 4 mg / l ) , respectively , and in 19% ( 18/95 ) resistance to metronidazole . amongst these 95 isolates two were resistant to metronidazole in combination with intermediate susceptible to tetracycline , and one strain was resistant to metronidazole and clarithromycin . the placebo group had an mic90 for clarithromycin of 0.085 mg / l , for metronidazole > 256 mg / l , for tetracycline 0.341 mg one h. pylori strain was resistant to clarithromycin and metronidazole , and intermediate susceptible to tetracycline and amoxicillin . no difference was found in h. pylori resistance rates between men and women ( p = 0.217 ) or between patients who used gastroprotective agents and who those did not ( p = 0.25 ) . in the eradication group , two strains were resistant to clarithromycin and three to metronidazole . we report the results of a study on the efficacy of a test and treat strategy for h. pylori in rheumatology patients of the netherlands who were positive for anti - h . the main findings in the studied patient population were : ( 1 ) a 7-day ppi - triple eradication therapy either with clarithromycin or metronidazole was efficacious with eradication rates of 87% ( 95% ci 8091% ) without prior testing for susceptibility of the infecting strain ; ( 2 ) in 21% of the patients in the placebo group , the positive h. pylori serology test could not be confirmed by positive culture or histology ; ( 3 ) compliance was an important factor for successful eradication of h. pylori ; and ( 4 ) prevalence of antibiotic resistance in h. pylori was low . the main reason for not performing endoscopy at baseline was that this was not feasible in everyday rheumatology practice ; therefore , serology was done to test for h. pylori . the reliability of serological kits for h. pylori infection has been widely confirmed , contributing to the reputation of serology as a simple , minimally invasive and inexpensive diagnostic and screening test . the best available serology test at the time of the study was the pyloriset an eia - g from orion diagnostica , espoo , finland , with a specificity of 7991% as assessed in previous studies in the netherlands , including patients on acid suppressive therapy [ 2224 ] . this specificity correlates well with our finding that in the placebo group , h. pylori could not be confirmed by culture or histology in 21% of the igg positive patients . ppi usage ( in this study 37% of the population ) can result in false negative invasive and non - invasive diagnostic tests , such as culture , histology and 13-c urea breath , and should be stopped two weeks before testing . 13-c urea breath tests have better accuracy ( > 90% ) , but the serology test used in this study was less expensive and in all study centres easily available . on the other hand , we must not overlook that conditions during transport of biopsies are critical for successful isolation of h. pylori . possibly , the antibacterial effect of nsaids , as has been suggested in in vitro studies , might also partly explain a false positive rate of serology of 21% [ 2729 ] . however , in a randomized clinical trial of 122 patients , aspirin in combination with a standard 7-day course oac eradication was not significantly different compared to the standard therapy . based on culture and histology findings we conclude that one fifth of our patients were treated superfluously , with possible risk of side - effects of the eradication medication . resistance to antibiotics in h. pylori is of particular concern because it is one of the major determinants in the failure of eradication regimens . resistance rates for metronidazole and clarithromycin found in this study were similar as previously observed in other studies in the netherlands in the years 199798 and 19972002 [ 11 , 12 , 30 , 31 ] . to our knowledge , there are no recent data available on h. pylori antibiotic primary resistance rates in the netherlands . in addition , in this study , compliance played a crucial role in success of eradication of h. pylori , i.e. , treatment failure was as high as 50% in the non compliant group of patients . possibly , the high number of tablets that has to be consumed during h. pylori eradication therapy is a contributing factor for non - compliance in this group of elderly patients who were also on other medications . in conclusion , serology driven test and treat strategy eradication of h. pylori with a 7-day ppi - triple therapy is successful in the majority of patients . success of eradication is , also in this group of rheumatology patients , to a great extent determined by compliance .

kikuchi - fujimoto disease ( kfd ) is a rare , benign , self - limiting cervical lymphadenitis . the skin manifestations of our case mimicked systemic lupus erythematosus ( sle ) , but immunofluorescence and serology were negative for it . a paucity of reports in literature with unusual skin manifestations has prompted us to report this case . a 30-year - old female presented with intermittent high - grade fever , weakness , pain in knee , wrist , and ankle joints , oral ulcers , erythematous rashes on face and upper chest , hemorrhagic crusting on lips and multiple , firm , tender enlarged bilateral cervical lymph nodes distributed mainly in the posterior cervical region of 2-month duration . multiple , variable size maculopapular lesions on face with hemorrhagic crusting on lips routine hemogram showed anemia , thrombocytopenia , and mild leukocytosis . antinuclear antibody and anti - double - stranded dna ( dsdna ) antibody were negative for sle . mantoux test , widal , rapid dengue antibody test for igm and igg , rapid malaria test for plasmodium vivax , hepatitis b surface antigen , hepatitis c virus , human immunodeficiency virus 1 ( hiv-1 ) , venereal disease research laboratory , rheumatoid factor , and chest x - ray were normal . histopathological evaluation of left cervical lymph node revealed lymph nodal tissue with paracortical , well - circumscribed necrotic lesions , with karyorrhectic debris , and fibrin deposits . there was a proliferation of phagocytic foamy histiocytes and infiltration of plasmacytoid monocytes , histiocytes , and lymphocytes surrounding karyorrhectic debris [ figures 2 and 3 ] . no follicular hyperplasia and no atypia were noted . staining for acid - fast bacilli was also negative . proliferation of phagocytic foamy histiocytes and infiltration of plasmacytoid monocytes ( h and e , 10 ) plasmacytoid monocytes , histiocytes , and lymphocytes surrounding karyorrhectic debris ( h and e , 100 ) the patient was treated with antibiotics , nonsteroidal anti - inflammatory drugs , and oral steroids for 10 days after which the lymph nodes regressed and other symptoms subsided . kfd , also known as histiocytic necrotizing lymphadenitis , was initially described by both kikuchi and fujimoto independently in 1972 . although it has been reported worldwide , it still remains a poorly recognized clinicopathological entity and is confused with lymphoma and sle . barr virus , hiv , human herpes virus 6 , human t - cell lymphotropic virus , dengue virus , and cytomegalovirus . yersinia enterocolitica and other bacterial agents such as bartonella and brucella have also been implicated . kfd most often presents with cervical lymphadenopathy which may be tender and can be accompanied by fever , upper respiratory tract symptoms . less common symptoms include arthralgia , skin rashes , weakness , weight loss , diarrhea , anorexia , chills , nausea , and vomiting . about 30% of patients with kfd these cutaneous manifestations can be nonspecific and can present as morbilliform and rubella - like eruptions , reddish plaques which resemble lymphoma , erythematous , and acneiform eruptions on face other nonspecific finding includes thrombocytopenia and pancytopenia , and 25% of patient may present with atypical blood lymphocytes . diagnosis of kfd is based on histopathological findings of lymph node biopsy as no specific diagnostic laboratory tests are available . biopsy of lymph node shows focal or complete loss of follicular architecture associated with necrosed cortical and paracortical areas . the extensive infiltrate consists of immunoblast cells , small lymphocytes , macrophages , and so - called plasmacytoid t - cells . clinically , kfd may resemble sle or lymphoma ( especially t - cell non - hodgkins lymphoma ) as both these diseases can present with lymphadenopathy and fever . furthermore , the skin lesions of kfd can mimic those seen in sle . according to some literature a histological feature that helps in the differentiation of kfd from sle is almost total absence of plasma cells in the involved nodal tissue in kfd . neutrophils are also rarely seen in kfd , which is also important differentiating feature from sle . moreover , diagnostic serologic and immunofluorescence tests can differentiate sle as antinuclear and anti - dsdna antibodies were done in our patient , and they were negative . histological feature that helps in the differentiation of kfd from lymphoma includes incomplete architectural effacement with patent sinuses , presence of numerous reactive histiocytes , relatively low mitotic rates , and absence of reed sternberg cells [ table 1 ] . histopathological comparison of kikuchi - fujimoto disease , systemic lupus erythematosus , and lymphoma no specific treatment is available for kfd . in supportive treatment , nonsteroidal anti - inflammatory drugs may be used to alleviate lymph node tenderness and fever . the disease runs a benign course usually , and natural history is for spontaneous resolution in several weeks to months . one should remain alert to the possibility of kfd when a young female patient presents with complaints of developing neck lymphadenopathy . skin as well as clinical manifestation of kfd may mimic other systemic disease , specially lymphoma and sle so careful histopathological examination has importance at clinching the diagnosis in such cases . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . certain skin features , such as the erythematous facial lesions , hemorrhagic crusting on lips , and oral ulcers , as evidenced in our patient , should be considered possible cutaneous manifestations of kikuchi - fujimoto disease . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . certain skin features , such as the erythematous facial lesions , hemorrhagic crusting on lips , and oral ulcers , as evidenced in our patient , should be considered possible cutaneous manifestations of kikuchi - fujimoto disease . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . certain skin features , such as the erythematous facial lesions , hemorrhagic crusting on lips , and oral ulcers , as evidenced in our patient , should be considered possible cutaneous manifestations of kikuchi - fujimoto disease .

the methods of recruiting young men into the thai military have been described previously ( 3,4 ) . approximately 50,00060,000 men , mostly 21 years of age , are selected each year by lottery in their home province ( the province in which they are listed on the house registration ) . the system produces a representative national sampling of thai men . because of their young age , hiv prevalence in these annual recruit classes may serve as a proxy for hiv incidence . induction into the military occurs in may ( m ) and november ( n ) of each year . at the time when blood is collected , recruits provide information about the location of their main residence ( including province and district ) during the previous 2 years ( 3 ) . although the actual locations where infections occurred are unknown , residential data enables analysis of the association between hiv prevalence and this key geographic marker . to refine the hiv prevalence analyses , geographic localization uses districts as the first administrative subunit of provinces . when data were analyzed by using annual classes grouped at the district level , calculations of the percentage testing positive for hiv were statistically unreliable because the number of men tested in some rural districts was so small . therefore , we merged data in two ways to decrease variability attributable to the small sample size of the prevalence figures : classes were combined across time , and districts were combined across space . sixteen classes of men recruited from 1991 to 2000 were combined temporally into four larger classes , each representing discrete 2-year periods : 1 ) n91 , m92 , n92 , m93 ; 2 ) n94 , m95 , n95 , m96 ; 3 ) n96 , m97 , n97 , m98 ; and 4 ) n98 , m99 , n99 , m00 . data on classes recruited from the m91 , n93 , m94 , and n00 lotteries were not available for analysis ( m91 , before full implementation ; n93 and m94 , protocol under revision ; n00 , completed after dataset closed ) . however , even after combining classes into these 2-year periods , a number of districts still had numbers too low for statistical reliability . we also merged some districts with neighboring districts so that each had a minimum denominator of 20 in the hiv prevalence calculation . numbers of > 20 persons provide minimal , but acceptable , reliability in the percentage - positive calculations . districts with < 20 were combined with other districts according to the following protocol , following a sequence of priorities : we combined districts if they were in the same province , had historic connections ( formerly part of single larger district ) , had similarly small numbers tested , had similar demographics , and had similar topography or other geographic features . for the gis analysis , data tables provided in excel files ( microsoft corp . , redmond , wa ) by the armed forces research institute of medical sciences were joined to district - level gis maps obtained from the thai environmental institute and national statistical office . we used arcview 3.2a software ( esri , redlands , ca ) to create dot density and choropleth maps . ( a choropleth map uses shades or colors to demonstrate the geographic distribution of a range of values . ) each of the 10,043 dots represents a recruit who tested positive for hiv infection ( positive for hiv-1 by enzyme immunoassay and western blot ) among the 442,923 recruits tested . arcview software uses an algorithm that allows the random placement of dots within a geographic area ( in this case a district ) . by creating these dots at the district level but presenting them at the province level , we show the vivid geographic pattern of infection while illustrating the human impact of the epidemic . nevertheless , these absolute numbers provide important information regarding the potential hiv - related impact on the healthcare system and various other social structures . dot density map of young men who tested positive for hiv at time of entry into the royal thai army , thailand , november 1991may 2000 . the evolution of the hiv epidemic over the course of the decade is seen in the four maps shown in figure 2 . these maps use gradients of color shading to distinguish the percentage of men who tested positive for hiv during each 2-year period . the initial high prevalence of hiv in men from the upper north region is illustrated , as is the decline in prevalence over time ( which likely occurred as a result of public health interventions ) . the relative persistence of hiv prevalence in some districts in the far south of the country is also shown , suggesting that locale - specific features of the epidemic need to be well understood in that area , potentially leading to changes in public health interventions . choropleth maps of hiv prevalence in four classes of young men at time of entry into the royal thai army , thailand , 19912000 . prevalence is stratified by color and localized to district or group of districts so that calculations are based on > 20 men . previous analyses conducted at the province level identified regional variation quite clearly , but at a less detailed level ( 3,4 ) . the maps from our study indicate considerable variation even to the district level of analysis , which suggests important local factors may be at work in determining rates of transmission . the hiv epidemic in thailand seems to have largely missed some districts and devastated others , even within the same province . chiang mai and chiang rai provinces are examples of this phenomenon . these maps also show that , although the growth of the epidemic has slowed in most parts of the country , the epidemic has not decreased in some districts in south thailand . both types of imagery are required to describe and understand the epidemic and to suggest locales in which public health initiatives are needed . in areas where the intensity of the epidemic is highest , health and social services may require additional resources to serve persons with the disease . in areas where the prevalence rates are highest or are increasing , socially and culturally appropriate interventions these gis maps are the most accurate maps available to date and are built upon the unique hiv prevalence datasets collected over a decade by the royal thai army . more sophisticated methods of multivariate gis visualization could enhance this analysis even more but would require the royal thai army to assess recruits at induction on other variables , especially risk factors , or to use recruits home addresses rather than districts or provinces of residence . nonetheless , changes in time and place of chronic viral infection can be added to the recognized areas of gis application in epidemiology , transmission of vector - borne and water - borne diseases , and environmental health ( 7 ) . the maps also illustrate the point that political borders may have no relationship to epidemiologic boundaries . differences in hiv prevalence often occur , not only between regions and provinces but also within provinces and across provincial borders . when an epidemic can be visualized at this level of detail and accuracy , researchers can formulate and address questions regarding the bases of these differences . at the same time , policy makers can better direct intervention strategies and more finely assess the outcomes of these interventions . coordination of data collection and joint gis analysis by neighboring countries would enhance regional understanding of the emergence and spread of hiv epidemics and monitoring of control programs .

sensitive skin , also designated as hyperreactive skin , is a common condition.1 according to epidemiological studies performed in france,2 the uk,3 and the us,4 more than 50% of the population experience sensitive skin . this condition is characterized by symptoms such as pricking , burning , pain , a tight sensation , and pruritus . in some subjects visible signs of inflammation are present . environmental factors like ultraviolet radiation , heat , cold , wind , or pollution can trigger this condition as can stress and cosmetics . after wounding and injury , skin is extremely sensitive and irritable during the healing phase until regular barrier function is reestablished.5,6 although initially described in the facial area , sensitive skin can also occur in other regions of the body , with the scalp being among the most affected.5,7 scalp sensitivity is mainly provoked by the use of inappropriate shampoos.7 the sensitive scalp reacts with irritation to harsh surfactants or other additives such as certain perfumes or colorants which can be components of shampoos . to successfully treat a sensitive scalp , an effective shampoo needs to comprise an extra mild surfactant system to protect the natural skin barrier and lipid system . finally , the shampoo needs to be free of potentially irritating additives . against this background , a special shampoo for the sensitive and problematic scalp was developed , based on an extremely mild surfactant system and containing bisabolol , an anti - irritant and anti - inflammatory ingredient of chamomile . the shampoo is free of additives such as perfumes , silicones , colorants , parabens , paraffins , and betaine . since skin can remain hyperreactive after wounding or postoperatively,6 we selected a challenging model system to test this new shampoo formulation : the status after hair transplantation ( figure 1 ) . especially after hair transplant , gentle but efficient cleansing of the scalp starting 2 days after the operation is essential to prevent crusting and graft dislodgment in patients to ensure a good surgical result.8,9 in this study , we investigated the efficacy and compatibility of a rinse - off shampoo specifically developed for the sensitive scalp . following hair transplantation , regular use of the test shampoo , starting 2 days after surgery until stitch removal , resulted in a significant reduction in the extent of scabbing and erythema . data showed an excellent skin compatibility and product efficacy of the test shampoo , especially for the specific requirements after hair transplant . additionally , the plastic surgeons highly recommended the further use of the test shampoo after hair transplant . the test shampoo utilized in the course of the study ( eucerin dermocapillaire hypertolerant shampoo , beiersdorf ag , hamburg , germany ) contained decyl glucoside , sodium myreth sulfate , peg-8 , peg-200 hydrogenated glyceryl palmate , disodium peg-5 laurylcitrate sulfosuccinate , peg-90 glyceryl isostearate , and bisabolol . forty - five subjects ( 42 male and three female ; 2065 years ) qualified for study participation after a previous hair transplantation , which was performed at the moser medical , clinics for aesthetic and plastic surgery ( vienna , austria ) . the aim of this study was to determine the effects of the newly developed shampoo for use in patients after hair transplantation . in this application study , the recommendations of the current version of the declaration of helsinki and the guidelines of the international conference on harmonization good clinical practice ( ich gcp ) were observed as applicable to a nondrug study . medical control of the dermatological status of the study subjects was performed by trained personnel of the moser medical , clinics for aesthetic and plastic surgery . the study investigators monitored subjects for unexpected skin reactions and surveyed possible subjective perceptions of discomfort . also , all subjects were recruited by the moser medical , clinics for aesthetic and plastic surgery . approximately 2 days after hair transplant , volunteers started to use the test shampoo and continued until their stitches were removed ( between 7 and 23 days ) . they were instructed to utilize a realistic amount of shampoo and to wash their scalp at least once a day with it . during the study , subjects were required to refrain from using other shampoos as well as other scalp treatments . the test area was the scalp , in particular , the area where hair follicles were transplanted . directly after hair transplant ( t1 ) and also after removal of stitches ( t2 ) , the plastic surgeons carried out dermatological scalp examinations with respect to the severity of scabs and extent of erythema using the following 5-point rating scale : 0= none , 1= slight , 2= moderate , 3= substantial , 4= extreme . also , at stitch removal , scalp compatibility in every subject was assessed based on the following 5-point rating scale : 0= very poor ; 1= poor ; 2= moderate ; 3= good ; 4= very good . after stitch removal , the plastic surgeons were asked to answer the question : from a dermatological point of view , would you further recommend this product to this patient ? with a yes or no . at the time of stitch removal , volunteers completed a self - assessment questionnaire to rate shampoo efficacy and scalp compatibility based on the following 8-point rating scale : 0= can not be assessed , 1= does not apply at all , 2= does not apply , 3= does rather not apply , 4= neutral , 5= rather applies , 6= applies , 7= applies completely . particularly , subjects established the agreement with the following statements : ( a ) the test shampoo has a fine and creamy foam , ( b ) the test shampoo has a good cleansing performance , ( c ) the test shampoo can easily be washed out , ( d ) the test shampoo cares for my hair , ( e ) the test shampoo is suitable for use after hair transplant , ( f ) the test shampoo gently removes scabs , ( g ) the test shampoo calms the scalp , ( h ) the test shampoo alleviates itching , ( i ) the test shampoo does not have a burning sensation on the scalp , ( j ) the test shampoo is particularly mild and gentle to the scalp , and ( k ) the test shampoo is particularly skin tolerable . a significance level of 0.05 ( ) was chosen for statistical analysis , based on two - sided hypothesis testing . for analysis , microsoft excel 2007 ( microsoft corporation , redmond , wa , usa ) and the sas software ( sas institute inc . , cary , nc , usa ) package for windows v9.2 were used . the dermatological assessment regarding scabs and erythema was conducted using a comparison among points in time employing the wilcoxon s signed rank test . questionnaires were evaluated using a test for relevance of the inquired statement utilizing a binomial test for null hypothesis proportion p0=0.5 . for analysis of self - assessment , absolute and relative frequencies were represented by tables and graphs based on the original data . the relevance of the inquired statements was tested using a binominal test for null hypothesis proportion p0=0.5 . due to rounding errors , the test shampoo utilized in the course of the study ( eucerin dermocapillaire hypertolerant shampoo , beiersdorf ag , hamburg , germany ) contained decyl glucoside , sodium myreth sulfate , peg-8 , peg-200 hydrogenated glyceryl palmate , disodium peg-5 laurylcitrate sulfosuccinate , peg-90 glyceryl isostearate , and bisabolol . forty - five subjects ( 42 male and three female ; 2065 years ) qualified for study participation after a previous hair transplantation , which was performed at the moser medical , clinics for aesthetic and plastic surgery ( vienna , austria ) . the aim of this study was to determine the effects of the newly developed shampoo for use in patients after hair transplantation . in this application study , the recommendations of the current version of the declaration of helsinki and the guidelines of the international conference on harmonization good clinical practice ( ich gcp ) were observed as applicable to a nondrug study . medical control of the dermatological status of the study subjects was performed by trained personnel of the moser medical , clinics for aesthetic and plastic surgery . the study investigators monitored subjects for unexpected skin reactions and surveyed possible subjective perceptions of discomfort . also , all subjects were recruited by the moser medical , clinics for aesthetic and plastic surgery . approximately 2 days after hair transplant , volunteers started to use the test shampoo and continued until their stitches were removed ( between 7 and 23 days ) . they were instructed to utilize a realistic amount of shampoo and to wash their scalp at least once a day with it . during the study , subjects were required to refrain from using other shampoos as well as other scalp treatments . the test area was the scalp , in particular , the area where hair follicles were transplanted . directly after hair transplant ( t1 ) and also after removal of stitches ( t2 ) , the plastic surgeons carried out dermatological scalp examinations with respect to the severity of scabs and extent of erythema using the following 5-point rating scale : 0= none , 1= slight , 2= moderate , 3= substantial , 4= extreme . also , at stitch removal , scalp compatibility in every subject was assessed based on the following 5-point rating scale : 0= very poor ; 1= poor ; 2= moderate ; 3= good ; 4= very good . after stitch removal , the plastic surgeons were asked to answer the question : from a dermatological point of view , would you further recommend this product to this patient ? with a yes or no . at the time of stitch removal , volunteers completed a self - assessment questionnaire to rate shampoo efficacy and scalp compatibility based on the following 8-point rating scale : 0= can not be assessed , 1= does not apply at all , 2= does not apply , 3= does rather not apply , 4= neutral , 5= rather applies , 6= applies , 7= applies completely . particularly , subjects established the agreement with the following statements : ( a ) the test shampoo has a fine and creamy foam , ( b ) the test shampoo has a good cleansing performance , ( c ) the test shampoo can easily be washed out , ( d ) the test shampoo cares for my hair , ( e ) the test shampoo is suitable for use after hair transplant , ( f ) the test shampoo gently removes scabs , ( g ) the test shampoo calms the scalp , ( h ) the test shampoo alleviates itching , ( i ) the test shampoo does not have a burning sensation on the scalp , ( j ) the test shampoo is particularly mild and gentle to the scalp , and ( k ) a significance level of 0.05 ( ) was chosen for statistical analysis , based on two - sided hypothesis testing . for analysis , microsoft excel 2007 ( microsoft corporation , redmond , wa , usa ) and the sas software ( sas institute inc . , cary , nc , usa ) package for windows v9.2 were used . the dermatological assessment regarding scabs and erythema was conducted using a comparison among points in time employing the wilcoxon s signed rank test . questionnaires were evaluated using a test for relevance of the inquired statement utilizing a binomial test for null hypothesis proportion p0=0.5 . for analysis of self - assessment , absolute and relative frequencies were represented by tables and graphs based on the original data . the relevance of the inquired statements was tested using a binominal test for null hypothesis proportion p0=0.5 . due to rounding errors , figure 2 illustrates that immediately after surgery , the surgeon assessed the severity of scabs as 1.670.64 . when stitches were removed , this value was significantly decreased to 1.000.64 ( p<0.001 ) . postoperatively , the extent of erythema was rated as 1.640.68 . following removal of stitches , this score value had significantly declined to 0.130.40 ( p<0.001 ) . in all subjects , the extent of scabbing as well as the extent of erythema was significantly lower during the removal of stitches than immediately after surgery . when stitches were removed , the plastic surgeon assessed the scalp compatibility of the test shampoo as good to very good ( 97% ; table 1 ) . also , based on dermatological scalp examination , the plastic surgeon recommended further use of the test shampoo after hair transplant to all study participants ( 100% ; p<0.001 ) . to assess the efficacy of the test shampoo after hair transplant , subjects completed a dermatological questionnaire at the time of stitch removal ( figure 3a and b ) . an answer belonging to category 04 was valued as no agreement and an answer belonging to category 57 was ranked as agreement : applies . two self - assessment questionnaires were lacking isolated values because these volunteers did not answer all questions . in the self - assessments , the volunteers significantly confirmed the reduction of itching , a scalp - calming effect , good product mildness , and cleansing performance as well as good hair care effects after shampoo treatment . with respect to the application of the shampoo , no incompatibility reactions were reported in volunteers self - assessments . figure 2 illustrates that immediately after surgery , the surgeon assessed the severity of scabs as 1.670.64 . when stitches were removed , this value was significantly decreased to 1.000.64 ( p<0.001 ) . postoperatively , the extent of erythema was rated as 1.640.68 . following removal of stitches , this score value had significantly declined to 0.130.40 ( p<0.001 ) . in all subjects , the extent of scabbing as well as the extent of erythema was significantly lower during the removal of stitches than immediately after surgery . when stitches were removed , the plastic surgeon assessed the scalp compatibility of the test shampoo as good to very good ( 97% ; table 1 ) . also , based on dermatological scalp examination , the plastic surgeon recommended further use of the test shampoo after hair transplant to all study participants ( 100% ; p<0.001 ) . to assess the efficacy of the test shampoo after hair transplant , subjects completed a dermatological questionnaire at the time of stitch removal ( figure 3a and b ) . an answer belonging to category 04 was valued as no agreement and an answer belonging to category 57 was ranked as two self - assessment questionnaires were lacking isolated values because these volunteers did not answer all questions . in the self - assessments , the volunteers significantly confirmed the reduction of itching , a scalp - calming effect , good product mildness , and cleansing performance as well as good hair care effects after shampoo treatment . with respect to the application of the shampoo , no incompatibility reactions were reported in volunteers self - assessments . sensitive skin is a common condition of the scalp , and the most common symptoms associated with this condition are pruritus ( approximately 60% ) and prickling ( approximately 30%).5,10 misery et al10 report that the incidence of a self - declared hyperreactive scalp is increasing with increasing age . the authors speculate that this could be due to an age - related loss of nerve endings or a chronic use of irritants present in regularly applied shampoos . however , the underlying pathophysiology of sensitive skin is still unclear . a tendency to barrier impairment and an increased permeability of the stratum corneum accordingly , skin hyperreactivity to water - soluble irritants could be induced by a larger quantity of irritants that penetrate the skin.1,11 nonspecific inflammation may also be associated with the release of prostaglandin e2.12 as a treatment strategy for sensitive skin , the use of well - tolerated cosmetics or the use of cosmetics with soothing effects has been recommended.13 in this context , a new shampoo for the treatment of sensitive scalp was developed containing an extremely mild surfactant system for a gentle cleansing to avoid irritation . additionally , skin soothing bisabolol , a sesquiterpene alcohol found in chamomile , was incorporated into the formulation.14 this active compound has been demonstrated to decrease proinflammatory cytokine production.15 also , in experimental models of acute dermatitis , this agent exerted topical anti - inflammatory effects . furthermore , bisabolol may be effective in the treatment of skin inflammatory disorders such as atopic or contact dermatitis as well aspsoriasis.16 finally , the test shampoo was free of irritating additives . since symptoms of a hyperreactive scalp are primarily of a subjective nature , may vary , and can be triggered by a multitude of factors,8 it is not easy to establish uniform clinical conditions for the test of such shampoos . therefore , we chose the status after hair transplant as a highly challenging model system to test this new shampoo . this technique is currently considered the gold standard in hair transplantation . in the course of fut , a large section of the scalp tissue is removed for follicular unit harvest , grafts are dissected and stored , the recipient area is prepared with slits , and finally , grafts are positioned . especially after hair transplantation , the scalp is very sensitive and needs special care to avoid graft dislodgment and to ensure a good surgical result in patients . gentle but efficient shampooing of the scalp to prevent crusting , starting 2 days postoperation , done once per day is highly recommended.8,9 considering these prerequisites , the new shampoo reduced scab and erythema . this correlation was proven by comparison of the dermatological ratings of the plastic surgeon directly after hair transplant and at the time of stitch removal . it is crucial to gently remove scabs since avoidance of crusting reduces the occurrence of graft dislodgment during the first 7 days after hair transplant . also , adherent scab removal at 25 days after the operation may result in lost grafts.17 although the scalp is well vascularized and , hence , serious infections are scarce , they can occur with insufficient hygiene or elevated crust formation.18 the study results indicate that the use of the test shampoo facilitates the healing process and soothes the scalp . at stitch removal , the plastic surgeon performed a dermatological scalp examination and assessed the skin compatibility of the test shampoo as good to very good . the test shampoo was very well - tolerated by the study population over the entire duration of usage . this finding was further corroborated by the fact that the surgeon recommended the further use of the test shampoo after hair transplant to all study participants ( 100% ) . since symptoms of scalp sensitivity are predominantly subjective , volunteers were asked to fill out questionnaires when stitches were removed to rate various efficacies and scalp compatibility statements . they stated examples with significant agreement that the shampoo gently removes scabs and alleviates itching . both characteristics are extremely important after hair transplantation to ensure a smooth healing process and a satisfactory outcome of the transplantation . in conclusion , our data show that the application of the newly developed shampoo containing an extremely mild surfactant system and the calming ingredient bisabolol to the sensitive scalp after hair transplant results in excellent skin compatibility and product efficacy . also , the test shampoo signifi - cantly reduces the extent of scabs and erythema . therefore , it is ideally suited for use after hair transplant and also for general treatment of sensitive scalp .

fumonisins are a group of mycotoxins that are produced by fusarium verticillioides , f. proliferatum , f. nygamai and alternaria alternate f. sp . fumonisins cause serious diseases such as equine leukoencephalomalacia ( elem ) , porcine pulmonary edema ( ppe ) and hydrothorax in pigs , liver cancer in rats and esophageal cancer in humans and neural tube defects in infants . international agency for research on cancer ( iarc ) has listed fumonisins as a category 2b carcinogen ( 4 ) . the recommended maximum level of fumonisins in human foods is 24 ppm according to food and drug administration ( fda ) and iranian maximum tolerated level is 1 mg / kg ( = ppm ) for total fumonisins ( 2 , 3 ) . this toxin is produced in corn , wheat , sorghum , asparagus , rice , beer and beans ( 3 ) . aflatoxins are most toxic mycotoxins and can be found in peanuts , nuts , copra , soya , corn , rice , cotton , grain , wheat , grains , fruits and red pepper ( 1 , 4 , 5 ) . the main aflatoxins include b1 , b2 , g1 and g2 that are produced by aspergillus flavus , a. parasiticus and a. nomius . afm1 and afm2 are oxidative metabolites of afb1 and afb2 and are produced by liver microsomal enzymes of some species of mammals such as dairy cattle that consume contamination feedstuffs with aflatoxins ( 6 , 7 ) . this toxin listed in group i carcinogen by iacr ( 8) . the recommended maximum level of aflatoxins in human foods is 4 ppb according to european community and codex alimentarius ( 9 ) . iranian maximum tolerated level is 15 ng / g ( = ppb ) for total aflatoxin ( 8) . of 51 maize samples that was collected from four provinces in iran ( fars , kermanshah , khuzestan , mazandaran ) , afb1 was detected in 80% of the maize samples obtained from mazandaran provinces ( 5.67 4.75 g / kg ) ( 10 ) . in other study of 70 samples of flour and wheat that were tested , 17 samples ( 24.28% ) had too much pollution with aflatoxin and ochratoxin ( 11 ) . elisa method in human food samples showed that 2% of samples were contaminated with fumonisin and 3% were contaminated with aflatoxin ( 12 ) . of 43 cereal samples in india , 21(48% ) samples were contaminated with total fumonisin ( 13 ) . since mazandaran region provide a very good conditions for the growth of fungi and occurrence of mycotoxins such as fumonisin and aflatoxin have a important role in human health and since biscuit and cookie are extensively used among all ages of humans , purpose of this study was the analysis of total aflatoxin and total fumonisin in biscuit and cookie samples in babol city ( mazandaran province , northern iran ) . in this study , 30 biscuit ( n=15 ) and cookie ( n=15 ) samples were randomly collected from supermarkets in babol city in winter 2011 and total aflatoxin ( b1 , b2 , g1 and g2 ) and total fumonisin ( b1 , b2 and b3 ) contamination were analyzed in this samples separately ( due high price of kits and lack of supporting of institution and easy access to kits and economic problems , we had confined ourselves to 30 samples ) . samples were grounded to powder and then 20 g of each sample was mixed with 100 cc of 70% methanol in blender and then was shaken in erlenmeyer flask continuously for 3 min . after settled , the extract was filtered with whatman no . 1 ( 3 ) . contamination in the samples was measured in mycology laboratory of islamic azad university , babol branch in iran using competitive elisa method and agraquant total aflatoxin and total fumonisin assay kits ( taken from romer singapore company ) . therefore , 200 l of enzyme - conjugated solutions were added to uncoated- antibody microplate wells and then 100 l of each standard solutions and samples extract were added to it . of these 100 l solutions were transferred to coated - antibody microplate wells and were incubated at room temperature in 2025c for 10 min . toxins in samples and control standards competed with enzyme conjugate for binding to solid phase antibody . after washing step , 100l of enzyme substrate was added to wells and incubated at room temperature for 5 min . then 100 l of stop solution was added to wells and blue color changed into yellow . toxin concentration in the samples was compared with standard concentrations and absorption by using of a standard curve . then information was analyzed by analysis of variance ( anova ) by using of spss software package ( p<0.05 ) . in this study , 30 biscuit ( n=15 ) and cookie ( n=15 ) samples were randomly collected from supermarkets in babol city in winter 2011 and total aflatoxin ( b1 , b2 , g1 and g2 ) and total fumonisin ( b1 , b2 and b3 ) contamination were analyzed in this samples separately ( due high price of kits and lack of supporting of institution and easy access to kits and economic problems , we had confined ourselves to 30 samples ) . samples were grounded to powder and then 20 g of each sample was mixed with 100 cc of 70% methanol in blender and then was shaken in erlenmeyer flask continuously for 3 min . after settled , the extract was filtered with whatman no . 1 ( 3 ) . contamination in the samples was measured in mycology laboratory of islamic azad university , babol branch in iran using competitive elisa method and agraquant total aflatoxin and total fumonisin assay kits ( taken from romer singapore company ) . therefore , 200 l of enzyme - conjugated solutions were added to uncoated- antibody microplate wells and then 100 l of each standard solutions and samples extract were added to it . of these 100 l solutions were transferred to coated - antibody microplate wells and were incubated at room temperature in 2025c for 10 min . toxins in samples and control standards competed with enzyme conjugate for binding to solid phase antibody . after washing step , 100l of enzyme substrate was added to wells and incubated at room temperature for 5 min . then 100 l of stop solution was added to wells and blue color changed into yellow . toxin concentration in the samples was compared with standard concentrations and absorption by using of a standard curve . then information was analyzed by analysis of variance ( anova ) by using of spss software package ( p<0.05 ) . out of 15 biscuit samples , 14 ( 93.4% ) were contaminated with total fumonisin with < 2ppm . the highest contamination rate of total fumonisin was 2.3 ppm in biscuit samples ( mean se : 0.67 0.16 ) . also 14 ( 93.4% ) of 15 cookie samples were contaminated with total fumonisin < 2 ppm and only 1 ( 6.6% ) sample was infected with 24 ppm of this toxin and such as biscuit samples , none of the cookie samples were contaminated with > 4 ppm of this toxin and highest contamination rate in samples was 2.1 ppm for cookie samples ( mean se : 0.71 0.17 ) . out of 30 biscuit and cookie samples 28 ( 93.4% ) were contaminated with < 2 ppm of total fumonisin and 2 ( 6.6% ) showed infection with 24 ppm of this toxin . the highest contamination rate among the all biscuit samples was 2.3 ppm that was higher than iran standard institute ( mean se : 0.69 0.12 ) . despite the contamination of samples , there was no contamination higher than the standard limit of eu . ( acceptable limits of total fumonisin are 24 ppm and 1 mg / kg according to who , institute of standards , and industrial research of iran ( isiri ) respectively ) ( table 1 ) . also of 15 biscuit samples 13 ( 86.7% ) were contaminated with < 4 ppb of total aflatoxin and 2 ( 13.3% ) were contaminated with 4 ppb that were more than acceptable limit in eu . the highest level of aflatoxin contamination in biscuits was 7.9 ppb ( mean se : 1.65 0.57 ) . in 15 cookie samples 13 ( 86.7% ) were contaminated with < 4 ppb of total aflatoxin and 2 ( 13.3% ) were positive for contamination ( 4 ppb ) that were more than acceptable limit in eu and highest rate of infection was 4.1 ppb with this toxin in cookies ( mean se : 1.45 0.33 ) . from 30 samples 26 ( 86.7% ) were contaminated with < 4 ppb of total aflatoxin and 4 ( 13.3% ) were positive for contamination with this toxin . the highest contamination rate was found 7.9 ppb in biscuit samples ( mean se : 1.55 0.32 ) . however , there was no contamination higher than the standard limit of iran . ( acceptable limit of total aflatoxin is 4ppb and 15 ng / g according to codex alimentarius and isiri , respectively ) ( table 2 ) . in addition , there was a significant relationship between biscuit samples and contamination ( p<0.05 ) . fungal metabolites have toxic , mutagenic and teratogenic effects and have a considerable role in human health ( 8) . fumonisins are mycotoxins mainly produced by fungi of the genus fusarium and have been shown to be hepatocarcinogenic and nephrocarcinogenic in animals . aflatoxins are a group of mycotoxins produced by strains of aspergillus and are most potent hepatocarcinogen in mammals . these toxins contaminated agricultural and food products especially cereals and cereal products ( 8 , 14 ) . a study in malaysia by elisa method in grains samples showed that from 80 samples 100% were contaminated with fb1(15 ) . in our study out of 15 biscuit samples 93.4% were contaminated with total fumonisin < 2 ppm and 6.6% showed 24 ppm of contamination . 93.4% cookie samples were contaminated with total fumonisin < 2 ppm and only 6.6% sample was infected with 24 ppm of this toxin . in iran ( mazandaran province ) in 42 samples of wheat flour , 7.1% were contaminated with total fumonisin ( 04.5 ppm ) ( 3 ) . in other study from 38 corn samples , fb1 was between 1.1912.95 mg / kg ( 14 ) . in 11 corn grain samples , all of the samples were contaminated with fb1 ( 12703980 ng / g ) , fb2 ( 1901175 ng / g ) and fb3 ( 155960 ng / g ) ( 16 ) . different reasons such as environmental factors including different geographical areas , temperature , humidity and rainfall prior to and during preharvest and harvest periods influence on levels of fumonisin might be involved ( 17 ) . a research conducted by using of liquid chromatography - mass spectrometry ( lc - ms ) method on bread made of wheat flour and corn at portuguese bakeries showed that of 80 samples 24 ( 30% ) were contaminated with fb1 ( 0448 g / g ) , 25 ( 30.1% ) with fb2 ( 0270 g / g ) and other types of fumonisin ( 0550 g / g ) ( 4 ) . in our study , contamination in biscuit samples was higher than contamination in cookie samples . fumonisin was reduced after cooking extrusion at 190 c for 60 min approximately 5070% ( 18 , 19 ) . in addition , hygienic condition in the bakeries and confectioneries and workers health in these environments can affect on the levels of contamination in different foods ( 3 ) . also from 15 biscuit samples , 86.7% were contaminated with < 4 ppb of total aflatoxin and 13.3% were contaminated with 4 ppb , which were more than acceptable limit . the highest level of aflatoxin in biscuits was 7.9 ppb . in 15 cookie samples , 86.7% were contaminated with < 4ppb of total aflatoxin and 13.3% were positive for contamination ( 4ppb ) . of 30 samples , 26 samples were contaminated with < 4ppb of total aflatoxin and 4 were positive for contamination with 4ppb of total aflatoxin and highest contamination was founded 7.9ppb in biscuit samples and was higher than the eu standard . researchers in croatia by elisa showed that one of six wheat samples was infected by total aflatoxin ( 02.6 g / kg ) and total fumonisin was not detected in samples ( 20 ) . in our study none of the samples was infected by > 4ppm of total fumonisin but total aflatoxin was higher than total fumonisin . in iran from 35 pre - harvest maize samples , incidence of afb1 , afb2 and total aflatoxins in maize samples were 66% , 54% and 63% with mean of 9.5 16.3 , 1.7 2.6 and 10.4 18.4 ng / g , respectively ( 21 ) . physical properties of samples and presence of different species spores and toxin - producer species can affect on the type and percent of fungi and toxins in our study samples in compared to other studies ( 1 , 22 , 23 ) . in different cereal processed products such as wheat - based crisp bread , total aflatoxin contamination in our samples was 86.7% and is comparable with this study . in pakistan in 70 samples of processed foods such as biscuit ( 10 samples ) , afb1 was observed in 3 samples of biscuits ( 0.31 0.01 g / kg ) ( 25 ) . in 200 wheat flour samples in iran , total aflatoxin levels of samples were 0.82 and 1.99 ng / g in summer and winter , respectively . afb1 levels were detected in 3.1% , 7.4% over permissible limits by worldwide regulations in samples collected in summer and winter , respectively ( 8) . different months for analyzes make different amounts of toxin in the samples ( 3 , 15 , 24 ) . for example , aflatoxin contamination rate in cold months is higher than hot months or fusarium species are not capable to survive in the low oxygen and low ph in environment ( 2527 ) . food commodities are often contaminated with more than one mycotoxin , because mold species produced different mycotoxins at the same time ( 24 ) . some mycotoxins can be produced by more than one mold species or some fungal species are capable to produce several types of mycotoxins ( 28 ) . different analytic methods that are used by researchers to analyze mycotoxins are another reason for discrepancy between the results of different researches . in result , in end , by using of different methods in agriculture and protection of farm products in harvest or after harvest , storage and packaging food , applying fungicides and hygienic condition can reduce amount of fungi growth and mycotoxin in the environment ( 3 , 30 ) . none of the samples was contaminated by > 4ppm of total fumonisin and 13.3% samples were positive for total aflatoxin with 4ppb . fumonisin and aflatoxin contamination in biscuit and cookie is harmful for humans health , so toxicology , prevention and control of these toxins should be considered . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

malignant mixed mullerian tumor ( mmmt ) is a very rare tumor , accounting for less than 1% of all ovarian cancers . we present a 64-year - old woman with stage iii mmmt of ovary that was treated with platinum - based chemotherapy after optimal cytoreductive surgery . after 25 months of being disease free , she had a pelvic recurrence and a good response to chemoradiotherapy . optimal cytoreductive surgery and chemotherapy may be the best treatment in mmmt but more discussion and experiences are needed regarding the effectiveness of radiotherapy . malignant mixed mullerian tumor ( mmmt ) of ovary is a rare tumor , accounting for less than 1% of all ovarian cancers , that most commonly occurs in postmenopausal women with low parity ( 1 ) . it is a biphasic tumor , composed of both epithelial and stromal components ( 2 ) . known risk factors for mmmt include obesity , nulliparity , exogenous estrogen , and long - term tamoxifen use ( 3 ) . stage of disease is the most important survival - determining factor in patients with mmmt ( 4 ) . although there is no standard regimen for chemotherapy , it is advised in patients with stage ii or higher ( 1 ) . , we introduce a case of prolonged survival of a patient with pelvic recurrence of ovarian mmmt after chemoradiotherapy . in january 2007 , we admitted a 64-year - old woman with acute abdominal pain , nausea and vomiting in ghaem hospital , mashhad university of medical sciences , iran . she had low appetite and inability in passing stool and gas in the recent week . on physical examination , she had a distended abdomen and on abdominal radiography , there were multiple air - fluid levels , suspected of bowel obstruction . at laparotomy with a large midline incision , there was adhesion of omentum and bowels on a large heterogeneous pelvic mass . cytoreductive surgery by transabdominal hysterectomy and bilateral salpingoopherectomy ( tah + bso ) and complete surgical staging was done . histopathologic examination of hematoxylin - eosin stained tissue samples revealed mmmt in right ovary ( figures 1 and 2 ) with metastasis to the left adnexa and omentum . according to the international federation of gynecology and obstetrics ( figo ) staging system ( 5 ) , the patient was in stage iiia . the serum ca-125 was elevated ( 386 iu / ml ) but the level of cea and ca19 - 9 were within normal limits ( 3.5 iu / ml and 9 iu / ml , respectively ) . the patient received six courses of chemotherapy consisting of carboplatin 6auc and paclitaxel ( 270 mg / m ) regimen every three weeks . after six courses of chemotherapy , abdominopelvic computed tomography ( ct ) findings were normal and there was no evidence of residual tumor in the pelvic cavity . the patient was followed up every three months by pelvic examination , abdominal and pelvic sonography , and ca-125 tumor marker . after 31-month disease - free survival , a nodule of about 1.5-cm diameter was palpated in presacral region on rectal and vaginal examination . in july 2010 , a round , hypoechoic solid mass of 16 18 mm at the posterior right part of the rectum was found in transvaginal ultrasonography . magnetic resonance imaging confirmed the existence of a mass of 17-mm diameter , compatible with a lymph node . we recommended transrectal ultrasound - guided biopsy but the patient did not accept . in may 2011 , a ct scan showed persist lymphadenopathy of 21 11 mm in the presacral region . in june 2011 and after 42 months from the primary surgery , a ct - guided biopsy of the presacral mass was performed and histopathologic examination revealed a metastatic carcinoma . the patient underwent chemotherapy with primary regimen again ( carboplatin 6 auc and 270-mg / mpaclitaxel ) every three weeks and 50.4 gy radiotherapy in 28 fractions to a standard pelvic box field with photon of 15 mv . after 26 months follow - up , she became progression free with normal abdominopelvic ct findings and normal levels of tumor marker ( ca-125 ) ( table 1 ) . mmmt is a rare and very aggressive cancer of female genital tract , seen mostly in the uterus and very rarely on the ovaries ( ovarian carcinosarcoma , ocs ) ( 6 ) . on histopathologic examination , these tumors are characterized by a benign or atypical epithelial component and an invariably , low - grade , malignant stromal component ( 7 ) . the median age at diagnosis of ocs is more than that of epithelial ovarian cancer ( eoc ) ( 8) . risk factors for mmmt include obesity , nulliparity , exogenous estrogen , and long - term tamoxifen use ( 3 ) . the survival for both early - stage and late - stage carcinosarcoma is shorter than serous tumors ( 9 ) . we have presented a 64-year - old woman with none of the known risk factors who was diagnosed with stage iii ovarian mmmt . the pelvic recurrence was diagnosed 42 months later and was successfully treated by chemotherapy and radiotherapy . at the time of writing the report , she was tumor free for 24 months , which was a predominantly good survival for a metastatic case of mmt . in the most recent reports , platinum - based chemotherapy is recommended following primary surgical debulking ( 10 , 11 ) . however , there is controversial about the effectiveness of adjuvant radiotherapy ( 12 ) . our patient was treated with the platinum - based chemotherapy as well . in a review article that was published in 2011 , it was believed that the benefits of radiation therapy in the treatment of ocs lacks enough data and is mostly based on reports ; in addition , the role of this treatment modality remains unknown and only useful in single , isolated pelvic recurrence ( 13 ) . the most common site of distant recurrence was the peritoneal cavity and they reported that adjuvant pelvic radiotherapy after surgery would decrease the risk of pelvic recurrence and might delay the appearance of distant metastasis ( 14 ) . reported that radiotherapy may be appropriate for patients with chemotherapy refractory , recurrent , or persisted disease restricted to the pelvic ( 15 ) . in our patient , the pelvic recurrence was successfully treated by pelvic radiotherapy and she remained tumor free for 26 months . there is also controversy regarding the role and prognostic significance of tumor markers ( cea , ca125 , ca14 - 4 , ca15 - 3 ) ( 16 ) . in a retrospective study , it was reported that tumor markers had no significant role ( 13 ) . there are still different results in the literatures that consider ca125 as a useful marker for following therapy in ovarian mmmt ( 17 ) . in our patient , ca125 was useful in follow - up and had significant rise after recurrence and decreased after successful treatment . some studies have reported that the presence of heterologous sarcomatous elements is associated with a poor prognosis ( 18 , 19 ) . in our patient with relatively long survival tumor , the pathology report was high - grade mmm with homologous predominance of spindle cells sarcomatous component ( fibrosarcoma ) ( figure 2 ) ; the carcinomatous component was glandular ( adenocarcinoma ) . ovarian mmmt is very rare and there is significant controversy over the treatment modalities , especially regarding the role of radiotherapy and the best time for its prescription . we have reported a patient with stage iii ocs who was treated with complete surgery and platinum - based chemotherapy and then with chemoradiotherapy after recurrence .

pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage . pain of dental extraction produces moderate to severe pain which is routinely treated with nonsteroidal anti - inflammatory drugs ( nsaids ) for 23 days.[24 ] the nsaids have the advantage of being analgesic as well as anti - inflammatory and are therefore the rational choice for pain associated with inflammation . at the same time the nsaid 's are liable to produce gastrointestinal adverse effects such as gastritis and peptic ulcer and thus should be used cautiously . analgesics such as tramadol , which are non - nsaids , can be tried on such patients . dental extraction pain is an excellent clinical model for acute pain , especially third molar extraction pain . this study was designed to compare the analgesic efficacy of ketorolac with tramadol for pre and postoperative pain relief as well as their side effect in patients undergoing dental extraction . this prospective randomized , placebo - controlled study was conducted on patients for 1 year ( jan 2010jan 2011 ) attending the dental outpatient department of teerthanker mahaveer medical college and research centre , moradabad , up . seventy four patients undergoing tooth extraction were recruited according to the inclusion and exclusion criterion . they were divided into six groups , i.e. , to receive tramadol , ketorolac , or placebo pre or postoperatively . patients with history of acid peptic diseases , hemorrhagic diathesis , known hypersensitivity of nsaids or tramadol , and epileptic patients were excluded . a total of 74 patients were recruited during the study , 25 each in the tramadol and ketorolac groups and 24 in the placebo group . both the investigator and patient were blind . each drug was coded and packed into identical appearing packets , with only the patient number and whether to be given pre- or postoperatively indicated on the label . drugs were given by the dental surgeon per orally ( po ) as per table 1 . analgesic drugs administration protocol a verbal rating scale ( vrs ) was chosen to grade pain for this study because it is very reliable and easy to administer . the first assessment of the pain intensity / relief was done half an hour after the end of the procedure . the 2 , 3 , and 4 assessments done at 2 , 4 , and 6 h postextraction . we devised a modified vrs for grading pain , taking into consideration the use of a rescue analgesic ( ibuprofen 400 mg ) , and it is shown in table 2 . modified verbal rating scale used as placebo is included in this study , it was ethically incorrect not to include the rescue analgesic pain scores of patients receiving same drug preoperatively or postoperatively as well as other drug groups were compared using mann - whitney 's u test . age of patients , the amount of rescue medication taken , and time after extraction when rescue medication was taken were compared by anova followed by post - hoc dunnett 's t3 test . number of males and females in different groups were compared by chi - square test , p value . adverse effects were compared using chi - square test or fischer 's exact test as appropriate . out of the 74 patients recruited , 12 patients each were in preoperative group for tramadol , ketorolac , and placebo group ; 13 patients each in postoperative group of tramadol and ketorolac ; and 12 patients were in postoperative group of placebo . the mean age of the patients in the study was 31.57 years , ranging from 1865 years , with 92% of the patients in the 2545 year age group . the mean dose of the local anesthetic was 2.4 ml ranging from 2 to 8 ml . the mean duration of the procedure was 15.93 min , ranging from 2 to 70 min . for 59 patients ( 78% ) it took less than 20 min for the completion of the procedure . for two patients ( 1.5% ) , it took 70 min . the indications for the third molar extractions were impacted teeth , infected teeth , or both . of the 74 patients , 51 patients ( 69% ) had impacted teeth , 7 patients ( 9.5% ) had infected teeth , and 16 patients ( 21.5% ) had both . all the six patients group were similar in terms of gender distribution , average age , the amount of local anesthetic administered , the antibiotic coverage given , the position of the molar extracted , and the duration of the procedure . the results of the analysis of the primary end point namely mean pain scores show that the analgesics ( ketorolac and tramadol ) were more efficacious in reducing the pain as compared to placebo [ figure 1 ] . mean combined pain score for tramadol plus ketorolac versus placebo at 30 min was 1.261 and 1.312 , respectively , and at 6 h was 2.132 and 2.884 , respectively . when the pain scores of analgesic group were compared against those of the placebo group separately , the analgesic group was found to have lower maximum pain scores at each time point as compared to placebo , indicating that the analgesics were superior to placebo in producing pain relief . change in mean pain scores ( preoperative and the postoperative group combined ) for tramadol , ketorolac and placebo over the 6 hour study period . ( x - axis time , y - axis pain scores ) on comparison of the pain scores of the preoperative group versus the postoperative group of tramadol , postoperative administration was found to reduce the pain score more than preoperative administration at each time point . mean pain score for preoperative tramadol and postoperative tramadol at 30 min was 1.4 and 1.1 , respectively , and at 6 h it was 2.42 and 1.875 , respectively [ figure 1 ] . when preoperative and postoperative ketorolac were compared , there was no significant difference between the maximum pain score ( mean pain score for preoperative ketorolac and postoperative ketorolac at 30 min was 1 and 1.545 , respectively , and at 6 h it was 2.416 and 1.818 , respectively [ figure 1 ] . when tramadol was compared with placebo ( both preoperative and postoperative ) , the analgesic group needed less rescue drug than the placebo group . the average time taken by the patient from the start of the procedure to the time when pain was severe enough for the patient to feel the need to self administer the rescue analgesic was more in the tramadol than in the placebo group , which indicates that more prolonged pain relief was seen in the analgesic group as compared to those receiving placebo ( meantime for rescue analgesic in the tramadol and ketorolac group 366 min , in the placebo group 240 min [ figure 2 ] . on comparison of preoperative placebo with preoperative tramadol , there appeared to be no significant difference in need for rescue analgesic . p = 0.241 ( mean time for rescue in the preoperative placebo group 245 min and mean time for rescue in the preoperative tramadol group 313 min ) . however , on comparison of preoperative placebo and preoperative ketorolac , the need for rescue was significantly lower in the ketorolac group . p = 0.006 ( mean time for rescue in the preoperative placebo group 245 min and mean time for rescue in the preoperative ketorolac group 407 min ) . need for rescue analgesic in the preoperative versus postoperative groups of tramadol , ketorolac and placebo as indicated by mean time ( in minutes ) at which the rescue drug was self administrated . ( x - axis drug , y - axis mean time ) when postoperative placebo was compared with postoperative tramadol , the need for rescue analgesic was significantly lower in the tramadol group . p = 0.012 ( mean time for rescue in the postoperative placebo group 233 min and mean time for rescue analgesic in the postoperative tramadol group 372 min ) . on comparison of postoperative placebo with postoperative ketorolac , the need for rescue analgesic was significantly lower in the ketorolac group . p = 0.008 ( mean time for rescue in the postoperative placebo group 233 min and mean time for rescue in the postoperative ketorolac group 373 min ) . when preoperative tramadol was compared with postoperative tramadol , there was no significant difference in the need for rescue analgesic . p = 0.217 ( mean time for rescue in the preoperative tramadol group 313 min and mean time for rescue in the postoperative tramadol group 372 min ) . when preoperative ketorolac was compared with postoperative ketorolac also , there was no significant difference in the need for rescue analgesic between the two . p = 0.766 ( mean time for rescue in the preoperative ketorolac group 407 min and mean time for rescue in the postoperative ketorolac group 373 min ) . only 8 patients ( 16.3 % ) of the total of 49 reported side effects . of these eight patients that reported side effects , seven belonged to the tramadol group and one to the placebo group . one patient each from the tramadol group and the ketorolac group felt weakness / tiredness . two patients in the ketorolac group and one patient in the tramadol group felt nausea / vomiting . comparison of adverse effect reportage of tramadol versus placebo groups showed that tramadol was significantly more likely to produce adverse effects compared to placebo . comparison of adverse effect reportage of ketorolac versus placebo groups showed that ketorolac that was significantly more likely to produce adverse effects compared to placebo . comparison of ketorolac and tramadol for adverse effects showed that there was no significant difference between the two . for purposes of analysis , occurrence of adverse effects was represented as nominal data that is either any adverse effect occurred or no adverse effect occurred . pearson 's chi - square test ( without yale 's continuity correction ) showed a significant difference between groups . nsaid 's are known to produce side effects such as gastric irritation leading to ulceration and bleeding disorders . despite the inflammatory component , one of the drugs used in this study was a non - nsaid , so that the side effects produced by the anti - inflammatory analgesics could be avoided . the analgesic efficacy of non - nsaids versus nsaid 's to placebo was compared in this study . we also attempted to compare the adverse effects of non - nsaid and nsaid with placebo , when given as single dose . another aspect , which was looked at in this study , was pre - empting the expected pain by preoperative administration versus countering pain after it sets in by postoperative administration . this study has succeeded in demonstrating the analgesic efficacy of single oral doses of either tramadol or ketorolac for impacted third molar extraction with an acceptable incidence and severity of side effects , over the first 6 h following extraction . the pre- and postoperative administration of ketorolac was found to be equally effective in controlling dental pain . we looked at whether there is any need for analgesics for postextraction pain as the patient is already receiving a local anesthetic prior to surgery and found that a significant proportion of patients do experience pain severe enough to require analgesics . single dose of either tramadol or ketorolac was more efficient than placebo in relieving pain , over the first 6 h. other studies corroborate this finding . keeping the concept of pre - emptive analgesia in mind , we expected preoperative administration of analgesics to be more efficacious in relieving the pain than postoperative administration . our results show that postoperative administration of tramadol appears to be more efficacious than preoperative administration in terms of pain relief . analgesic effect of tramadol begins within 1 h and reaches a peak in approximately 23 h. it is likely that the postextraction pain reaches a peak in the first 24 h. pharmacodynamics and pharmacokinetics of most administered drugs are time dependent . opioid activity of tramadol is due to both low - affinity binding of the parent compound and higher affinity binding of the o - demethylated metabolite to opioid receptors . as it is extensively metabolized by a number of pathways , the bioavailability of the active metabolite is high . the analgesic effect of tramadol begins within 1 h and reaches a peak in approximately 23 h. though tramadol was given preoperatively with the idea of preempting the expected pain , it appears that it was given too early to be of any benefit and the peak analgesic effect and the time of maximum pain after tooth extraction may not have coincided with each other . even though the preoperative administration of ketorolac was found to be effective , it was not significantly better than postoperative administration . ketorolac inhibits the enzyme cyclo - oxygenase , thereby inhibiting the synthesis of prostaglandins , which is a mediator of inflammation and pain . it is a potent nsaid with a plasma half - life of approximately 4.55.5 h. the dose of ketorolac ( 20 mg ) administered in this study was possibly not enough to preempt the pain produce by third molar extraction or that the time of administration was too close to the extraction for the drug to produce any effect . third molar extraction produces a fair amount of injury to the surrounding tissue , leading to release of arachidonic acid , which is converted into prostaglandin by cyclo - oxygenase . inhibition of cyclo - oxygenase even before the release of arachidonic acid from injured tissue ensures that the synthesis of prostaglandins is blocked and pain and inflammation is preempted . tramadol being an opioid and norepinephrine modulator , more central nervous system - related side effects as expected was found as six patients is tramadol group had cns - related side effects and none in placebo group reported . comparison of tramadol and ketorolac did not show any significant difference in side effect reportage ; however , the organ system affected differed in each case . ketorolac has been reported to produce nausea , vomiting , dyspepsia , peptic ulceration , and prolongation of bleeding time , whereas tramadol effects on the gastrointestinal tract are fairly minor and its cns effects are more prominent . some of the reported side effects include anxiety , confusion , euphoria , sleep disorders , visual disturbance , and dependence . some studies assessed the utility of 10 indices ( including the verbal rating scale ) in the subjective experience of acute pain . in other studies,[1215 ] it has been observed that pain intensity is a relatively easy dimension of pain experience for patients to report , most self - report measures of pain intensity are strongly related to one another , and so can probably be used interchangeably in many situations . as telephonic interviews were needed for the assessment of postoperative pain , patient not accessible by telephone were not included in the study . the fact that this was a single - dose study and pain was assessed only over the first 6 h was another limitation . ideally , the duration of a study for assessing the analgesic efficacy of drugs is postextraction pain , which should be for a period of 23 days , with multiple dosing . this study demonstrated that postoperative administration of tramadol is equally effective as traditional nsaid 's in relieving pain in the first 6 h after molar extraction , and therefore , it can be tried in patients who are intolerant to nsaids . a firm conclusion regarding the time of intervention ( i.e. , pre and postextraction ) for optimal pain control is a point for clarification and needs further analysis .

despite global improvements in caries status , dental caries remains to be the most common chronic childhood disease world - wide . teeth with deep pits and fissures are shown to be more vulnerable to develop caries . in the other hand , fissure sealant application is proved to be able to block pits and fissures in order to prevent occlusal caries development . any sign of microleakage in sealants is considered as the weak point eventually leading to failure as the inability to isolate pit and fissures would enhance the retention of bacteria , nutrients , and their acidic metabolic products . the necessity of tooth preparation prior to the sealant application and its effect on microleakage has different perceptions among the researchers . believes that lower microleakage rate is associated with teeth following bur preparation while some studies illustrate opposing results . pumice prophylaxis has long been used prior to the sealant application with its effect on microleakage being mostly reported as beneficial . in the other hand , the use of acid etching alone or associated with one of the other preparatory methods has shown little to no difference in microleakage level of the teeth . the aim of this investigation was to compare the degree of microleakage at the enamel - sealant interface when prepared by a fissurotomy bur or pumice prophylaxis before the acid etchant being applied . occlusal fissures of the specimens in group a were prepared with a micro stf fissurotomy bur ( ss white burs inc . , lakewood , nj , usa ) on a high - speed hand piece . for each specimen , the bur was moved along all the occlusal fissures with a gentle force applied . the occlusal surface of the specimens in the group b received a thorough prophylaxis for 20 s with water - based slurry of pumice , using a prophylaxis brush fitted on a slow - speed hand piece . all teeth were then etched for 20 s using 35% phosphoric acid gel ( 3 m espe , usa ) , washed and dried for 20 s each . each sample received an adhesive ( adper single bond plus , 3 m espe , usa ) prior to fissure sealant placement . a light - cured sealant ( clinpro sealant , 3 m espe , usa ) was then applied to all the prepared fissure through a syringe type tip . polymerization was performed using an led demetron ii ( kerr , usa ) light - curing unit with an output of 800 mw / cm for 20 s. specimens were then subjected to a thermo cycle machine with 1000 cycles between 5c and 55c with 20 s dwell time . apices of each sample tooth was then covered with a layer of sticky wax followed by the application of two layers of nail varnish to the surface of each tooth leaving 2 mm around the sealant borders . specimens were then immersed in a 3% basic fuchsin dye solution for 72 h. sample teeth were subsequently washed under the tap water for 1 min to remove excess dye from the surfaces . each was then embedded in acrylic blocks and sectioned in the bucco - lingual plane through the sealant for two identical sections using a water - cooled diamond disk on an isomet saw ( buehler ltd . three prepared sections with four surfaces from each sample were examined under 10 magnifications using the xtd series stereomicroscope ( blue light inc . all the sections were blindly subjected to an assessment process using a four point scoring system suggested by overbo and raadal . dye penetration scale employed was as : ( 0 ) no dye penetration ; ( 1 ) dye penetration restricted to the outer half of the enamel - sealant interface ; ( 2 ) dye penetration in the inner half of the enamel - sealant interface ; ( 3 ) dye penetration into the underlying fissure . data were analyzed using kruskall - wallis analysis of variance and mann - whitney u - tests for comparing microleakage levels among and between groups . mean standard deviation of microleakage score of the cases in each group was separately calculated [ table 1 ] . the highest frequency of the microleakage in all groups was for score 0 and 1 , with fissurotomy 80% , pumice prophylaxis 79.2% and control group 66.7% . surprisingly , score 3 was not observed in any of the groups [ figure 1 ] . meansd of microleakage score of different preparatory groups dye penetration scores in three different tooth preparation methods kruskall - wallis analysis of variance showed significant differences among microleakage levels of different preparation methods ( p = 0.003 ) . mann - whitney u - test indicated that teeth in fissurotomy bur and pumice prophylaxis groups had significantly reduced level of microleakage than those in acid etch alone ( p = 0.005 and p = 0.003 , respectively ) ; however , it showed no significant differences of micloleakage level between fissurotomy bur and pumice prophylaxis groups ( p = 0.83 ) . sealing pits and fissures have been proved to have an effective role on preventing teeth from fissure caries in childhood . there are several preparatory methods introduced prior to the placement of the fissure sealant with varying degrees of efficacy on the adhesiveness of material to the prepared surfaces . however , the necessity of the tooth surface preparation prior to the sealant application is still under scrutiny . as a possible choice , the currently available and specifically designed fissurotomy bur could conservatively widen the fissures providing a larger surface for the adhesion of sealant material . a similar concept has also been reported using fine diamond bur in order to reduce microleakage of the sealants . fissurotomy bur has been suggested to have a significantly high influence on sealant retention when used along with acid etch . as indicated earlier it seems that bur preparation can provide greater surface area , inducing better sealant adhesion as well as an increased chance for the flow of sealant into the depth of fissures improving its wear resistance . reported that quarter round bur preparation with acid etch has a better effect than acid etching alone in reducing microleakage ; however , several other studies are also raising doubts on the effectiveness of bur preparation . the diversity of the materials and methods used to assess the microleakage may explain the contradictions between the results along with the use of dye agents such as methylene blue or basic fuchsin . results of the current investigation indicated that pumice prophylaxis along with acid etch as superior to acid etching alone in reducing microleakage around the sealants , supporting earlier reports by ansari et al . despite the introduction of pumice prophylaxis as one of the essential steps of fissure sealing by cueto and bunocore , this application has later been proved ineffective in promoting the sealant effectiveness by lowering the microleakage . the retention of sealant is believed to be enhanced when adhesive bonding agents are applied prior to the sealant . however , there are also reports suggesting no improvement for microleakage reduction when these adhesive agents are applied . the invasive use of bur to open the fissures has not been widely accepted due to its damaging effect on the teeth requiring fissure sealant . interestingly , there was no case with score three dye penetration ( to the full depth of the fissure ) indicating reasonable flow of the sealant into the depth of fissures . findings of this study indicate that the use of fissurotomy bur along with etchant gel enhances adhesiveness of sealant to the tooth . the use of prophylaxis paste would be advantageous in microleakage reduction to the use of etching alone .

human protothecosis is an algal infection due to environmentally ubiquitous achlorophyllic algae : prototheca species . prototheca wickerhamii and prototheca zopfii are the species reported in human infections , and infection occurs predominantly in the immunocompromised , for whom outcomes are often poor . the optimal management of cases remains unclear with in - vitro minimum inhibitory concentrations ( mics ) of antimicrobials difficult to interpret , and no clinical breakpoints established . published case reports to date are relatively uncommon , thus reports of risk factors , clinical presentation and outcomes of potential therapies are valuable for future research into therapy . a 63-year - old male presented with two weeks of right lower limb swelling , erythema and ulceration . the patient recalled frequent use of a home hot - tub over the preceding months before presentation . his medical history included dermatomyositis with associated pulmonary fibrosis , type 2 diabetes mellitus , peripheral neuropathy , stage iii chronic kidney disease , dilated congestive cardiac failure with moderate systolic dysfunction , atrial fibrillation , and compensated chronic liver disease due to primary biliary cirrhosis . he was not known to have hiv infection but testing was not performed for this . in addition to his chronic co - morbidities , the patient had a history of abdominal wall mycobacterium chelonae infection requiring debridement and multidrug therapy 3 years previously ; and disseminated mycobacterium haemophilum requiring prolonged antimicrobials 2 years previously . medications on presentation included long term prednisolone 30 mg daily , in addition to intermittent higher doses up to 35 mg over the preceding 6 months for presumed flares of his inflammatory lung disease . other medications included beta - blockers , diuretics , digoxin , ursodeoxycholic acid and subcutaneous insulin . on examination at presentation to hospital ( day 0 ) , the patient was febrile at 38.5 celsius , haemodynamically stable and normoglycemic on presentation . 1 ) revealed a 15 cm region of erythema and warmth with central ulceration and several surrounding dark bullae . laboratory results revealed raised inflammatory markers with a leukocyte count of 12.610/l with a left shift , and a c - reactive protein of 32 mg / l . renal function indices were abnormal with an egfr of 37 ml / min/1.73 m. the patient was provisionally diagnosed with a right lower limb soft tissue infection and acute on chronic renal failure . swabs of the ulcer were sent for general microscopy and culture , with no organisms seen on gram stain . a biopsy of the ulcer edge was performed and sent for mycobacterial and fungal cultures . direct microscopy on the biopsy showed no fungal elements , and no acid - fast bacilli were demonstrated on ziehl - neelsen stain . biopsy sample pcr for methicillin - resistant staphylococcus aureus ( mrsa ) was positive and renally dose - adjusted vancomycin was commenced . one week following presentation there was no improvement of the right lower limb soft tissue infection . new bullae and ulceration had formed on the medial aspect of the leg . at this time yeast - like organisms were grown from biopsy enrichment cultures ( cooked meat broth ) , along with mrsa , with no growth on primary plates ( horse blood columbia agar and chocolate agar ) . subcultures of the yeast - like organisms produced creamy colonies on sabouraud 's dextrose agar ( sda ) and pale purple colonies 12 mm in diameter at 2 days incubation on candida chromagar ( chromagar , france ) . these were gram - positive and had morula forms , characteristic of prototheca spp . on wet preparation ( fig . 2 ) . rapid yeast plus ( thermo fisher scientific remel products , usa ) provided an adequate identification for prototheca wickerhamii and api 20c aux ( biomrieux , france ) provided an excellent identification for prototheca wickerhamii . antifungal susceptibilities were performed by broth microdilution using the sensititre yeast - one test panel ( trek diagnostic systems , usa ) . the mics for fluconazole , itraconazole , voriconazole , posaconazole , caspofungin , anidulafungin , micafungin , 5-fluorocytosine , and amphotericin b were 128 , 0.5 , 0.25 , 0.25 , > 8 , > 8 , > 8 , > 64 and 0.25 mg / liter , respectively . susceptibility testing for isavuconazole was performed in a 96-well round - bottom plate using standard broth microdilution assay , according to the clinical and laboratory standards institute ( clsi ) document m27-a3 for yeast . results were obtained by reading the plates visually after 48 and 72 h incubation at 37 c . the mic of isavuconazole was read as the lowest concentration of the drug at which there was 100% inhibition of growth . the mic obtained for isavuconazole against prototheca was 0.5 mg / l after both 48 and 72 h incubation periods . the patient was diagnosed with cutaneous protothecosis and intravenous liposomal amphotericin was commenced at 3 mg per kg based on ideal body weight . other antibiotics were discontinued and prednisone therapy was reduced to 20 mg . by day three of amphotericin ( day 10 post admission ) , however , renal function had deteriorated with an egfr of 22 ml / min/1.73 m and liposomal amphotericin was ceased . oral itraconazole 200 mg twice daily was commenced with recovery of renal function to baseline over the following week before discharge . over the next three months , the ulcers improved slowly and a repeat biopsy of the residual lateral ulcer was did not isolate prototheca species on cultures or microscopy . the right lower limb ulceration had healed with a minor residual area of granulation and superficial desquamation . prototheca species are achlorophyllic algae found in soil and water . human infections due to prototheca species are rare , with only 160 cases ( as described by todd et al . ) up to mid-2011 , since the first report in 1964 . since this report , a further 41 cases have been reported , perhaps suggesting human protothecosis is being increasingly diagnosed . all human cases of protothecosis , where organisms were identified to species level , have been caused by either p. wickerhamii or p. zopfii species . inoculation is thought to occur via occult or overt skin trauma , especially in the context of contaminated water , . this patient in this case reported frequent hot - tub exposure and this was felt to be the likely environmental exposure , although a sample of the hot - tub water was not available for microbiological testing . hot tubs have been associated with other cutaneous and systemic infections , particularly with mycobacteria , and pseudomonas aeruginosa . confirmed or suspected protothecosis acquisition from hot - tub use has not been described before . clinical presentations of protothecosis may be broadly divided into the categories of cutaneous and systemic ( including disseminated disease ) . the spectrum of cutaneous protothecosis is broad and constitutes the majority of cases reported , with wide range of findings including ulcers ( as with our case ) , erosions , crusting , papules , plaques and pustules . a wide range of systemic manifestations has been reported , including abdominal infections , pneumonia , bloodstream infection and central nervous system infection , , , . disseminated disease is reported generally in the immunosuppressed , frequently with exogenous steroid use , as with this case . non - iatrogenic immunosuppressing risk conditions include chronic renal failure and diabetes mellitus , , the latter which was seen in this case . culture and prototheca species typically form creamy yeast - like colonies on sabouraud 's media , , as was seen in this case . on wet preparation , they can be distinguished from yeast by the absence of budding , and presence of small sporangia with endospores forming characteristic spoked wheels on microscopy of cultures , as illustrated in fig . 2 diagnosis may also be made in histopathologic sections , with morulae often visible with stains used to detect fungus , such as periodic acid schiff stain or grocott 's modification of gomori methenamine silver . commercial systems which may correctly identify prototheca include the api 20c or api 20c aux ( bio - mrieux , marcy l'etoile , france ) as well as vitek 2 ( bio - mrieux ) , but the api 32c ( bio - mrieux ) does not include these organisms in its database . molecular techniques have been used to identify prototheca species in human clinical samples , including ribosomal internal transcribed spacer ( its ) sequencing and 26s rrna gene sequencing . successful identification with mass spectrometry has been reported but , as clinical isolates of prototheca spp . are uncommon , these are not well represented in current databases , leading to potential for failed identification or misidentification , . the natural history of isolated skin disease is an indolent progressive course , though there are reports of spontaneous resolution , . in the immunosuppressed such prognoses underscore the need for prompt diagnosis and rapid treatment of even apparently isolated skin disease , particularly in the context of immunosuppression , . medical treatment for protothecosis most commonly includes antifungal agents and/or tetracyclines , . reported treatment success rates are higher for cutaneous disease ( 73% ) and olecranon bursitis ( 83% ) and low for disseminated disease ( 33% ) there are no established antimicrobial breakpoints for susceptibility or resistance but prototheca species generally appear to be susceptible to amphotericin in - vitro , with variable susceptibility to triazoles . in - vitro synergy has been reported for tetracycline , and recently in - vitro susceptibility to miltefosine and terbinafine , with resistance to echinocandins reported . intravenous amphotericin , either alone or in combination with other agents , has the highest reported treatment success rates to date : 77% and 86% , respectively . triazoles , especially itraconazole , have been reported as successful treatments , but failure with these is commonly reported , including with cutaneous disease , which prompted our induction with amphotericin therapy for this case . to our knowledge , there are no reports of isavuconazole susceptibility testing for prototheca spp . we found a relatively low mic of 0.5 mg / l in our clinical isolate , suggesting this agent may merit further study as a treatment option for protothecosis . reduction of immunosuppression is also a consideration , as corticosteroids in particular appear to increase susceptibility to prototheca infection . in our case , we believe prompt microbiological diagnosis , delivery of amphotericin followed by itraconazole therapy and reduction of immunosuppression contributed to the successful outcome . the patient was cautioned about hot - tub use as a potential source of protothecosis or mycobacterial disease .

the taiwan national health insurance program was established on march 1 , 1995 , by the bureau of national health insurance . more than 99% of the taiwanese population is enrolled into this program.14 the national health research institutes was commissioned to national health insurance research databases ( nhirds ) for research proposals . the nhirds consist of comprehensive health care data provided to researchers , including ambulatory care records , inpatient care records , registration files , catastrophic illness files , and various data regarding drug prescriptions . in this study , we used the longitudinal health insurance database , which is a sub data set of nhirds and contains 1 million beneficiaries randomly selected from those enrolled in the insurance program . the longitudinal health insurance database contained insurant information , outpatient and inpatient visits , and medical treatment records between january 1 , 1996 , and december 31 , 2010 . the nhri reported that there were no statistically significant differences in age or sex between the randomly sampled group and all beneficiaries of the nhi program . to improve claims data accuracy , the nhri invited expert reviews on a random sample of every 50 to 100 ambulatory and inpatient claims in each hospital , and clinic routine practice of performing cross - checks and validations of medical claims ensures the accuracy of the nhirds diagnostic coding . we selected patients with the diagnosis of endometriosis ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 617.x ) from january 1 , 1997 , to december 31 , 2000 , as the case cohort . accordingly , each patient in the case cohort was matched on the basis of age , sex , and index year to 8 randomly identified beneficiaries without endometriosis to build the control cohort . to minimize the influence of possible reverse causation,15 we excluded those subjects with a diagnosis of endometrial cancer before the diagnosis of endometriosis . the date of the initial diagnosis of endometriosis was assigned as the baseline date for each patient . to improve data accuracy , the endometriosis selection criteria required that all case icd-9 codes are assigned by a gynecologist and the patients must have the diagnosis of endometriosis for at least 2 times in the same year in outpatient clinic record . selection criteria for endometrial cancer patients ( icd-9-cm code 182 ) were assigned by a gynecologic oncologist . we selected endometrial cancer cases in this study only if they received 2 or more endometrial cancer diagnoses for ambulatory care visit or 2 or more diagnoses for inpatient care . all study subjects were followed from the baseline date to the first event , which was defined as occurrence of endometrial cancer up to the end of 2010 . patients diagnosed with endometrial cancer before or after the study period were excluded from both cohorts . we also identified relevant comorbidities , including hypertension ( icd-9-cm 401.x-405.x ) , diabetes mellitus ( icd-9-cm 250.x ) , and hyperlipidemia ( icd-9-cm 272.x ) for both the case cohort and the control cohort . for the investigation of urbanization , all 365 townships in taiwan were stratified into 7 levels according to the standards established by the taiwanese nhri based on a cluster analysis of the 2000 taiwan census data , with 1 referring to the most urbanized area and 7 referring to the least urbanized . the criteria on which these strata were determined included the population density ( persons per square kilometer ) , the number of physicians per 100,000 people , the percentage of people with a college education , the percentage of people older than 65 years , and the percentage of agricultural workers . because levels 4 , 5 , 6 , and 7 contained few endometriosis cases , they were combined into a single group and were recoded as level 4 . all data processing and statistical analyses were performed with spss 20 ( spss , chicago , il ) and sas 8.2 ( sas system for windows ; sas institute , cary , nc ) . the pearson test was used to compare differences in geographic location , monthly income , and urbanization level of patients residences between the case and control cohorts . event occurrence ( defined as occurrence of endometrial cancer ) was analyzed using the kaplan - meier method . the elapsed period was calculated for the patients who had endometriosis until the occurrence of endometrial cancer or the end of the study period ( december 31 , 2010 ) , whichever came first . after adjusting for urbanization level , monthly income , resident region , and comorbidities as potential confounders , we performed a cox proportional hazards analysis stratified by age at first diagnosis of endometriosis to investigate the risk for developing endometrial cancer during the 10-year follow - up period in both cohorts . hazard ratio and 95% confidence interval ( ci ) were calculated to quantify the risk for developing endometrial cancer . the results of comparisons with a 2-sided p value of less than 0.05 were considered to represent statistically significant differences . insurance reimbursement claims adopted in this study were from taiwan s nhirds , which are available for research purposes . this study was also evaluated and approved by the institutional review board of taipei veterans general hospital . the taiwan national health insurance program was established on march 1 , 1995 , by the bureau of national health insurance . more than 99% of the taiwanese population is enrolled into this program.14 the national health research institutes was commissioned to national health insurance research databases ( nhirds ) for research proposals . the nhirds consist of comprehensive health care data provided to researchers , including ambulatory care records , inpatient care records , registration files , catastrophic illness files , and various data regarding drug prescriptions . in this study , we used the longitudinal health insurance database , which is a sub data set of nhirds and contains 1 million beneficiaries randomly selected from those enrolled in the insurance program . the longitudinal health insurance database contained insurant information , outpatient and inpatient visits , and medical treatment records between january 1 , 1996 , and december 31 , 2010 . the nhri reported that there were no statistically significant differences in age or sex between the randomly sampled group and all beneficiaries of the nhi program . to improve claims data accuracy , the nhri invited expert reviews on a random sample of every 50 to 100 ambulatory and inpatient claims in each hospital , and clinic routine practice of performing cross - checks and validations of medical claims ensures the accuracy of the nhirds diagnostic coding . we selected patients with the diagnosis of endometriosis ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 617.x ) from january 1 , 1997 , to december 31 , 2000 , as the case cohort . accordingly , each patient in the case cohort was matched on the basis of age , sex , and index year to 8 randomly identified beneficiaries without endometriosis to build the control cohort . to minimize the influence of possible reverse causation,15 we excluded those subjects with a diagnosis of endometrial cancer before the diagnosis of endometriosis . the date of the initial diagnosis of endometriosis was assigned as the baseline date for each patient . to improve data accuracy , the endometriosis selection criteria required that all case icd-9 codes are assigned by a gynecologist and the patients must have the diagnosis of endometriosis for at least 2 times in the same year in outpatient clinic record . selection criteria for endometrial cancer patients ( icd-9-cm code 182 ) were assigned by a gynecologic oncologist . we selected endometrial cancer cases in this study only if they received 2 or more endometrial cancer diagnoses for ambulatory care visit or 2 or more diagnoses for inpatient care . all study subjects were followed from the baseline date to the first event , which was defined as occurrence of endometrial cancer up to the end of 2010 . patients diagnosed with endometrial cancer before or after the study period were excluded from both cohorts . we also identified relevant comorbidities , including hypertension ( icd-9-cm 401.x-405.x ) , diabetes mellitus ( icd-9-cm 250.x ) , and hyperlipidemia ( icd-9-cm 272.x ) for both the case cohort and the control cohort . for the investigation of urbanization , all 365 townships in taiwan were stratified into 7 levels according to the standards established by the taiwanese nhri based on a cluster analysis of the 2000 taiwan census data , with 1 referring to the most urbanized area and 7 referring to the least urbanized . the criteria on which these strata were determined included the population density ( persons per square kilometer ) , the number of physicians per 100,000 people , the percentage of people with a college education , the percentage of people older than 65 years , and the percentage of agricultural workers . because levels 4 , 5 , 6 , and 7 contained few endometriosis cases , they were combined into a single group and were recoded as level 4 . all data processing and statistical analyses were performed with spss 20 ( spss , chicago , il ) and sas 8.2 ( sas system for windows ; sas institute , cary , nc ) . the pearson test was used to compare differences in geographic location , monthly income , and urbanization level of patients residences between the case and control cohorts . event occurrence ( defined as occurrence of endometrial cancer ) was analyzed using the kaplan - meier method . the elapsed period was calculated for the patients who had endometriosis until the occurrence of endometrial cancer or the end of the study period ( december 31 , 2010 ) , whichever came first . after adjusting for urbanization level , monthly income , resident region , and comorbidities as potential confounders , we performed a cox proportional hazards analysis stratified by age at first diagnosis of endometriosis to investigate the risk for developing endometrial cancer during the 10-year follow - up period in both cohorts . hazard ratio and 95% confidence interval ( ci ) were calculated to quantify the risk for developing endometrial cancer . the results of comparisons with a 2-sided p value of less than 0.05 were considered to represent statistically significant differences . insurance reimbursement claims adopted in this study were from taiwan s nhirds , which are available for research purposes . this study was also evaluated and approved by the institutional review board of taipei veterans general hospital . the case cohort contained 15,488 patients diagnosed with endometriosis , whereas 123,904 patients were included in the control cohort . distributions of demographic characteristics and comorbidities for both the case and the control cohort are shown in table 1 . hyperlipidemia ( p < 0.001 ) , diabetes mellitus ( p < 0.001 ) , and obesity ( p < the case cohort also harbored a greater tendency to earn a lower monthly income ( p = 0.014 ) , reside in the southern area of taiwan , and reside in the middle levels of urbanization communities ( p = 0.003 ) compared with the control cohort . in contrast , the control cohort has a higher rate of hypertension ( p < 0.001 ) . demographic characteristics for the recruited participants , stratified by presence / absence of endometriosis from 1997 to 2000 in total , there were 392 participants who were newly diagnosed with endometrial cancer during the 10-year follow - up , with 104 in the case cohort ( 0.7% ) and 288 in the control cohort ( 0.2% ) . the kaplan - meier survival curves demonstrate significantly lower event - free rates in the case cohort than in the control cohort ( p = 0.001 , log - rank test ) ( fig . 1 ) . moreover , the incidence density was also higher in the case cohort ( 0.68 per 1000 patient - years ) than in the control cohort ( 0.23 per 1000 patient - years ) . distribution of endometrial cancer free rates between the case and the control cohort from 1997 to 2000 . the crude hazard ratio for event occurrence of endometrial cancer was 2.91-fold higher for the case cohort than for the control cohort ( 95% ci , 1.545.48 ; univariable cox regression analysis ) . furthermore , after adjusting for potential confounders , the hazard ratio did not change significantly , with a 2.83-fold greater risk in the case cohort than that in the control cohort ( 95% ci , 1.495.35 ; multivariable cox regression analysis ) ( table 2 ) . hazard ratios of endometrial cancer among endometriosis patients during the 10-year follow - up period from the index ambulatory visits or inpatient care from 1997 to 2000 next , we asked whether age at diagnosis of endometriosis posed any moderator effect on the occurrence of endometrial cancer . age at diagnosis of endometriosis was divided into 2 groups ( 40 years vs > 40 years ) . when the age at diagnosis of endometriosis is 40 years or younger , there is a non statistically different 1.42-fold higher adjusted hazard ratio in the case cohort than in the control cohort ( 95% ci , 0.553.70 ) . whereas when the age at diagnosis of endometriosis is older than 40 years , there is a statistically different 7.08-fold higher adjusted hazard ratio in the case cohort than in the control cohort ( 95% ci , 2.3321.55 ) ( table 3 ) . hazard ratios for endometrial cancer among the case cohort and the control cohort by age group lastly , we evaluated whether there is a difference for stage distribution between the case cohort and the control cohort . for the occurrence of endometrial cancer , there is no difference with respect to stage distribution between the case cohort and the control cohort ( table 4 ) . the result of our work indicates that patients with endometriosis harbor a higher risk for developing endometrial cancer . although endometriosis is a benign disease , still , multiple lines of evidence suggest that endometriosis could be viewed as a neoplastic process , including the increased susceptibility to develop some subtypes of epithelial ovarian cancer as well as molecular similarities between endometriosis and cancer.16 it is estimated that ovarian endometriosis has a 0.7% malignant transformation risk17 and 4.2 times greater risk for developing ovarian cancer.18 furthermore , a study by zaino et al13 showed that endometriosis was identified in approximately 30% of the cases with synchronous endometrioid type endometrial and ovarian cancers . although the association between endometriosis and ovarian cancer has been extensively investigated , in contrast , the association between endometriosis and endometrial cancer has rarely been reported.1821 the results of our work show discrepancy with the published articles . the potential reasons may include selection bias ( because most endometriosis cases are underdiagnosed ) , different study design ( cohort study design of our work vs case - control design of the published articles ) , different ethnic group , and different baseline demographics . the underlying molecular mechanisms by which these 2 disorders are connected remain uncertain . however , there are 2 putative shared mechanisms : estrogen stimulation and chronic inflammation . for the first mechanism , like uterine or breast cancer , endometriosis behaves as an estrogen - dependent disorder , specifically adapting to estrogen - induced signaling , by increased local production of estrogen through enhanced expression of aromatase cytochrome p450 expression but deficient 17-hydroxysteroid dehydrogenase type 2 expression ( which impairs inactivation of potent estradiol to less - potent estrone).22 evidence has further shown that there is pathological overexpression of estrogen receptor ( er ) in endometriotic stromal cells , resulting from deficient methylation of the er promoter , which also suppress estrogen ( er ) expression.23 studies in endometrial carcinoma has demonstrated paralleled high er-to - er ratio , with the amount of er messenger rna significantly lower in poorly differentiated endometrial cancer.2426 furthermore , progesterone receptor has been viewed as a classic er target gene . high er-to - er ratio in endometriotic stromal cells might contribute to the suppressed progesterone receptor and thus causes progesterone resistance , which might contribute to progesterone treatment failure in patients with endometriosis and/or endometrial cancer.23 for the second mechanism , the impact of chronic inflammation may play another critical role . endometriosis tissue is associated with overproduction of prostaglandins , cytokines , and chemokines.27 among these , cyclooxygenase 2 ( cox-2 ) , a rate - limiting enzyme in the biosynthesis of prostaglandin e2 , increased in both endometriosis and endometrial cancer patients.28 prostaglandin e2 promotes the initial carcinogenesis process and further consolidates tumor progression by increasing cell proliferation and neoangiogenesis while decreasing in situ immune performance.29 the impact of estrogen stimulation and chronic inflammation may not be mutually exclusive . the process regulates cox-2 in a positive feedback loop.30,31 fowler et al28 found aromatase expression in 65% of endometrial cancer patients compared with nonexpression in normal endometrium ; the results showed no difference in different histology groups . a study by collins et al25 found a high ratio of cox-2 detected in patients with er - positive endometrial cancer . on the basis of the above descriptions , the interconnection between cox-2 , er , and aromatase is close and might exert a synergetic effect , and , as such , there is association between endometriosis and endometrial cancer via the link by chronic inflammation . the strengths of our study include its use of a population - based database that is highly representative of the general population . first , we selected patients only by icd code ; potential bias including patient selection and diagnosis criteria might be present . second , the nhirds data set does not contain detailed information regarding parity , menstrual status , and hormonal use , all of which may be potential risk factors of endometriosis and/or endometrial cancer . these unmeasured variables may produce a confounding bias if it is associated with the studied exposure and disease simultaneously.32 third , the evidence derived from a retrospective cohort study is generally lower in research quality than that from randomized trials . to this end , further prospective cohort studies with adequate sample size are needed to verify the temporal association between endometriosis and endometrial cancer . to summarize , the findings in this study included increased association of endometriosis with endometrial cancer . the pathogenesis in endometriosis and endometrial cancer is complicated and the etiopathogenesis of both disorders is multifactorial , but there may exist a yet unidentified common link . however , much work is still needed to fully explain the exact mechanisms between these 2 disorders .

diabetes is one of the chronic diseases that substantially causing to disability , functional deterioration , morbidity , mortality and increased health costs ( 1 , 2 ) . diabetic patients especially those with disease complications present adverse quality of life and serious impairments in global functioning ( 1 ) . it appears to be also that burden of diabetes in iranian population is very high ( 3 ) . self - management behaviors are the cornerstone of blood sugar control and preventing the complications in type ii diabetes ( 4 ) . self - management is the day to day managing actions of diabetes inclined to taking drugs , adherence to regimen , weight loss planning , blood glucose monitoring , perform the regular physical activity and foot care by patients themselves ( 5 ) . adverse psychosocial agents and inappropriate therapeutic factors have negative effect in diabetes self - management ( 911 ) . also , socioeconomic conditions such as lower literacy and minus income hinder the excellent diabetes self - management ( 12 ) . socio - structural factors such as low support or assistance in family and poor health system agents such as displeasure with medical care as well have prominent impacts on diabetes self - management ( 1315 ) . insufficient participatory decision - making and weak physician - patient communication destroy the diabetes self - management behaviors ( 16 , 17 ) . diabetes- related factors including deficient education about diabetes , lower health literacy for diabetes management and demographic characteristics such as elderly may be decline diabetic self - management behaviors ( 1820 ) . also , diabetes- related literacy , patient s beliefs , self - efficacy , access to the healthcare system , and surrounding circumstances of family and relatives affects on diabetes self - management behaviors ( 21 , 22 ) . hence , individual factors and social , environmental and health care backgrounds influencing on diabetes self - management ( 23 , 24 ) . the sociostructural determinants in this study included provider - patient communication , health care satisfaction , health care access , duration of diabetes , treatment type , diabetes severity , retinopathy , neuropathy , nephropathy , cardiovascular disease , income , insurance , social support , marriage status , educational level , life network , cigarette smoking , diabetes knowledge , job , age , and sex . the patient s beliefs system included diabetes self - efficacy , belief of the efficiency of the treatment , belief of disease certainty , and motivation to treatment . this model established upon the magnitude of preceding studies . in this model , sociostructural determinants and collaborative decision - making are exogenous factors and patient s beliefs system and diabetes self - management are endogenous factors . it was assumed that among these determinants , patient s beliefs system would be the factor directly affecting diabetes self - management and sociostructural determinants would both directly influence diabetic self - management and indirectly influences diabetic self - management through collaborative decision - making and patient s beliefs system . also , it was assumed that collaborative decision - making would directly impact diabetes self - management and indirectly impact diabetes self - management through patient s beliefs system ( fig.1 ) . the objective of this research was to examination an assumed model depicting impacts of both sociostructural determinants and collaborative decision - making on diabetes self - management with mediating role of patient s beliefs system in iranian adults with type 2 diabetes . in this cross - sectional descriptive study , among adult patients with type ii diabetes who attended the outpatient clinics of the shariati hospital at tehran university of medical sciences during the dec 2010 to mar 2010 , 500 ( 245 men and 255 women ) patients as a sample were selected by convenience sampling . this sampling size selected dependent upon the participant s ratio to the model parameter s ratio , statistical population , subgroup analysis , data errors and possible missing data . the inclusion criteria were diagnosis of type ii diabetes at least one year s age , providing informed content by participants to participation in the research ; and participants attending to physicians and recording a medical history in diabetic outpatient s clinics of the shariati hospital . the exclusion criteria were diagnosis of type 1 diabetes ; currently acute diabetes complications ; other chronic diseases farther on than diabetes complications ; severe psychological disorders ; and recently diabetes diagnosis lessen than one year s age . the diabetes self - management scale was developed dependent upon the summary of diabetes self - care behaviors and diabetes self - management scale ( 8,25 ) . this scale is composed of seven diabetes regimen facets including diet , medication , glucose testing , exercise , control of increase or decrease in blood sugar , foot care , and attending to physicians for impediment of diabetes complications . this scale contained fourteen items in three parts of weekly self - management actions ( nine items ) , monthly self - management actions ( two items ) and annual self - management actions ( three items ) . cronbach s alpha ( n = 500 ) for weekly , monthly , annual , and total scale of self - management behaviors was .95 , .86 , .67 and .95 respectively . also , four weeks interval test - retest reliability on 34 patients for weekly , monthly and annual self - management behaviors was .92 , .90 and .97 respectively that pointed out the higher reliability of scale ( 26 ) . the diabetes self - efficacy scale was constructed dependent upon self - efficacy scale for patients with type ii diabetes ( 23,27 ) , theoretical foundations , and related issues of iranian culture . this scale consisted of 10 items including eight aspects of diet , exercise , glucose testing , medication adherence , foot care , prevention in increased or decreased blood sugar , and management of decreased or increased blood sugar . this instrument scored at 11-point likert - scale and ranged from zero to 100 that larger scores suggested higher self - efficacy in doing diabetes self - management . in this research , also , four weeks test - retest reliability in 34 patients was .94 that demonstrated high test - retest reliability over time . the beliefs of treatment effectiveness scale included items become adjusted from the personal models of diabetes questionnaire and beliefs of treatment effectiveness scale ( 28 , 29 ) . this instrument has nine items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 90 and higher scores indicated immense perceived belief that self - management behaviors could restrain diabetes and impede diabetic complications . in this research , one monthly of test - retest reliability on 34 patients was .94 for this scale that indicated outstanding test - retest reliability of this scale . the illness certainty scale ( ics ) has nine items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) that developed on the basis of illness uncertainty scale ( ius ; 30 ) . this scale appraises the illness certainty regarding to prognosis , medical care and coping with disease . the score ranged from zero to 90 that higher scores showed higher certainty regarding disease conditions and efficiency of treatment for prevention of diabetic complications . in this research , the cronbach s alpha ( n=500 ) was 0.92 and content validity has confirmed by a board of diabetes professionals . also , this scale has suitable test - retest reliability by four weekly of test - retest reliability on 34 ( r=0.92 ) . the treatment motivation scale ( tms ) was developed in terms of the treatment motivation scale and treatment self - regulation questionnaire ( 30 , 31 ) . this tool consisted of 6 items in 11-point likert - scale from 0% ( never ) to 100% ( always ) . the score ranged from zero to 60 in that higher scores point out excellent motivation for self - management and treatment . in this research , cronbach s alpha ( n = 500 ) was .85 that showed good internal consistency . in addition , the scale content validity has approved by a team of diabetes professionals and the scale test - retest reliability in one monthly period on 34 patients was suitable ( r=.91 ) . the provider - patient communication scale was developed based on the communication subscale of the interpersonal processes of care and provider - patient communication scale ( 23 , 32 ) . this scale is composed of eight items that evaluates clearly talking by physicians , explanation the medical care for patients , and responding to patients concerns . the scale items have an 11-point likert scale from 0% ( never ) to 100% ( always ) that ranged from zero to 80 and higher scores point out more desirable exchange of information or ideas between physicians and their patients . in this research , cronbach s alpha ( n=500 ) was .88 and content validity has confirmed by a board of diabetes professionals . furthermore , there is good test - retest reliability over time for this scale ( r= .93 ) by one monthly of test - retest reliability on 34 patients . the diabetes knowledge scale ( dkn ) was structured on the basis of the diabetes knowledge scale ( 33 ) , the diabetes knowledge questionnaire ( 23 ) , and general information package of diabetes specific to iranian patients . this scale has 10 items with 11-point likert spectrum from 0% ( never ) to 100% ( always ) and scores have range extended from 0 to 100 that higher scores showed higher degree of diabetes knowledge . this scale has cronbach s alpha equal with .91 in a sample of diabetes patients ( 33 ) . furthermore , the scale content validity has approved by a team of diabetes professionals and one monthly of test - retest reliability on 34 patients ( r=.95 ) was appropriate . the patient satisfaction to health care scale has eight items regarding satisfaction to medical care that patients obtained by healthcare systems for past one year age . this scale constructed by researchers and items included 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 80 that higher scores suggested greater satisfaction regarding to health care . in this research , content validity too has approved by a panel of diabetes professionals and four weekly of test - retest reliability on 34 patients was .94 that indicated good test - retest reliability . the access to health care scale was constructed on the basis of one subscale of the general practice assessment survey ( 34 ) and one item about cost of treatment . this scale items included 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 80 that higher scores point out better access to health care . cronbach s alpha ( n=500 ) was .90 for the access to health care scale . in addition , content validity has verified by a team of diabetes professionals and test - retest reliability in period of four weekly on 34 patients ( r=.93 ) was excellent . the social support from family members scale ( ssfms ) was established upon the family and friends support subscale of the chronic illness resources survey and social support scale ( 35 , 23 ) . the scale comprised 7 items including accepted emotional support , informational support , appraisal by their family members and tangible aids in the past 3 months that whole of the items express beneficially family support . this measure was an 11-point likert scale from 0% ( never ) to 100% ( always ) that scores ranged from zero to 70 and higher scores point out greater support from family individuals . cronbach s alpha ( n=500 ) was .92 for this scale . also , the content validity has confirmed by a board of diabetes professionals and four weekly of test - retest reliability on 34 patients was .93 that showed excellent test - retest reliability . the collaborative decision - making scale was developed based on the collaborative care planning scale ( ccps ; 36 ) and rochester participatory decision - making scale ( 37 ) . this scale be composed of 12 items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 120 in that higher scores point out improved collaborative decision - making . in this research , excessively , diabetes professional s team has approved the scale content validity and four weekly of test - retest reliability on 34 patients showed excellent test - retest reliability ( r=.93 ) . the demographical information , social- economical status and diabetic history questionnaire is researchers - developed tool that comprise three facets of participants demographical information including age , gender , and marital status ; social - economical status such as education level , employment , income , types of insurance , and whom living with ; and diabetes history including diabetes duration , diabetes severity , diabetes complications , diabetes types and diabetes treatment ( insulin therapy and oral agents therapy ) . this research was executed at the outpatient clinics of shariati hospital in tehran by individual procedure . also , research performed with pay attention to prominent issues of research including informed consent , confidentiality and protection of human participants . in this cross - sectional descriptive study , among adult patients with type ii diabetes who attended the outpatient clinics of the shariati hospital at tehran university of medical sciences during the dec 2010 to mar 2010 , 500 ( 245 men and 255 women ) patients as a sample were selected by convenience sampling . this sampling size selected dependent upon the participant s ratio to the model parameter s ratio , statistical population , subgroup analysis , data errors and possible missing data . the inclusion criteria were diagnosis of type ii diabetes at least one year s age , providing informed content by participants to participation in the research ; and participants attending to physicians and recording a medical history in diabetic outpatient s clinics of the shariati hospital . the exclusion criteria were diagnosis of type 1 diabetes ; currently acute diabetes complications ; other chronic diseases farther on than diabetes complications ; severe psychological disorders ; and recently diabetes diagnosis lessen than one year s age . the diabetes self - management scale was developed dependent upon the summary of diabetes self - care behaviors and diabetes self - management scale ( 8,25 ) . this scale is composed of seven diabetes regimen facets including diet , medication , glucose testing , exercise , control of increase or decrease in blood sugar , foot care , and attending to physicians for impediment of diabetes complications . this scale contained fourteen items in three parts of weekly self - management actions ( nine items ) , monthly self - management actions ( two items ) and annual self - management actions ( three items ) . cronbach s alpha ( n = 500 ) for weekly , monthly , annual , and total scale of self - management behaviors was .95 , .86 , .67 and .95 respectively . also , four weeks interval test - retest reliability on 34 patients for weekly , monthly and annual self - management behaviors was .92 , .90 and .97 respectively that pointed out the higher reliability of scale ( 26 ) . the diabetes self - efficacy scale was constructed dependent upon self - efficacy scale for patients with type ii diabetes ( 23,27 ) , theoretical foundations , and related issues of iranian culture . this scale consisted of 10 items including eight aspects of diet , exercise , glucose testing , medication adherence , foot care , prevention in increased or decreased blood sugar , and management of decreased or increased blood sugar . this instrument scored at 11-point likert - scale and ranged from zero to 100 that larger scores suggested higher self - efficacy in doing diabetes self - management . in this research , also , four weeks test - retest reliability in 34 patients was .94 that demonstrated high test - retest reliability over time . the beliefs of treatment effectiveness scale included items become adjusted from the personal models of diabetes questionnaire and beliefs of treatment effectiveness scale ( 28 , 29 ) . this instrument has nine items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 90 and higher scores indicated immense perceived belief that self - management behaviors could restrain diabetes and impede diabetic complications . in this research , one monthly of test - retest reliability on 34 patients was .94 for this scale that indicated outstanding test - retest reliability of this scale . the illness certainty scale ( ics ) has nine items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) that developed on the basis of illness uncertainty scale ( ius ; 30 ) . this scale appraises the illness certainty regarding to prognosis , medical care and coping with disease . the score ranged from zero to 90 that higher scores showed higher certainty regarding disease conditions and efficiency of treatment for prevention of diabetic complications . in this research , the cronbach s alpha ( n=500 ) was 0.92 and content validity has confirmed by a board of diabetes professionals . also , this scale has suitable test - retest reliability by four weekly of test - retest reliability on 34 ( r=0.92 ) . the treatment motivation scale ( tms ) was developed in terms of the treatment motivation scale and treatment self - regulation questionnaire ( 30 , 31 ) . this tool consisted of 6 items in 11-point likert - scale from 0% ( never ) to 100% ( always ) . the score ranged from zero to 60 in that higher scores point out excellent motivation for self - management and treatment . in this research , cronbach s alpha ( n = 500 ) was .85 that showed good internal consistency . in addition , the scale content validity has approved by a team of diabetes professionals and the scale test - retest reliability in one monthly period on 34 patients was suitable ( r=.91 ) . the provider - patient communication scale was developed based on the communication subscale of the interpersonal processes of care and provider - patient communication scale ( 23 , 32 ) . this scale is composed of eight items that evaluates clearly talking by physicians , explanation the medical care for patients , and responding to patients concerns . the scale items have an 11-point likert scale from 0% ( never ) to 100% ( always ) that ranged from zero to 80 and higher scores point out more desirable exchange of information or ideas between physicians and their patients . in this research , cronbach s alpha ( n=500 ) was .88 and content validity has confirmed by a board of diabetes professionals . furthermore , there is good test - retest reliability over time for this scale ( r= .93 ) by one monthly of test - retest reliability on 34 patients . the diabetes knowledge scale ( dkn ) was structured on the basis of the diabetes knowledge scale ( 33 ) , the diabetes knowledge questionnaire ( 23 ) , and general information package of diabetes specific to iranian patients . this scale has 10 items with 11-point likert spectrum from 0% ( never ) to 100% ( always ) and scores have range extended from 0 to 100 that higher scores showed higher degree of diabetes knowledge . this scale has cronbach s alpha equal with .91 in a sample of diabetes patients ( 33 ) . furthermore , the scale content validity has approved by a team of diabetes professionals and one monthly of test - retest reliability on 34 patients ( r=.95 ) was appropriate . the patient satisfaction to health care scale has eight items regarding satisfaction to medical care that patients obtained by healthcare systems for past one year age . this scale constructed by researchers and items included 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 80 that higher scores suggested greater satisfaction regarding to health care . in this research , content validity too has approved by a panel of diabetes professionals and four weekly of test - retest reliability on 34 patients was .94 that indicated good test - retest reliability . the access to health care scale was constructed on the basis of one subscale of the general practice assessment survey ( 34 ) and one item about cost of treatment . this scale items included 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 80 that higher scores point out better access to health care . cronbach s alpha ( n=500 ) was .90 for the access to health care scale . in addition , content validity has verified by a team of diabetes professionals and test - retest reliability in period of four weekly on 34 patients ( r=.93 ) was excellent . the social support from family members scale ( ssfms ) was established upon the family and friends support subscale of the chronic illness resources survey and social support scale ( 35 , 23 ) . the scale comprised 7 items including accepted emotional support , informational support , appraisal by their family members and tangible aids in the past 3 months that whole of the items express beneficially family support . this measure was an 11-point likert scale from 0% ( never ) to 100% ( always ) that scores ranged from zero to 70 and higher scores point out greater support from family individuals . also , the content validity has confirmed by a board of diabetes professionals and four weekly of test - retest reliability on 34 patients was .93 that showed excellent test - retest reliability . the collaborative decision - making scale was developed based on the collaborative care planning scale ( ccps ; 36 ) and rochester participatory decision - making scale ( 37 ) . this scale be composed of 12 items in 11-point likert spectrum from 0% ( never ) to 100% ( always ) . the score ranged from zero to 120 in that higher scores point out improved collaborative decision - making . in this research , excessively , diabetes professional s team has approved the scale content validity and four weekly of test - retest reliability on 34 patients showed excellent test - retest reliability ( r=.93 ) . the demographical information , social- economical status and diabetic history questionnaire is researchers - developed tool that comprise three facets of participants demographical information including age , gender , and marital status ; social - economical status such as education level , employment , income , types of insurance , and whom living with ; and diabetes history including diabetes duration , diabetes severity , diabetes complications , diabetes types and diabetes treatment ( insulin therapy and oral agents therapy ) . this research was executed at the outpatient clinics of shariati hospital in tehran by individual procedure . also , research performed with pay attention to prominent issues of research including informed consent , confidentiality and protection of human participants . the mean age of the participants were 44.04 years ( sd=6.59 ) and age ranged 25 to 55 . regarding to treatment kind , 259 participants consumed oral drugs only to managing diabetes ( 51.8% ) and 241 participants consumed insulin alone ( 48.2% ) . regarding to diabetes severity , 217 participants ( 43.4% ) had mild hba1c , 157 participants ( 31.4% ) had moderate hba1c and 126 participants ( 25.2% ) had severe hba1c . the correlation between variables , mean , and sd are demonstrated in the table 1 . at the beginning , the examination of the hypothetical model exhibited these fit indices : x(624,n=500)2=3692.64 , p<.001 , goodness - of - fit index ( gfi)=.71 , comparative fit index ( cfi)=.78 , adjusted gfi ( agfi)=.68 , root - mean - square error of approximation ( rmsea)=.09 , normed fit index ( nfi)=.74 , parsimonious gfi ( pgfi)=.063 , akaike information criterion ( aic ) = 3850.64 . gfi and cfi over .90 , rmsea less than .80 , and non - significant chi - square / df ratio ranged 1 to 5 are desired and indicate an excellent fitness of the model . rmsea ranged .08 to .10 and gfi , nfi , cfi with agfi ranged .85 to .90 is satisfactory . according to the results , except rmsea value that was below .10 , the other indices do nt arrive at the appropriate value . x / df ratio were 5/91 that positioned out of standard range and do not point out a suitable fitness of the model . according to the fig . 2 , in structural model exclude direct path coefficient between collaborative decision - making and patient s beliefs system , other coefficients was significant . also , in measurement model , except path coefficients between diabetes duration , job , age , and life network to sociostructural determinants , other paths were significant . dependent upon these results , the hypothetical model do nt fit the observed data in an iranian patients with type ii diabetes . therefore , model modification was essential on the basis of both statistical results and theoretical rationality . initially , on the basis of statistical results of hypothetical model , the path from diabetes duration , job , age , and life network to sociostructural determinants were removed in measurement model . also , from a theoretical standpoint , the paths from marriage status , educational level , cigarette smoking , income , treatment type , diabetes severity , retinopathy , neuropathy , nephropathy , cardiovascular disease to sociostructural determinants were removed . in addition , the paths from treatment agreement and patient involvement in treatment to collaborative decision - making , the paths from belief of disease certainty and treatment motivation to patient s beliefs system and the path from dietary adherence to diabetes self - management were removed . eventually , in terms of theoretical standpoint and results of goodness - of - fit indices of the first model , the direct paths from sociostructural determinants and collaborative decision - making to diabetes self - management were logical to be removed in the structural model . according to the examination of the hypothetical model , these variables directly not sufficient to bring about self - management actions but probably by mediating patients beliefs indirectly impact on self - management . the results of the revised model demonstrated improved model fitness : ( 115,n=500)=490.97 , p= .000 , gfi= .90 , agfi=.86 , cfi=.99 , rmsea=.080 , nfi=.98 , pgfi=.67 , aic= 566.97 . rmsea , gfi , cfi , nfi and agfi all arrived at the appropriate value of model fitness . also , x2/df ratio were 4/26 that positioned in usual range and showed a established goodness - of - fit for model . these statistics suggested that the modified model had a desirable fitness to the observed data . according to fig . 3 , the coefficients between factors in the revised model were too becomes better and whole of paths were significant . patients with higher self - efficacy and who is confident to therapy efficiency were more probably to carry out diabetes self - management . hence , figure 3 showed that the standardized coefficient between patient s beliefs system and diabetes self - management was excellent ( =.99 , p<.001 ) . in the ultimate model , results revealed that sociostructural determinants significantly influencing collaborative decision - making ( = .96 , p < .001 ) and patient s beliefs system ( =1.71 , p<.001 ) , which in turn , impacted diabetes self - management ( fig . , sociostructural determinants did not have significant influence on diabetes self - management directly ; but , sociostructural determinants affect on diabetes self - management indirectly via collaborative decision - making and patient s beliefs system . also , in the ultimate model collaborative decision - making did not directly influencing diabetes self - management merely affect on patient s beliefs system ( = .75 , p < .001 ) which in turn , resulted in diabetes self - management ( = .99 , p < .001 ) . the results of the present investigation on the basis of the goodness - of - fit indices show that the first hypothetical model failed to obtain admirable fitness with the observed data . afterwards , according to the modified model totally goodness - of - fit indices were improved and obtained the suitable values . also , the paths were significant for structural and measurement model . the hypothetical model was established upon preceding evidence , in what the associations between these determinants and self - management were frequently inspected using regressions analysis that could not discover any indirect influences between factors . direct and indirect associations were likely the fundamental mechanism in what manner these numerous factors influencing diabetes self - management . it may be concluded that the hypothetical relationships in the first model on the basis of external studies did not entirely convey to iranian patients with type ii diabetes supposedly due to cultural differences . in fact , iranian patients with diabetes may combine their own insight and experience into the diabetes self - management . it can be said if diabetic patients have different opinions about physician s advices dependent upon insight or their own experience ; they reject physician s advices and do not pursue the therapy . therefore , the belief s systems in the iranian culture may directly influence on diabetes self - management . alike to former inquiries the results of this research demonstrated that patient s beliefs system directly impacted diabetes self - management in this iranian sample ( 6 , 3840 ) . particularly , type 2 diabetic patients who have stronger belief in the efficiency of therapy and higher self - efficacy to manage diabetes and hinder related complications were more likely to carry out diabetes self - management than those who had a fragile beliefs system . according to the result of this study , sociostructural determinants similar to prior studies did not directly impact on diabetes self - management in this structural equation modeling ( 17 , 23 , 4143 ) . sociostructural determinants have indirectly impact on diabetes self - management via collaborative decision - making and patient s beliefs system in iranian patients with type 2 diabetes . in terms of prior studies , some sociostructural determinants like knowledge are essential however not adequate for diabetes self - managements and there are other agents joining between knowledge and consequences ( 23 , 40 ) . bains and egede suggested that sufficient knowledge is influential so that to enhance diabetes self - management , however other psychosocial factors as well are engaged in self - management ( 12 ) . in this study , collaborative decision - making and particularly the patient s beliefs system were connections between sociostructural determinants and diabetes self - management , that is to improve diabetes self - management , interventions for sociostructural factors should be planned to enhance knowledge and make better patient s beliefs system . social support as indicator for sociostructural determinants is one origin for diabetes self - efficacy in which might promote or reduce self - efficacy ( 44 ) . also , to promote patients adjustment of diabetes self - management , coordination between patients beliefs and clinicians beliefs concerning their diabetes and therapies must be produced . therefore , better therapeutic relationship between the physician and the patient may promote patients diabetes self - management via patient s beliefs modification about self - care behaviors . family members with providing social support reinforce carrying out the diabetes self - management excessively may be impact on patient s beliefs about diabetes management that result in improved diabetes management . bohlen and colleagues mentioned existence of therapeutic impediments and troublesome in access to medical care might result in adverse provider - patient relationships and lower health litracy that eventually reduced diabetes self - management ( 46 ) . also , yeaw , aagren , & christensen think to be true for some patients upward economic burden of medical care services affect on the patient s beliefs that all right negatively impacted diabetes self - management ( 11 ) . similar to prior studies , this study showed that sociostructural determinants have indirectly impact on diabetes self - management via collaborative decision - making ( 23 , 47 ) . one clarification for this finding is collaborative decision - making influenced by some sociostructural determinants such as age , gender , social support and health literacy . therefore , these agents have considerable influence on collaborative decision - making that in turn along with patient s beliefs system affect on the diabetes self - management . according to the findings of this structural equation modeling , collaborative decision - making indirectly impact on type ii diabetes self - management via patient s beliefs system . this result approves the findings informed by rose , harris , ho , and jayasinghe ( 39 ) , and lee and lin ( 45 ) . in one probable explanation , can be suggested collaborative decision - making raise the confidence s patient to therapist together with patient s self - efficacy for diabetes self- management and finally patient carry out better self - management . lee and lin mentioned patients with higher confidence to physicians probably have higher self - efficacy and positive outcome expectations that together with desirable adherence to therapy and worthwhile therapeutic consequences ( 45 ) . kellow , savige , & khalil quoted patient s engagement in treatment improve self - efficacy and go together with enhancement in treatment adherence and health consequences ( 48 ) . in other explanation , collaborative decision - making forming the foundation for the patient s beliefs system and finally lead to diabetes self- management . in spite of the significant results , this study definitely has particular restrictions . the cross - sectional study was restricting the power to recognize causal relationships between factors . convenience sampling method and selected target sample restricted the generalization of the findings . utilizing self - reported instruments some associations between these factors may necessity to be examined and confirmed in the future studies . additional adjustment in this research domain may donate to the enhancement of diabetes self - management and medical consequences in diabetic patients . hence , mixed design to assessing diabetes self - management , research replication with other community , and examining other kinds of sociostructural factors such as workplace conditions are recommended . this study showed that patient s beliefs system has direct influence on diabetes self - management and sociostructural determinants and collaborative decision - making via patient s beliefs system indirectly impact on diabetes self - management in iranian patients with type ii diabetes . therefore , to foster diabetes self - management and alteration of patient s entire lifestyle , extensive intervention agendas are necessity to make better sociostructural setting such as patients improved literacy , and improving collaborative decision - making which may increase patients opinions about the efficiency of the medical care and self - efficacy and then encourage diabetes self - care . in this study , authors completely observed the ethical issues such as informed consent , misconduct , data falsification or fabrication , plagiarism , redundancy , double submission or publication , etc .

obesity is increasingly prevalent . cheap , calorie - dense foods and relatively inactive lifestyles create long - term imbalances between energy uptake and expenditure , leading to adipocyte hypertrophy and subsequently hyperplasia . excess adipose tissue functions as an endocrine organ , producing bioactive molecules such as il-6 , tnf - alpha , and paf-1 which are implicated in many disease states including diabetes and atherosclerosis . it also produces the peptide hormone leptin , which transmits a satiety signal to the hypothalamus and acts as a negative feedback loop of the lipostat . leptin effects on food intake and neuroendocrine functions involve intermediate hypothalamic neuropeptides such as proopiomelanocortin ( pomc ) and neuropeptide - y ( npy ) in the hypothalamus which regulate appetite , energy expenditure , and sympathetic nervous system outflow [ 13 ] . in obesity , the commonly found high leptin levels are not associated with appetite suppression , giving rise to the concept of central leptin resistance or insensitivity [ 4 , 5 ] . this resistance may be caused by mutations affecting leptin transport , receptor affinity , or signal transduction . the majority of cases , however , probably reflect physiological de - sensitisation caused by excessive adipose tissue and leptin levels . restoring leptin sensitivity and hypothalamic appetite control presents an exciting new target in obesity management . the pregnane glycosides in hoodia exert appetite - suppressant effects via enhanced hypothalamic signaling , but this plant risks extinction . we studied caralluma fimbriata , a traditional indian famine food with no history of adverse effects , which also contains pregnane glycosides . this study evaluated the extract of c. fimbriata ( cfe , slimaluma . ) for appetite suppressing , antiobesogenic and antiatherogenic properties in the dio rat model . male wistar rats ( 200220 g ) , bred from a stock obtained from the central animal facility , indian institute of science , bangalore , india , were used in the study . animals were housed individually in polypropylene cages for at least 1 week of acclimation before the experiment started . animals were cared for in accordance with the principles of animal ethics guidelines , and the study was approved by the institutional animal ethics committee ( iaec ) . the rats were maintained under standard laboratory conditions at a room temperature at 23 1c with relative humidity : 50 10% and 12 h light / dark cycle . the c. fimbriata extract ( cfe ) was prepared and gifted by greenchem , bangalore , india . to produce cfe , the aerial parts of the plant were extracted with alcohol to obtain a 25% solution of pregnane glycosides , which was lyophilized to powder . the animals were randomly divided into three groups ( n = 6 ) : ( i ) untreated control , ( ii ) control for cafeteria diet ( ca ) , and ( iii ) cafeteria diet fed + cfe treated . rats in the untreated control group were fed standard pellet chow ad libitum , while rats in the ca and ca + cfe treatment groups received both pellet chow and cafeteria diet . cfe was administered by gavage , at three different doses , 25 , 50 , 100 mg / kg / day , for 90 days . obesity was induced by providing modified versions of the harris ( 1993 ) high - fat cafeteria diet . it consisted of 3 variants : ( i ) condensed milk + bread + peanuts + pellet chow ( 4 : 1 : 4 : 1 ) , ( ii ) chocolate + biscuits + dried coconut + pellet chow ( 3 : 2 : 4 : 1 ) , and ( iii ) cheese + boiled potatoes + beef tallow + pellet chow ( 4 : 2 : 4 : 1 ) . the appetite suppressing activity of cfe was calculated by monitoring ( a ) food intake for 90 days , ( b ) animal 's bodyweight at baseline , weekly and at term , and ( c ) the weights of fat pads ( perirenal , mesenteric , and , epididymal ) and liver at term . serum obesity indicators including cholesterol ( tc ) , triglycerides ( tg ) , and high - density lipoprotein ( hdl ) were measured using a commercial kit ( span diagnostic ltd , india ) in semiautomatic analyzer ( micro lab-200 ) . low - density lipoprotein ( ldl ) and very low - density lipoprotein ( vldl ) levels were calculated adopting the prescribed formula . at term , blood was collected by cardiac puncture under mild sodium pentothal anesthesia ; the serum was separated and stored at 20c until analysis . leptin was determined using a commercial rat enzyme - linked immunosorbent assay ( elisa ) kit ( biovendor , czech republic ) . sections of aorta from aortic arch to thoracic aorta were removed and fixed in 10% formalin neutrally buffered solution . 3 mm segments taken at 5 mm distance from the bifurcation of the left subclavian artery were embedded in paraffin wax . 5 m thick cross - sections were cut and stained with hematoxylin and eosin to determine intimal thickening . all the values were expressed as the mean sem and analyzed by one - way analysis of variance ( anova ) using the brown - forsythe statistic followed by games - howell post hoc comparisons tests in order to test differences between groups . these options were chosen as they do not require any assumptions of homogeneity of variance . the level of statistical significance was set at p < .05 . the statistical analyses were run using spss for windows ( version 16 , chicago , il ) . feeding behavior was monitored in the test animals for 90 days ( figure 1 ) . as expected , food intake was significantly greater in ca - fed groups than in the group given pellet chow ( p < .05 ) . concurrent administration of 25 , 50 , and 100 mg / kg / day of cfe with ca reduced food intake considerably compared to both ca and pellet chow groups ( p < .05 ) . both the degree and time course of the reduction in food intake were dose dependent . the anorexigenic effects of the lowest dose ( 25 mg / day ) were apparent by the end of week 7 ( p < .05 ) , the intermediate dose ( 50 mg / kg / day ) emerged by the end of week 4 , and the effects of the highest dose ( 100 mg / kg / day ) emerged were already evident at the beginning of the third week . animals in the untreated control group increased body weight by 147.50% ( 297.66 2.78 g ) at the end of the experiment ( figure 2 ) . those in the ca group increased weight by 172.30% ( 350.34 2.13 g ) , significantly more than the untreated control ( p < .05 ) . rats in all three ca + cfe groups gained weight at or close to the lower rate of the untreated control group . there was a hint of a dose - response curve . weight gain in the ca + 25 mg / kg / day cfe group was 142.46% ( 312.12 3.21 g ) , ca + 50 mg / kg / day cfe group was 136.53% ( 291.25 3.11 g ) , and ca + 100 mg / kg / day cfe group brought to 136.00% ( 288.50 2.11 g ) . these weight gains were all significantly less than the ca - fed animals ( p < .05 , figure 1).50 mg / kg / day of cfe appears to be the optimal antiobesogenic dose . table 1 shows that administration of the ca diet significantly increased the weight of perirenal ( p = .0001 ) , epididymal ( p = .0001 ) , and mesenteric fat pads ( p = .0001 ) compared to the animals fed pellet chow . in animals fed ca + cfe , weight gain in all fat pads was considerably lower than in the ca group , with the 50 and 100 mg / kg / day doses giving results close to the untreated control group . the ca diet resulted in fatty liver and increased the liver mass by 132.66 % compared to the untreated animals ( p < .0001 ) . in rats given ca + cfe , liver weight gain was reduced in a dose - related manner . at 25 mg / kg / day cfe , liver weight was reduced effectively to the untreated control value , that is , 105.14% . at 50 mg / kg / day cfe , liver weight was below the untreated control value , at 78.57% , and at 100 mg / kg / day cfe liver weight was reduced to 75.14% . the reduced liver weight in the two higher - dose groups reflects the reduced calorific intake and reduced body weight gain at these doses ; there were no signs of hepatotoxicity . the cafeteria diet produced the predicted changes in all serum obesity indicators , all of which were positively modified by cfe in a generally dose - dependent manner , as indicated in table 2 . table 2 shows that total cholesterol in the ca group increased by 125.90% ( 74.78 0.46 mg / dl ) compared to untreated controls ( p < .0001 ) . coadministration of cfe at 25 mg / kg / day had comparatively little effect ( 119.26% ; 89.19 1.61 ; p = .001 ) ; at 50 mg / kg / day the increase was restricted to 110.79% ( 82.86 1.73 ; p = .025 ) ; at 100 mg / kg / day total cholesterol was effectively normalized , at 102.01% ( 76.30 1.29 ) with no statistical differences between this group and the controls ( p = .797 ) . serum triglycerides in the ca group increased by 207.76% ( 150.9 1.61 ) compared to untreated controls ( p < .0001 ) . coadministration of 25 , 50 , and 100 mg / kg / day cfe restricted this increase to 161.59% ( 117.30 0.92 ; p = .0001 ) , 149.29% ( 108.36 1.51 ; p = .0001 ) , and 132.08% ( 95.9 0.75 ; p = .0001 ) , respectively . ldl levels in the ca group were significantly increased compared to untreated controls ( p = .0001 ) . while coadministration of 25 mg / kg / day cfe did not have a statistically significant impact ; 50 ( p = .0001 ) and 100 mg / kg / day cfe ( p = .0001 ) effectively prevented any increase in ldl levels . serum hdl levels fell in the ca group compared to untreated controls ( p = .0001 ) . coadministration of 50 mg / kg / day cfe progressively reduced the fall in hdl levels ( p = .001 ) , while 100 mg / kg / day cfe effectively prevented any decline in hdl levels ( p = .0001 ) . vldl levels were significantly elevated in ca - fed rats compared to the untreated controls ( p = .0001 ) . coadministration of 25 ( p = .001 ) , 50 ( p = .0001 ) , and 100 mg / kg / day ( p = .0001 ) cfe significantly inhibited this increase in a dose - dependent manner . table 2 also shows that in the ca group , the serum leptin concentration was dramatically higher than in untreated animals ( p = .0001 ) . concurrent administration of cfe effectively prevented this increase ( p = .0001 ) at all doses . figure 3(a ) shows a section of healthy aorta taken from a rat fed on lab chow . feeding rats a cafeteria diet with high fat content caused deposition of lipids in the intimal region of the aortic arch , as shown in figure 3(b ) . treating rats concurrently with different doses of cfe completely prevented lipid deposition and restored normal aortic characteristics as shown in figure 3(c ) which is representative of all three cfe groups . feeding rats a cafeteria , high - fat diet inevitably causes hyperphagia resulting in increased body weight compared to pellet chow fed animals and is a widely accepted model for clinical obesity . this gain in body weight is largely due to increased fat mass as a result of preadipocyte proliferation and differentiation and , to an extent , accumulation of lipids in the liver [ 11 , 12 ] . our results demonstrate that cfe has pronounced dose - dependent appetite suppressant and antiobesogenic effects in this model . these effects were reflected in the feed intake , body weight , liver weight , fat pad mass , and serum lipid profiles of the rats in our various treatment groups . fifty mg / kg / day of cfe appears to be the optimal dose for preventing ca diet - induced changes in body weight , hormones , fat pads , and liver . slight negative changes in liver and kidney function induced by the cafeteria diet were reduced by cfe in a dose - dependent manner and approached normal values at the intermediate dose level . the null findings are not detailed here ; two clinical trials of the food extract cfe also found no adverse effects [ 13 , 14 ] . the decline in food intake may reflect direct intervention in appetite control at the level of the hypothalamus , where the pregnane glycosides are known to act . there is also evidence that the pregnane glycosides act directly on adipose tissue , by inhibiting adipocyte proliferation and differentiation [ 1517 ] . an alternative hypothesis is that cfe may downregulate ghrelin synthesis in the stomach and subsequently neuropeptide - y in the hypothalamus , with ultimately the same effect of appetite suppression [ 1821 ] . finally , and very much in keeping with the favorable metabolic effects listed above , we showed for the first time that cfe has potent antiatherogenic properties in a rodent model . concurrent administration with cfe in ca - fed rats completely prevented the accumulation of lipids in the intima of the thoracic aorta , which otherwise develops in these animals . we speculate that the antiatherogenic effects may be mediated at least in part via improved plasma lipid profiles , with additional protection possibly conferred by cfe 's antioxidant and anti - inflammatory properties . this small scale study suggested that caralluma fimbriata extract showed pronounced dose - dependent appetite suppressant and antiobesogenic effects on a sample of rats fed a cafeteria diet . these data , combined with existing cfe clinical trial findings [ 13 , 14 ] , indicate that cfe has the potential to curb obesity and the pathologies linked to obesity .

as of july 2010 , who has reported 501 confirmed human cases of avian influenza a/(h5n1 ) which resulted in 287 deaths worldwide . at the same time , the statistics for the h1n1 2009 outbreak has so far included 214 countries with a total reported number of infections and deaths of 419,289 and 18,239 , respectively . today , an ominous expectation exists that the next pandemic will be triggered by a highly pathogenic virus , to which there is little or no pre - existing immunity in humans . the nation 's ability to mitigate a pandemic influenza depends on the available emergency response resources and infrastructure , and , at present , challenges abound . predicting the exact virus subtype remains a difficult task , and even when identified , reaching an adequate vaccine supply can currently take up to nine months [ 4 , 5 ] . even if the existing vaccines prove to be potent , their availability will be limited by high production and inventory costs [ 6 , 7 ] and also will be constrained by the supply of antiviral drugs , healthcare providers , hospital beds , medical supplies , and logistics . hence , pandemic mitigation will have to be done amidst limited availability of resources and supporting infrastructure . this challenge has been acknowledged by who and echoed by the hhs and cdc [ 8 , 9 ] . the existing models on pandemic influenza ( pi ) containment and mitigation aims to address various complex aspects of the pandemic evolution process including : ( i ) the mechanism of disease progression , from the initial contact and infection transmission to the asymptomatic phase , manifestation of symptoms , and the final health outcome [ 1012 ] , ( ii ) the population dynamics , including individual susceptibility [ 13 , 14 ] and transmissibility [ 10 , 1517 ] , and behavioral factors affecting infection generation and effectiveness of interventions [ 1820 ] , ( iii ) the impact of pharmaceutical and nonpharmaceutical measures , including vaccination [ 2123 ] , antiviral therapy [ 2426 ] , social distancing [ 2731 ] and travel restrictions , and the use of low - cost measures , such as face masks and hand washing [ 26 , 3234 ] . recently , the modeling efforts have focused on combining pharmaceutical and nonpharmaceutical interventions in search for synergistic strategies , aimed at better resource utilization . most of such approaches attempt implementing a form of social distancing followed by application of pharmaceutical measures . for one of the most notable among these efforts is a 2006 - 07 initiative by midas , which cross - examined independent simulation models of pi spread in rural areas of asia [ 43 , 44 ] , usa and uk [ 45 , 46 ] , and the city of chicago , respectively . midas cross - validated the models by simulating the city of chicago , with 8.6 m inhabitants and implementing a targeted layered containment [ 48 , 49 ] . the research findings of midas and some other groups [ 12 , 33 ] were used in a recent modeling community containment for pandemic influenza these findings were also used in a pandemic preparedness guidance developed by cdc . at the same time , the iom report points out several limitations of the midas models , observing that because of the significant constraints placed on the models the scope of models should be expanded . the iom recommends to adapt or develop decision - aid models that can provide real - time feedback and include the costs and benefits of intervention strategies . our literature review yields a similar observation that most existing approaches focus on assessment of a priori defined strategies , and virtually none of the models are capable of learning , that is , adapting to changes in the pandemic progress , or even predicting them , to generate dynamic strategies . such a strategy has the advantage of being developed dynamically , as the pandemic spreads , by selecting a mix of available mitigation options at each decision epoch , based on both the present state of the pandemic and its predicted evolution . in an attempt to address the iom recommendations , we present a simulation optimization model for developing predictive resource distribution over a network of regional outbreaks . the underlying simulation model mimics the disease and population dynamics of each of the affected regions ( sections 2.1 and 2.2 ) . as the pandemic spreads from region to region , the optimization model distributes mitigation resources , including stockpiles of vaccines and antiviral and administration capacities ( section 2.3 ) . the model seeks to minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks , using measures of morbidity , mortality , and social distancing , translated into the cost of lost productivity and medical expenses . the methodology is calibrated and implemented on a sample outbreak in fla , usa with over 4 m inhabitants ( section 3 ) . the strategy is compared to the reactive myopic policy , which allocates resources from one actual outbreak region to the next , each time trying to cover the entire regional population at risk , regardless of the resource availability . the comparison is done at different levels of vaccine and antiviral availability and administration capacity . we also present a sensitivity analysis for assessing the impact of variability of some critical factors , including : ( i ) antiviral efficacy , ( ii ) social distancing conformance , and ( iii ) cdc response delay . the objective of our methodology is to generate a progressive allocation of the total resource availability over a network of regional outbreaks . the methodology incorporates ( i ) a cross - regional simulation model , ( ii ) a set of single - region simulation models , and ( iii ) an embedded optimization model . we consider a network of regions with each of which classified as either unaffected , ongoing outbreak , or contained outbreak ( figure 1 ) . the single - region simulation models mimic the population and disease dynamics of each ongoing region , impacted by intervention measures . the pandemic can spread from ongoing to unaffected regions by infectious travelers who pass through regional border control . at every new regional outbreak epoch , the optimization model allocates available resources to the new outbreak region ( actual distribution ) and unaffected regions ( virtual distribution ) . daily statistics is collected for each ongoing region , including the number of infected , deceased , and quarantined cases , for different age groups . as a regional outbreak a testbed illustration and a comparison of our strategy to the myopic policy is given in section 3 . the model is initialized by creating population entities and mixing groups , for each region . the details of the resulting regional contact dynamics and infection transmission are given in section 2.2 . as the infected cases start seeking medical help , a new regional outbreak is detected . the outbreak can spread to unaffected regions as some infectious travelers pass undetected through the border control . by tracing these travelers , the model determines which of the unaffected regions , if any , become new outbreaks . the single - region model subsumes the following components ( see figure 3 ) : ( i ) population dynamics ( mixing groups and schedules ) , ( ii ) contact and infection process , ( iii ) disease natural history , and ( iv ) mitigation strategies , including social distancing , vaccination , and antiviral application . the model collects detailed statistics , including number of infected , recovered , deceased , and quarantined cases , for different age groups . for a contained outbreak , the societal cost includes the cost of lost lifetime productivity of the deceased ; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined . each region is modeled as a set of population centers formed by mixing groups or places where individuals come into contact with each other during the course of their social interaction . examples of mixing groups include households , offices , schools , universities , shopping centers , entertainment centers , and so forth , . each individual is assigned a set of attributes such as age , gender , parenthood , workplace , infection susceptibility , and probability of travel , among others . each person is also assigned t time - discrete ( e.g. , t = 1 hour ) weekday and weekend schedules , which depend on : ( i ) person 's age , parenthood , and employment status , ( ii ) disease status , ( iii ) travel status , and ( iv ) person 's compliance to social distancing decrees . as their schedules advance , the individuals circulate throughout the mixing groups and come into contact with each other ( see section 2.2.2 ) . it is assumed that at any point of time , an individual belongs to one of the following compartments ( see figure 4 ) : susceptible , contacted ( by an infectious individual ) , infected ( asymptomatic or symptomatic ) , and recovered / deceased . in what follows , we present the infection transmission and disease natural history model , which delineates the transitions between the above compartments . infection transmission occurs during contact events between susceptible and infectious cases , which take place in the mixing groups . at the beginning of every t period ( e.g. , one hour ) , for each mixing group g , the simulation tracks the total number of infectious cases , ng , present in the group . it is assumed that each infectious case generates rg per t unit of time new contacts , chosen randomly ( uniformly ) from the pool of susceptibles present in the group . we also assume the following : ( i ) during t period , a susceptible may come into contact with at most one infectious case and ( ii ) each contact exposure lasts t units of time . once a susceptible has started her contact exposure at time t , she will develop infection at time t + t with a certain probability that is calculated as shown below . let li(t ) be a nonnegative continuous random variable that represents the duration of contact exposure , starting at time t , required for susceptible i to become infected . we assume that li(t ) is distributed exponentially with mean 1/i(t ) , where i(t ) represents the instantaneous force of infection applied to susceptible i at time t [ 5557 ] . the probability that susceptible i , whose contact exposure has started at time t , will develop infection at time t + t is then given as ( 1)p{li(t)t}=1ei(t)t . a schematic of the disease natural history is shown in figure 5 . during the incubation phase , the infected case stays asymptomatic . at the end of the latency phase , she enters the infectious phase [ 44 , 46 , 48 ] . she becomes symptomatic at the end of the incubation period . at the end of the infectious phase , she enters the period leading to a health outcome , which culminates in her recovery or death . mortality for influenza - like diseases is a complex process affected by many factors and variables , most of which have limited accurate data support available from past pandemics . furthermore , the time of death can sometimes be weeks following the disease episode ( which is often attributable to pneumonia - related complications ) . because of the uncertainty underlying the mortality process , we adopted an age - based form of the mortality probability of infected i , as follows : ( 2)mi=i(1i ) , where i is the age - dependent base mortality probability of infected i , i is her status of antiviral therapy ( 0 or 1 ) , and is the antiviral efficacy measured as the relative decrease in the base probability . we assume that a recovered case develops full immunity but continues circulating in the region . mitigation is initiated upon detection of a critical number of confirmed infected cases , which triggers resource distribution and deployment . vaccination is constrained by the allocated stockpile and administration capacity , measured in terms of the immunizer - hours . we assume that as some symptomatic cases seek medical help [ 62 , 63 ] , those at risk of them will receive an antiviral . the process is constrained by the allocated stockpile and administration capacity , measured in terms of the number of certified providers . both vaccination and antiviral application are affected by a number of sociobehavioral factors , including conformance of the target population , degree of risk perception , and compliance of healthcare personnel [ 6466 ] . the conformance level of the population at risk can be affected , among other factors , by the demographics and income level [ 6771 ] as well as by the quality of public information available . the degree of risk perception can be influenced by the negative experience developed during previous pharmaceutical campaigns [ 72 , 73 ] , as well as by public fear and rumors [ 74 , 75 ] . we adopted a cdc guidance , which establishes five categories of pandemic severity and recommends quarantine and closure options according to the category . the categories are determined based on the value of the case fatality ratio ( cfr ) , the proportion of fatalities in the total infected population . for cfr values lower than 0.1% ( category 1 ) , voluntary at - home isolation of infected cases is implemented . for cfr values between 0.1% and 1.0% ( categories 2 and 3 ) , in addition to at - home isolation , the following measures are recommended : ( i ) voluntary quarantine of household members of infected cases and ( ii ) child and adult social distancing . for cfr values exceeding 1.0% ( categories 4 and 5 ) , is affected by some of the behavioral factors listed above , we assume a certain social distancing conformance level . travel restrictions considered in the model included regional air and land border control for infected travelers . as presented in figure 2 , the optimization model is invoked at the beginning of every nth new regional outbreak epoch ( n = 1,2 , ) , starting from the initial outbreak region ( n = 1 ) . the objective of the model is to allocate some of the available mitigation resources to the new outbreak region ( actual distribution ) while reserving the rest of the quantities for potential outbreak regions ( virtual distribution ) . by doing so , the model seeks to progressively minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks , spreading from the ongoing locations . mitigation resources can include stockpiles of vaccines and antivirals , administration capacity , hospital beds , medical supplies , and social distancing enforcement resources , among others . the predictive mechanism of the optimization model is based on a set of regression equations obtained using single - region simulation models . in what follows , we present the construction of the optimization model and explain the solution algorithm for the overall simulation - based optimization methodology . we introduce the following general terminology and notation : : set of all network regions : set of regions in which pandemic is contained at the nth outbreak epoch ( n = 1,2 , ) : set of ongoing regions at the nth outbreak epoch : set of unaffected regions at the nth outbreak epoch : set of available types of mitigation resources ( r = { 1,2 , , r } ) : amount of resource i allocated to region k : available amount of resource i r at the nth outbreak epoch : set of age groups . the optimization criterion ( objective function ) of the model incorporates measures of expected societal and economic costs of the pandemic : the societal cost includes the cost of lost lifetime productivity of the deceased ; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined . to compute these costs , the following impact measures of morbidity , mortality , and quarantine are used , for each region k : : total number of infected cases in age group h who seek medical assistance : total number of infected cases in age group h who do not seek medical assistance : total number of deceased cases in age group h : total number of person - days of cases in age group h who comply with quarantine . to estimate these measures , we use the following regression models obtained using a single - region simulation of each region k : ( 3)xhk=hk0+irhkiqik+i , mr , imhkimqikqmk , where denotes the regression coefficient associated with resource i and is the regression coefficient for the interaction between resources i and m. similar models are used for yhk , dhk , and vhk . the above relationships between the impact measures and the resource distributions ought to be determined a priori of implementing a cross - regional scenario ( see section 3 ) . here we assume , however , that as the pandemic evolves , the disease infectivity will naturally subside . hence , the regression equations need to be re - estimated at every new outbreak epoch , for each region k c , using the single - region simulation models , where each simulation must be initialized to the current outbreak status in region k in the cross - regional simulation . as an alternative to using a computationally burdensome approach of re - estimating the regression equations , a modeler may choose to use a certain decay factor to adjust the estimates of the regional impact measures at every nth outbreak epoch , in the following way : ( 4)xhjn=nxhj , yhjn=nyhj , dhjn=ndhj , vhjn=nvhj . in addition , we use the following regression model to estimate the probability of pandemic spread from affected region l to unaffected region k , as a function of resources allocated to region l , which , in turn , impact the number of outgoing infectious travelers from the region : ( 5)plk=lk0+irlkiqil+i , mrimlkimqilqml , where denotes the regression coefficient associated with resource i , is the regression coefficient associated with interaction between resources i and m , and represents the intercept . consequently , the total outbreak probability for unaffected region k can be found as pk = lbplk . as in the case of the impact measures , the estimates of the regional outbreak probabilities need to be progressively re - estimated or adjusted using a scheme similar to ( 4 ) , as follows : finally , we calculate the total cost of an outbreak in region k at the nth decision epoch as follows : ( 7)tckn=h(mh+wh)xhkn+hwhyhkn+hw^hdhkn + hwhvhkn , where mh is total medical cost of an infected case in age group h over his / her disease period , wh is total cost of lost wages of an infected case in age group h over his / her disease period , w^h is cost of lost lifetime wages of a deceased case in age group h , and wh is daily cost of lost wages of a non - infected case in age group h who complies with quarantine . the model the optimization model has the following form . ( 8)minimize tcjn(q1j , q2j, ,qrj ) + scntcsn(q1s , q2s, ,qrs)psnsubject to qij+scnqispsnqin ir , qij , qis0 ir . the first term of the objective function represents the total cost of the new outbreak j , estimated at the nth outbreak epoch , based on the actual resource distribution { q1j , q2j , the second term represents the total expected cost of outbreaks in currently unaffected regions , based on the virtual distributions { q1s , q2s , , qrs } ( 7 ) and the regional outbreak probabilities ps ( 6 ) . the set of constraints assures that for each resource i , the total quantity allocated ( current and virtual , both nonnegative ) does not exceed the total resource availability at the nth decision epoch . note that both the objective function and the availability constraints are nonlinear in the decision variables . ,qrj ) + scntcsn(q1s , q2s, ,qrs)psnsubject to qij+scnqispsnqin ir , qij , qis0 ir . the first term of the objective function represents the total cost of the new outbreak j , estimated at the nth outbreak epoch , based on the actual resource distribution { q1j , q2j , , qrj } ( see ( 7 ) ) . the second term represents the total expected cost of outbreaks in currently unaffected regions , based on the virtual distributions { q1s , q2s , , qrs } ( 7 ) and the regional outbreak probabilities ps ( 6 ) . the set of constraints assures that for each resource i , the total quantity allocated ( current and virtual , both nonnegative ) does not exceed the total resource availability at the nth decision epoch . note that both the objective function and the availability constraints are nonlinear in the decision variables . the solution algorithm for our dynamic predictive simulation optimization ( dpo ) model is given below . initialize the sets of regions : a = , b = , c = s. select randomly the initial outbreak region j. set n = 1 . c{j}. solve the resource distribution model for region j. update the total resource availabilities . if b , do step 8 . else , do step 10 . for each ongoing region , implement a next day run of its single - region simulation . based on the outbreak probability values , determine if there is a new outbreak region(s ) j. if there is no new outbreak(s ) , go to step 7 . otherwise , go to step 9 . for each ongoing region , implement a next day run of its single - region simulation . update sets a and b , if needed . for each unaffected region , calculate its outbreak probability . based on the outbreak probability values , determine if there is a new outbreak region(s ) j. if there is no new outbreak(s ) , go to step 7 . otherwise , go to step 9 . for each new outbreak region j , update sets b b { j } and c c{j}. re - estimate regression equations for each region k b c using the single - region simulations , where each simulation is initialized to the current outbreak status in the region ( alternatively , use ( 4 ) and ( 6 ) ) . solve the resource distribution model for region j. update the total resource availabilities . c{j}. re - estimate regression equations for each region k b c using the single - region simulations , where each simulation is initialized to the current outbreak status in the region ( alternatively , use ( 4 ) and ( 6 ) ) . solve the resource distribution model for region j. update the total resource availabilities . to illustrate the use of our methodology , we present a sample h5n1 outbreak scenario including four counties in fla , usa : hillsborough , miami dade , duval , and leon , with populations of 1.0 , 2.2 , 0.8 , and 0.25 million people , respectively . a basic unit of time for population and disease dynamics models was taken to be t = 1 hour . regional simulations were run for a period ( up to 180 days ) until the daily infection rate approached near zero ( see section 3.3 ) . below , we present the details on selecting model parameter values . most of the testbed data can be found in the supplement . each infected person was assigned a daily travel probability of 0.24% , of which 7% was by air and 93% by land . the probabilities of travel among the four regions were calculated using traffic volume data [ 8083 ] , see table 1 . infection detection probabilities for border control for symptomatic cases were assumed to be 95% and 90% , for air and land , respectively . the instantaneous force of infection applied to contact i at time t ( ( 1 ) , ) was modeled as ( 9)i(t)=ln(1pi(t ) ) , where pi(t)=ii(t ) , where i is the age - dependent base instantaneous infection probability of contact i , i(t ) is her status of vaccination at time t ( 0 or 1 ) , and is the vaccine efficacy , measured as the reduction in the base instantaneous infection probability ( achieved after 10 days ) . the values of age - dependent base instantaneous infection probabilities were adopted from ( see table 2 ) . the disease natural history included a latent period of 29 hours ( 1.21 days ) , an incubation period of 46 hours ( 1.92 days ) , an infectiousness period from 29 to 127 hours ( 1.21 to 5.29 days ) , and a period leading to health outcome from 127 to 240 hours ( 5.29 to 10 days ) . base mortality probabilities ( i in ( 2 ) ) were found using the statistics recommended by the working group on pandemic preparedness and influenza response . this data shows the percentage of mortality for age - based high - risk cases ( hrc ) ( table 3 , columns 13 ) . mortality probabilities ( column 4 ) were estimated under the assumption that high - risk cases are expected to account for 85% of the total number of fatalities , for each age group . single - region simulation models were calibrated using two common measures of pandemic severity [ 35 , 45 , 46 ] : the basic reproduction number ( r0 ) and the infection attack rate ( iar ) . r0 is defined as the average number of secondary infections produced by a typical infected case in a totally susceptible population . iar is defined as the ratio of the total number of infections over the pandemic period to the size of the initial susceptible population . to determine r0 , all infected cases inside the simulation were classified by generation of infection , as in [ 33 , 43 ] . the value of r0 was calculated as the average reproduction number of a typical generation in the early stage of the pandemic , with no interventions implemented ( the baseline scenario ) . historically , r0 values for pi ranged between 1.4 and 3.9 [ 37 , 43 ] . to attain similar values , we calibrated the hourly contact rates of mixing groups ( original rates were adopted from ) . for the four regions , the average baseline value of r0 was 2.54 , which represented a high transmissibility scenario . mitigation resources included stockpiles of vaccines and antiviral and administration capacities ( section 3.4 ) . phi the vaccination risk group included healthcare providers , and individuals younger than 5 years ( excluding younger than 12 months old ) and older than 65 years . the risk group for antiviral included symptomatic individuals below 15 years and above 55 years [ 60 , 86 ] . the efficacy levels for the vaccine ( in ( 9 ) ) and antiviral ( in ( 2 ) ) we did not consider the use of antiviral for a mass prophylactic reduction of infection susceptibility due to the limited antiviral availability and the risk of emergence of antiviral resistant transmissible virus strains . the vaccination risk group included healthcare providers , and individuals younger than 5 years ( excluding younger than 12 months old ) and older than 65 years . the risk group for antiviral included symptomatic individuals below 15 years and above 55 years [ 60 , 86 ] . the efficacy levels for the vaccine ( in ( 9 ) ) and antiviral ( in ( 2 ) ) were assumed to be 40% [ 44 , 87 ] and 70% , respectively . we did not consider the use of antiviral for a mass prophylactic reduction of infection susceptibility due to the limited antiviral availability and the risk of emergence of antiviral resistant transmissible virus strains . npi a version of the cdc guidance for quarantine and isolation for category 5 was implemented ( section 2.2.4 , ) . once the reported cfr value had reached 1.0% , the following policy was declared and remained in effect for 14 days : ( i ) individuals below a certain age ( 22 years ) stayed at home during the entire policy duration , ( ii ) of the remaining population , a certain proportion stayed at home and was allowed a one - hour leave , every three days , to buy essential supplies , and ( iii ) the remaining ( 1 ) noncompliant proportion followed a regular schedule . a version of the cdc guidance for quarantine and isolation for category 5 was implemented ( section 2.2.4 , ) . once the reported cfr value had reached 1.0% , the following policy was declared and remained in effect for 14 days : ( i ) individuals below a certain age ( 22 years ) stayed at home during the entire policy duration , ( ii ) of the remaining population , a certain proportion stayed at home and was allowed a one - hour leave , every three days , to buy essential supplies , and ( iii ) the remaining ( 1 ) noncompliant proportion followed a regular schedule . an outbreak was considered contained , if the daily infection rate did not exceed five cases , for seven consecutive days . once contained , a region was simulated for an additional 10 days for accurate estimation of the pandemic statistics . a 2 statistical design of experiment was used to estimate the regression coefficient values of the significant decision factors and their interactions ( see section 2.3 ; the values of adjusted r ranged from 96.36% to 99.97% ) . the running time for a cross - regional simulation replicate involving over four million inhabitants was between 17 and 26 minutes ( depending on the initial outbreak region , with a total of 150 replicates ) on a pentium 3.40 ghz with 4.0 gb of ram . the performance of the dpo and myopic policies is compared at different levels of resource availability . table 4 summarizes the total vaccine and antiviral requirements for each region , based on the composition of the regional risk groups ( see section 3.3 ) . table 5 shows the per capita costs of lost productivity and medical expenses , which were adopted from and adjusted for inflation for the year of 2010 . comparison of the two strategies is done at the levels of 20% , 50% , and 80% of the total resource requirement shown in table 4 . figures 6(a ) and 6(b ) show the policy comparison in the form of the 95% confidence intervals ( ci ) for the average number of infected and deceased , respectively . figure 7 also shows the policy comparison using the 95% ci for the average total pandemic cost , calculated using the pandemic statistics , and the per capita costs from table 5 . for illustrative purposes , we also show the average number of regional outbreaks , for each policy , at different levels of resource availability , in the testbed scenario involving four regions , with the hillsborough as the initial outbreak region ( table 6 ) . it can be observed that the values of all impact measures exhibit a downward trend , for both dpo and myopic policies , as the total resource availability increases from 20% to 80% . an increased total resource availability not only helps alleviating the pandemic impact inside the ongoing regions but also reduces the probability of spread to the unaffected regions . for both policies , as the total resource availability approaches the total resource requirement ( starting from approximately 60% ) , the impact numbers show a converging behavior , whereby the marginal utility of additional resource availability diminishes . this behavior can be explained by noting that the total resource requirements were determined assuming the worst case scenario when all ( four ) regions would be affected and ought to provided with enough resources to cover their respective regional populations at risk . it can also be seen that on average , the dpo policy outperforms the myopic approach at all levels , which can attest to a more efficient resource utilization achieved by the dpo policy ( see also table 6 ) . the difference in the policy performance is particularly noticeable at the lower levels of resource availability , and it gradually diminishes , as the resource availability increases and becomes closer to be sufficient to cover the entire populations at risk in all regions . it can also be noted that the variability in the performance of the dpo strategy is generally smaller than that of the myopic policy . in general , for both strategies , the performance variability decreases with higher availability of resources . in this section , we assess the marginal impact of variability of some of the critical factors . the impact was measured separately by the change in the total pandemic cost and the number of deaths ( averaged over multiple replicates ) , resulting from a unit change in a decision factor value , one factor at a time . factors under consideration included : ( i ) antiviral efficacy , ( ii ) social distancing conformance , and ( iii ) cdc response delay . we have used all four regions , separately , as initial outbreak regions for each type of sensitivity analysis . due to limited space , we have opted to show the results for only one initial region , chosen arbitrarily , for each of the three types of sensitivity studies . while duval county was selected as the initial outbreak region to show the sensitivity results on antiviral efficacy , hillsborough and miami dade were used as the initial regions to show the results on , respectively , social distancing conformance and cdc response delay . figure 8 depicts the sensitivity of the average total cost and average total deaths to antiviral efficacy values between 0% and 80% . as expected , for both policies , the curves for the average number of deaths exhibit a decreasing trend which is almost linear for the values of between 0% and 40% . as the value of approaches 70% , the curves start exhibit a converging behavior . the curves for the average total pandemic cost exhibit a similar pattern for both policies . it can be noted that the performance of both policies is somewhat identical for low antiviral efficacy ( between 0% and 30% ) . however , the performance of the dpo policy improves consistently as increases which can be attributed to a more discretionary allocation of the antiviral stockpile by the dpo policy . reduction of the contact intensity through quarantine and social distancing has proven to be one of the most effective containment measures , especially in the early stages of the pandemic [ 27 , 30 , 31 , 41 ] . figure 9 shows the sensitivity of the average total cost and average total deaths to the social distancing conformance ranging between 60% and 80% . we observed that for both impact measures , the dpo policy demonstrated a better performance with the difference ranging from $ 3b to $ 26b in the total cost and from 1,400 to 20,000 in the number of fatalities . the biggest difference in performance was achieved at the lower - to - medium levels of conformance ( between 65% and 72% ) . as the conformance level approached 80% , the dominating impact of social distancing masked the effect of better utilization of vaccines and antivirals achieved by the dpo strategy . the cdc response delay corresponds to the interval of time from the moment an outbreak is detected to a complete deployment of mitigation resources . depending on the disease infectivity , cdc response delay may represent one of the most critical factors in the mitigation process . figure 10 shows how the performance of both policies was significantly impacted by this factor . the dpo policy showed a uniformly better performance with the difference ranging between $ 3b to $ 4b in the average total cost , and between 800 to 1,800 in the average number of mortalities , over the range ( 2472 hours ) of the response delay that we examined . for both policies , there was no significant difference when the delay was between 24 and 48 hours . however , for the delay values exceeding 48 hours , the average number of deaths and total cost increased at a high rate . as recently pointed by the iom , the existing models for pi mitigation fall short of providing dynamic decision support which would incorporate the costs and benefits of intervention . in this paper , we present a large - scale simulation optimization model which is attempted at filling this gap . the model supports dynamic predictive resource distribution over a network of regions exposed to the pandemic . the model aims to balance both the ongoing and potential outbreak impact , which is measured in terms of morbidity , mortality , and social distancing , translated into the cost of lost productivity and medical expenses . the model was calibrated using historic pandemic data and compared to the myopic strategy , using a sample outbreak in fla , usa , with over 4 million inhabitants . , the marginal utility of additional resource availability was found to be diminishing , as the total resource availability approached the total requirement . in the testbed scenario , the dpo strategy on average outperformed the myopic policy . as opposed to the dpo strategy , the myopic policy is reactive , rather than predictive , as it allocates resources regardless of the remaining availability and the overall cross - regional pandemic status . in contrast , the dpo model distributes resources trying to balance the impact of actual outbreaks and the expected impact of potential outbreaks . it does so by exploiting region - specific effectiveness of mitigation resources and dynamic reassessment of pandemic spread probabilities , using a set of regression submodels . hence , we believe that in scenarios involving regions with a more heterogeneous demographics , the dpo policy will likely to perform even better and with less variability than the myopic strategy . we also note that the difference in the model performance was particularly noticeable at lower levels of resource availability , which is in accordance with a higher marginal utility of additional availability at that levels . we thus believe that the dpo model can be particularly useful in scenarios with very limited resources . the simulation optimization methodology presented in this paper is one of the first attempts to offer dynamic predictive decision support for pandemic mitigation , which incorporates measures of societal and economic costs . our comparison study of the dpo versus myopic cross - regional resource distribution is also novel . additionally , our simulation model represents one of the first of its kind in considering a broader range of social behavioral aspects , including vaccination and antiviral treatment conformance . the simulation features a flexible design which can be particularized to a broader range of phi and npi and even more granular mixing groups . we also developed a decision - aid simulator which is made available to the general public through our web site at http://imse.eng.usf.edu/pandemics.aspx . the tool is intended to assist public health decision makers in implementing what - if analysis for assessment of mitigation options and development of policy guidelines . examples of such guidelines include vaccine and antiviral risk groups , social distancing policies ( e.g. , thresholds for declaration / lifting and closure options ) , and travel restrictions lack of reliable data prevented us from considering geo - spatial aspects of mixing group formation . we also did not consider the impact of public education and the use of personal protective measures ( e.g. , face masks ) on transmission , again due to a lack of effectiveness data . we did not study the marginal effectiveness of individual resources due to a considerable uncertainty about the transmissibility of an emerging pandemic virus and efficacy of vaccine and antiviral . for the same reason , the vaccine and antiviral risk groups considered in the testbed can be adjusted , as different prioritization schemes have been suggested . the form of social distancing implemented in the testbed can also be modified as a variety of schemes can be found in the literature , including those based on geographical and social targeting . effectiveness of these approaches is substantially influenced by the compliance factor , for which limited accurate data support exists . it will thus be vital to gather the most detailed data on the epidemiology of a new virus and the population dynamics early in the evolution of a pandemic , and expeditiously analyze the data to adjust the interventions accordingly .

aging is a highly complex process that affects various tissues and systems in the body , and which is regulated by many divergent pathways and on many levels . the cell culture does model intriguing aspects of aging , but it may be a little preliminary to call changes over time in culture aging per se . recently , some researchers have used long - term primary cultures of differentiated cells in aging studies [ 37 ] . it is widely thought that senescent changes are relatively more prevalent and severe in postmitotic cells . senescent cells show a series of morphological and physiological alterations , including a flat and enlarged morphology , mitochondrial alterations , chromatin condensation , and changes in gene expression pattern . replicative senescence is defined as a state where normal somatic cells lose their replicative capacity in vitro after prolonged division in culture , an occurrence which results in irreversible growth arrest . the brain contains large numbers of postmitotic cells particularly vulnerable to normal age - related changes , which affect its function . therefore , establishment of a model for neuronal cell aging in vitro may produce valuable information to explore aging at the cellular and molecular levels . several lines of evidence suggest that mitochondria play an important role in the aging process and are affected by aging . one of the hallmarks of age - related mitochondrial function is associated with decreased mitochondrial membrane potential ( m ) and increased reactive oxygen species ( ros ) levels in aging tissues [ 1821 ] or cells from animals of different ages . sa - beta - gal activity is a widely used marker for replicative cellular senescence in vivo and in vitro . recently , several studies have indicated that sa - beta - gal activity can be used as a biomarker for senescence of neurons in vivo and in vitro[2325 ] . previous studies have investigated the relationships between changes in m and [ ca]i during and after a toxic glutamate challenge in cultured rat hippocampal neurons . in addition , parihar et al . compared ros production while simultaneously monitoring m before and after glutamate treatment of live neurons from embryonic , middle - aged , and old rats . age - dependent changes of neuronal survival , protein oxidation , and creatine kinase bb expression in long - term hippocampal cell culture have been examined . in this study , we conducted a systematic parallel observation of the changes in m and ros levels and examined the senescence of neurons by -galactosidase staining . moreover , we have attempted to identify on which day in culture the ros , delta psi or beta - gal suggest irreversible or accelerated deterioration in long - term culture based on the findings . all other reagents were purchased from sigma - aldrich ( st . louis , mo , usa ) unless otherwise stated . primary cultures of hippocampal neurons were prepared according to the published protocols from the laboratories of nelson with modification . briefly , hippocampi were dissected from newborn ( p0 , 024 h ) sprague - dawley rats in ice - cold dissection solution containing sucrose / glucose / hepes ( disgh solution : 136 mm nacl , 5.4 mm kcl , 0.2 mm na2hpo4 , 2 mm kh2po4 , 16.7 mm glucose , 20.8 mm saccharose , 0.0012% phenol red , and 10 mm hepes , ph7.4 ) . isolated hippocampi were mechanically triturated , and then digested in solution containing 0.25% trypsin and 0.02% edta at 37 for 15 min . single cell suspension was obtained by repeated passages of the dissociated tissues through flame - polished pipette in plating medium ( dmem supplemented with 10% heat inactivated fbs and 1% penicillin - streptomycin ) . cells were finally plated on poly - l - lysine ( 0.1 mg / ml ) coated plates at optimal cell density . high density cultures ( 110 cells , 1000 cells / mm ) and lower density cultures ( 310 cells , 310 cells / mm ) , plated onto 6-well culture plates , were used for measuring mitochondrial function and cytochemical studies , respectively . the serum - containing plating medium was replaced by growth medium ( serum - free dmem / f12 medium supplemented with 2% b27 and 1% penicillin - streptomycin ) within 24 h after plating . more than 95% of cells were neurons on 10 day in vitro ( div ) , verified by positive staining of mouse monoclonal anti - nse ( neuron - specific enolase ) ( data not shown ) . all animal procedures were carried out with the approval of the local animal care and use committee . in brief , the monolayers of cells were washed 2 times with phosphate - buffered saline ( pbs ) , fixed with 3% formaldehyde for 35 min , washed 2 times in pbs , and then stained for 18 h at 37 in a co2-free atmosphere with fresh -galactosidase staining solution [ 1 mg / ml 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) , 40 mm citric acid / sodium phosphate , 5 mm potassium ferrocyanide , 5 mm potassium ferricyanide , 150 mm nacl , 2 mm mgcl2 , ph 6.0 ] . after staining , cells were washed twice with pbs and photographed . the percentage of sa--gal - positive cells was determined by counting the number of blue cells within a sample of 1000 cells . m of hippocampal neurons was measured by uptake of lipophilic cation rhodamine 123 ( rh123 ) into mitochondria . about 510 cells were collected at div 5 , 10 , 15 , 20 , 25 and 30 and incubated with 10 g / ml rh123 at 37 for 30 min . then the cells were washed twice with pbs and resuspended in 500 l pbs . intracellular ros production was measured by using a non - fluorescent compound 2 , 7-dichlorofluorescin diacetate ( dcfh - da ) , which can be converted to dcfh by esterases when taken up . dcfh reacts with ros to generate a new highly fluorescent compound , dichlorofluorescein , which can be analyzed with flow cytometry . about 510 cells at different ages were incubated with 20 m dcfh - da at 37 for 30 min , washed twice with pbs , and then measured with flow cytometry . data are presented as mean standard deviation ( sd ) calculated from at least 3 separate experiments . the data were analyzed by one - way anova followed by student - newman - keuls test using spss 11.0 software . all other reagents were purchased from sigma - aldrich ( st . louis , mo , usa ) unless otherwise stated . primary cultures of hippocampal neurons were prepared according to the published protocols from the laboratories of nelson with modification . briefly , hippocampi were dissected from newborn ( p0 , 024 h ) sprague - dawley rats in ice - cold dissection solution containing sucrose / glucose / hepes ( disgh solution : 136 mm nacl , 5.4 mm kcl , 0.2 mm na2hpo4 , 2 mm kh2po4 , 16.7 mm glucose , 20.8 mm saccharose , 0.0012% phenol red , and 10 mm hepes , ph7.4 ) . isolated hippocampi were mechanically triturated , and then digested in solution containing 0.25% trypsin and 0.02% edta at 37 for 15 min . single cell suspension was obtained by repeated passages of the dissociated tissues through flame - polished pipette in plating medium ( dmem supplemented with 10% heat inactivated fbs and 1% penicillin - streptomycin ) . cells were finally plated on poly - l - lysine ( 0.1 mg / ml ) coated plates at optimal cell density . high density cultures ( 110 cells , 1000 cells / mm ) and lower density cultures ( 310 cells , 310 cells / mm ) , plated onto 6-well culture plates , were used for measuring mitochondrial function and cytochemical studies , respectively . the serum - containing plating medium was replaced by growth medium ( serum - free dmem / f12 medium supplemented with 2% b27 and 1% penicillin - streptomycin ) within 24 h after plating . more than 95% of cells were neurons on 10 day in vitro ( div ) , verified by positive staining of mouse monoclonal anti - nse ( neuron - specific enolase ) ( data not shown ) . all animal procedures were carried out with the approval of the local animal care and use committee . in brief , the monolayers of cells were washed 2 times with phosphate - buffered saline ( pbs ) , fixed with 3% formaldehyde for 35 min , washed 2 times in pbs , and then stained for 18 h at 37 in a co2-free atmosphere with fresh -galactosidase staining solution [ 1 mg / ml 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) , 40 mm citric acid / sodium phosphate , 5 mm potassium ferrocyanide , 5 mm potassium ferricyanide , 150 mm nacl , 2 mm mgcl2 , ph 6.0 ] . after staining , cells were washed twice with pbs and photographed . the percentage of sa--gal - positive cells was determined by counting the number of blue cells within a sample of 1000 cells . m of hippocampal neurons was measured by uptake of lipophilic cation rhodamine 123 ( rh123 ) into mitochondria . about 510 cells were collected at div 5 , 10 , 15 , 20 , 25 and 30 and incubated with 10 g / ml rh123 at 37 for 30 min . intracellular ros production was measured by using a non - fluorescent compound 2 , 7-dichlorofluorescin diacetate ( dcfh - da ) , which can be converted to dcfh by esterases when taken up . dcfh reacts with ros to generate a new highly fluorescent compound , dichlorofluorescein , which can be analyzed with flow cytometry . about 510 cells at different ages were incubated with 20 m dcfh - da at 37 for 30 min , washed twice with pbs , and then measured with flow cytometry . data are presented as mean standard deviation ( sd ) calculated from at least 3 separate experiments . the data were analyzed by one - way anova followed by student - newman - keuls test using spss 11.0 software . dissociated cells from the hippocampus of newborn rat brain were plated at a cell density of 310 cells / mm . within the first 2 days after plating once stabilized , the number of surviving neurons remained constant up to 25 days and then started to decline . photomicrographs of rat hippocampal neurons at different ages in culture are shown in figure 1 . the first 15 days was a period of maturation of young neurons in culture , during which they formed an extensive network of processes and increased the size of their cell bodies ( figure 1a c ) . mature cells exhibited an increase in the size of the cell bodies , thick dendritic processes , and an extremely dense and extensive network ( figure 1c , d ) . neurons with vacuolated soma and beaded or fragmented neurites were observed after div 20 , and then the number of neurons decreased after div 25 ( figure 1e , f ) . senescent cells display visibly increased -galactosidase activity at ph 6.0 when measured in situ . in order to establish a link between senescence and culture time the proportion of the senescent cells steadily increased in neuron cultures ( figure 2a f ) . more than 90% of cells stained sa--gal positive at div 25 and div 30 ( figure 2 g ) . to analyze the alteration in m in long - term culture , we examined the m by using the mitochondria - specific dye rh123 at div 5 , 10 , 15 , 20 , 25 and 30 . as shown in figure 3 , there was an age - related decrease of m in neurons . the fluorescence intensity of rh123 of div 25 neurons and div 30 neurons decreased to 71% and 59% , respectively , of the levels of div 10 neurons . as shown in figure 4 , there was a time - associated increase of ros generation in neurons . the fluorescence intensity of dcfh of div 25 neurons and div 30 neurons increased to 178% and 215% , respectively , of the levels of div 10 neurons . dissociated cells from the hippocampus of newborn rat brain were plated at a cell density of 310 cells / mm . within the first 2 days after plating once stabilized , the number of surviving neurons remained constant up to 25 days and then started to decline . photomicrographs of rat hippocampal neurons at different ages in culture are shown in figure 1 . the first 15 days was a period of maturation of young neurons in culture , during which they formed an extensive network of processes and increased the size of their cell bodies ( figure 1a c ) . mature cells exhibited an increase in the size of the cell bodies , thick dendritic processes , and an extremely dense and extensive network ( figure 1c , d ) . neurons with vacuolated soma and beaded or fragmented neurites were observed after div 20 , and then the number of neurons decreased after div 25 ( figure 1e , f ) . senescent cells display visibly increased -galactosidase activity at ph 6.0 when measured in situ . in order to establish a link between senescence and culture time the proportion of the senescent cells steadily increased in neuron cultures ( figure 2a f ) . more than 90% of cells stained sa--gal positive at div 25 and div 30 ( figure 2 g ) . to analyze the alteration in m in long - term culture , we examined the m by using the mitochondria - specific dye rh123 at div 5 , 10 , 15 , 20 , 25 and 30 . as shown in figure 3 , there was an age - related decrease of m in neurons . the fluorescence intensity of rh123 of div 25 neurons and div 30 neurons decreased to 71% and 59% , respectively , of the levels of div 10 neurons . as shown in figure 4 , there was a time - associated increase of ros generation in neurons . the fluorescence intensity of dcfh of div 25 neurons and div 30 neurons increased to 178% and 215% , respectively , of the levels of div 10 neurons . furthermore , these results indicate that long - term culture primary hippocampal neurons may serve as a mitochondria dysfunction model associated with aging , and that div 25 neurons could possibly represent aging neurons in culture . there has been a certain similarity between whole animal neuron aging and neuronal aging in culture . long - term culture of cerebellar granule neurons showed changes in [ ca]i that were strikingly similar to the age - dependent changes recorded in cerebellar brain slice preparations . long - term culture of hippocampal neurons reproduced the pattern of changes in ca channel density observed in brain slices obtained from aging rats . the morphological changes of mitochondria in long - term primary neuronal culture were consistent with the results in vivo . in addition , parihar et al . monitored m simultaneously with measuring ros on an individual mitochondrial basis in single live hippocampal neuronal cells isolated from embryonic ( e18 ) , middle - aged ( 12 months ) , and old ( 24 months ) rats , and maintained in a uniform culture environment . the results showed an age - related increase in ros production and an age - related depolarization at rest in single somal and axonal / dendritic mitochondria . used fep ( fluorinated ethylene - propylene)-sealed culture dishes for culturing cells that maintains their health and sterility for over a year . they reduced or eliminated problems with infection and hyperosmolality , while maintaining ph and o2 homeostasis . using conventional techniques , increases in the osmotic strength of media due to evaporation are a large and underappreciated contributor to the gradual decline in the health of primary neuron cultures . we applied sa--gal staining to identify senescent hippocampal neurons in long - term culture , and our results show that sa--gal activity gradually increased . these results , in agreement with those obtained in other studies , support the use of sa - beta - gal activity as a biomarker for senescence of neurons in vivo and in vitro . depolarized m in aged cells appears to be one of the features of age - dependent alterations of mitochondrial function . one proposed mechanism to explain depolarized m in aged cells is the alteration in the activity of the respiratory chain complexes . the results showed that m depolarized in senescent neurons , which was in agreement with previous reports on exocrine cells , hepatocytes and neurons [ 22 39 ] . moreover , depolarized m produces more ros on cardiac myocytes , neurons , and hela cells . mitochondria , by virtue of their intense respiratory chain activity , are the major source of ros , and also constitute a main target of cumulative oxidative stress . a large body of experimental evidence suggests that ros production is increased in aging tissues . hence , mitochondria - generated ros have been implicated as a common feature that connects aging of organisms and age - related diseases . our data showed that ros levels increased with age in vitro , which is consistent with the observations in vivo . a decrease in m has been shown to be associated with an increase in ros in a variety of experimental models . in summary , our results demonstrate a decrease in mitochondrial function with days in culture . furthermore , we identify that div 25 neurons could be an ideal time point for modeling age - related mitochondrial dysfunction in culture under our experimental conditions , which may contribute to the exploration of relationship between mitochondria dysfunction and aging , and may be useful for future anti - aging studies .

loss of reduction following closed or open reduction of displaced supracondylar fractures of the humerus in children varies widely and is considered dependent on stability of the fracture pattern , gartland type , number and configuration of pins for fixation , technical errors , adequacy of initial reduction , and timing of the surgery . this study was aimed to evaluate the factors responsible for failure of reduction in operated pediatric supracondylar fracture humerus . we retrospectively assessed loss of reduction by evaluating changes in baumann 's angle , change in lateral rotation percentage , and anterior humeral line in 77 consecutive children who were treated with multiple kirschner wire fixation and were available for followup . the intraoperative radiographs were compared with those taken immediately after surgery and 3 weeks postoperatively . technical errors were significantly higher in those who lost reduction ( p = 0.001 ; odds ratio : 57.63 ) . lateral pins had a significantly higher risk of losing reduction than cross pins ( p = 0.029 ; odds ratio : 7.73 ) . other factors including stability of fracture configuration were not significantly different in the two groups . the stability of fracture fixation in supracondylar fractures in children is dependent on a technically good pinning . fracture pattern and accuracy of reduction were not important factors in determining the stability of fixation . supracondylar fractures in children are one of the commonest upper limb fractures in the first decade of life.1 the gartland type 3 displaced and many of the type 2 supracondylar fractures are stabilized with kirschner ( k ) wire fixation after reduction.12 the stability after fracture fixation has been the subject of much debate in the past.3 the quality of reduction , configuration of wire fixation , i.e. crossed or parallel , timing of surgery , medial or lateral entry for placement of pin , number of pins , and more recently fracture configuration and the inherent instability of the fracture pattern have all been considered as important factors contributing to the stability of fixation.37 the technical errors play an important role in the final outcome.67 some centers have guidelines on how to pin these injuries , and very often the fracture pattern is not taken into account even though some patterns are considered more unstable than others and deemed to require specific methods of fixation.3 while there are many articles dealing with various aspects of supracondylar fracture stability after fixation , there is very little written about the role of the fracture pattern and its effect on the stability of reduction.48 in this study , we have analyzed various factors including the bahk's4 fracture types and their effects with respect to the fracture stability following fixation , and the loss of fixation postoperatively . instability attributable to bahk 's fracture pattern has not been evaluated as a possible confounder when assessing stability characteristics of different fixation techniques . a retrospective analysis of ninety consecutive supracondylar fractures of humerus in children admitted from july 2004 to october 2009 operated by 15 different surgeons and trainees were included in this study . clinical data and radiographs were obtained from stored clinical records , computerized hospital information processing services ( chips ) , and picture archiving and communication system ( pacs ) . data related to age , gender , side , fracture configuration , surgical details , pin configuration , and timing of surgery were collected . gartland type 2 or 3 fractures treated with closed reduction without internal fixation , fractures with intercondylar extension and osteogenesis imperfecta were then excluded . the fracture patterns of all included children were classified using the preoperative and intraoperative images with bakh 's criteria [ table 1]4 and [ figure 1 ] . distribution of unstable fracture configurations types and their effect on stability ( modified from bahk et al . 2008 ) x - ray of elbow joint various fracture patterns as described by bahk et al . 2008 faulty technique was defined for this study as a ) the lack of fixation of one or more wires either in the distal or in the proximal fragment [ figures 2 and 3 ] b ) convergence of the pin in the far cortex [ figure 4 ] c ) pins crossing each other at the fracture site [ figure 5 ] . fixation of four cortices , two in the proximal and two in the distal fragment , with adequate separation to fix two columns was considered a good fixation for the purpose of this study [ figure 6 ] . ( a ) anteroposterior and lateral images of distal humerus demonstrating the reduction intraoperatively in a displaced supracondylar fracture of the humerus . ( b ) immediate postoperative radiograph nteroposterior and lateral views of distal humerus showing loss of reduction . ( c ) three dimensional computed tomogram showing that the lateral wire failed to get adequate purchase in distal fragment . the child was taken up for repeat surgery due to major loss of reduction ( a ) anteroposterior and lateral radiographs showing a low transverse gartland type 3 extension supracondylar fracture humerus . ( b ) immediate postoperative and followup radiographs showing malreduced fracture with no distal purchase ( marked with arrow ) . there was no loss of reduction despite technically poor pinning ( a ) anteroposterior and lateral radiographs showing type 3 gartland high transverse and sagittal oblique fracture pattern . ( b ) intraoperative anteroposterior and lateral radiographs showing acceptable reduction with technically poor pinning ( convergence of lateral entry pins ) . ( c ) followup radiographs at 4 weeks show maintenance of reduction in spite of biomechanically poor construct with inadequate spread ( a ) intraoperative anteroposterior and lateral images showing good reduction . the anteroposterior distal to proximal pin does not have a satisfactory hold in the proximal fragment . ( b ) there is minor loss of reduction with malalignment and rotation at the fracture site x - ray of elbow joint anteroposterior and lateral views showing ( a ) displaced supracondylar fracture humerus type iii extension low transverse fracture pattern . ( c ) maintenance of reduction and fixation at 4 weeks adequacy of intraoperative reduction and postoperative position was assessed using baumann 's angle , lateral rotation percentage , and anterior humeral line.35 the above were measured from intraoperative images or immediate postoperative radiographs and compared with radiographs taken at cast removal and the difference in the position recorded . loss of reduction was quantitated by criteria defined by skaggs et al . and it has been shown that a change in the baumann 's angle is consistent with a loss of reduction.9 greater than 12 is a major loss of reduction and 612 a moderate loss of reduction.10 for this study , we considered the moderate and major loss of reduction as defined by the above criteria.3 the stability of reduction was analyzed by assessing loss of reduction in the cast postoperatively . analysis was carried out using student 's t test for the effect of individual variables on loss of reduction . the effect of quality of initial reduction and technically sound pinning on the loss of reduction was analyzed by fisher 's exact test . all significant factors were further evaluated with a logistic multivariate regression analysis to eliminate the effect of confounding factors . of the 90 children with supracondylar fractures of the humerus , 1 child was lost to follow up , 1 was treated with dunlop traction , 1 had osteogenesis imperfecta , 1 had intercondylar extension , and 9 patients were having type 2 gartland fractures , which were managed with closed reduction and casting . the data from the remaining 77 children , who underwent operative fixation , were further analyzed . indication for pinning in a gartland type 2 fracture was either gross swelling or medial comminution . sixty four of the children underwent closed reduction and 13 underwent open reduction followed by pinning . in the second group , four were open fractures requiring wound debridement , two patients had vascular injury with ischemia and required saphenous vein grafting , eight patients had a median nerve palsy , and one child had a radial and ulnar nerve palsy documented at presentation . one child was documented to have radial and median nerve palsy and another had ulnar and median nerve palsy noticed after surgery . three children underwent a second reduction in the postoperative period because of a major loss of reduction . the fracture patterns were classified as low transverse in 62 , medial oblique in 3 , high sagittal oblique in 4 , lateral oblique in 5 , and high transverse in 3 cases [ table 1 , figure 1 ] . the 62 low transverse fractures were classified as stable and the other 15 as unstable fractures.4 loss of reduction occurred in 14 ( 18.2% ) of the cases . the children who lost reduction had a mean intraoperative lateral rotation percentage of 14.69% and after loss of reduction a change in the average lateral rotation percentage of 41.2% . a moderate to severe bowmann angle change of greater than 6 was seen in all except three of these children . in the three children who had a second reduction , the loss of reduction was diagnosed based on a change in the anterior humeral line intersection and lateral rotation percentage change varying from 25 to 48% . in other cases , eventually , however , of all the 14 who lost reduction , only one child had clinical cubitus varus . reduction was lost in 12 ( 19.4% ) of the 62 stable cases and 2 ( 13.3% ) of the 15 unstable cases . those with lateral pins only , namely , a ) two lateral pins b ) three lateral pin ; and others with both medial and lateral pins , namely , c ) crossed pins with one medial and one lateral entry pin and d ) two lateral and one medial entry pins . in the lateral entry configuration , ( a ) two lateral pins were used in 20 cases and ( b ) three lateral pins in 9 cases . of these 29 children with lateral entry pins , 10 ( 34.5% ) showed postoperative loss of reduction . configurations with at least one medial pin were ( c ) crossed pins in 28 cases and ( d ) two lateral pins and one medial pin in 20 cases . in the groups which included at least one medial pin , 4 ( 8.3% ) out of 48 cases lost reduction . a fischer 's exact test showed a statistically significant difference between the two groups ( p < 0.0046 ) . the cases with loss of reductions were analyzed to assess the effect of faulty techniques . thirty six cases with faulty techniques were identified based on the criteria defined earlier ; 14 ( 38.9% ) of these showed postoperative loss of reduction and all 41 cases who underwent technically sound pinning showed no loss of reduction . the technical errors in those who lost reduction in this study were no purchase in the distal fragment in five ( two lateral and three medial ) cases , no purchase in the proximal fragment cortex in two cases , and inadequate spread in seven cases . an analysis was done to find out if the presence of the third pin influenced the postoperative loss of reduction . four cases in the former and 10 in the latter group showed a postoperative loss of reduction . total number of cortical purchase by the pins used for fixation was classified as those with a ) four cortical purchases or b ) less than four cortical purchases . sixty six cases had four and 11 cases had less than four cortical purchases of pins . eleven cases in the former and three cases in the latter group had loss of reduction . forty cases were done in the night ( from 8 p.m. to 8 a.m. ) and 37 cases in the day ( from 8 a.m. to 8 p.m. ) . seven in each group lost reduction which was not statistically significant ( p = 1.00 ) . the quality of initial reduction was assessed based on baumann 's angle and anterior humeral line . anatomical reduction has been defined as anterior humeral line passing through middle of capitellum , restoration of baumann angle , and intact medial and lateral column.11 the acceptable reductions were those with minor degrees of malreduction in baumann angle 6 as per skagg 's criteria.8 there were 33 cases with anatomical initial reduction , of which 19 had technically sound and 6 had technically poor fixation . forty cases had acceptable initial reduction of which 19 had technically sound fixation and 16 had technically poor fixation . the quality of initial reduction did not affect the final result , provided the pinning was technically sound as shown by fisher 's exact test ( p = 0.342 ) . multivariate logistic regression analysis [ table 2 ] showed that lateral only pinning ( odds ratio : 7.73 ; 95% confidence interval ; 1.2348.60 ; p = 0.029 ) and technical errors ( odds ratio : 57.63 ; 95% confidence interval 4.72703.04 ; p = 0.001 ) were independently contributing to the postoperative loss of reduction . gartland type , number of pins , fracture pattern , timing of surgery , quality of initial reduction , and number of cortical purchase were not significantly responsible for the postoperative loss of reduction . the loss of reduction after k wire fixation for supracondylar fracture surgery varies widely from 1.6 to 33.3%.12 our series had a 18.2% loss of reduction and the causes and prevention of this are the main focus of this study . multiple factors such as the timing of the surgery , quality of reduction , pin configuration , and technique of pinning such as spread of the pins have been implicated in poor outcomes ; adequate pin spread to two columns and specific configurations are suggested for oblique and high fractures.3613 number of studies have emphasized the importance of stability which is influenced by the gartland type , loss of periosteal hinge , and comminution.8 recently , unstable fracture patterns have been defined on the basis of the site and obliquity of the fracture.4 these have been shown to have a high incidence of malunion , and specific pin configurations and greater monitoring are recommended.4 in this study , we were unable to detect a significant postoperative reduction loss in these unstable patterns when comparing them with the typical transverse supracondylar fractures , indicating that other factors were responsible for loss of reduction . multidirectionally unstable fracture has been defined recently where there is demonstrable flexion extension instability under fluoroscopy . intraoperative instability under fluoroscopy of this type , however , was not documented in our patients.8 we were also unable to find a significant difference in the loss of reduction in gartland type 2 and type 3 fractures . this may be due to a very small number of gartland type 2 fractures in the series or no real difference if the fixation provides adequate stability . there was no difference in the loss of reduction with respect to the timing of surgery in our series . this report is in concurrence with many previous studies which found no difference in those cases treated immediately within 8 h of injury and those treated more than 8 h after the injury.1319 it is suggested that the surgeons may not be at their best during night due to inadequately trained supporting personnel and nonavailablity of adequate instruments in the night , so that an optimal condition for operating this fracture may not be available.20 however , a child with grossly swollen , ecchymosed elbow or with neurovascular compromise should be operated at the earliest.21 in our tertiary care center , round - the - clock availability of consultants and adequately staffed operating room personnel are probably the reasons that there was no significant difference noted in the loss of reduction in the groups operated in the day and in the night in this series . a systematic review and a prospective series had elucidated that the chances of converting to open reduction increased with delay in closed reduction and pinning.2223 ramachandran et al . in an analysis of a retrospective case series concluded that delay in management of low energy supracondylar fracture humerus with gross swelling increased the risk of compartment syndrome.24 the analysis , however , showed that in selected cases , delay in pinning a type iii supracondylar fracture humerus did not adversely affect the outcome . the decision to delay can be made on a case - by - case basis after considering the logistic constraints like the capacity of the team and the urgency of the clinical situation . an important component of management of these fractures is an anatomical reduction.25 bloom et al . analyzed the effect of residual internal rotation after reduction in a biomechanical study on the stability of pin constructs.25 the study concluded that stability of supracondylar fractures malreduced in internal rotation ( within acceptable limits ) was compromised and a third pin configuration was advocated to increase the construct stiffness . analysis of our results concedes that even with acceptable malreduction , a technically sound pinning can avert a major loss of reduction . a systematic review12 has shown that cross pinning is superior to lateral entry pinning in maintaining reduction . our findings concur with the above and show significantly higher odds of loss of reduction , 7.73 times when the patient has lateral entry pins only constructs . biomechanical studies have shown that chances of rotational loss of reduction in lateral only constructs are high compared with cross pinning , indicating that cross pinning has greater torsional stability.2627 additional medial comminution in the fracture increases the chance of varus collapse if the fracture is rotationally unstable after lateral only pinning.11 in spite of a very clear association between loss of reduction and lateral entry pinning with two or three pins , one of the arguments for using a lateral pin construct is a documented higher incidence of ulnar nerve injury when a medial pin is used.28 proponents of cross pinning suggest that the injury to ulnar nerve can be avoided by a small medial incision and keeping the elbow extended during the pinning.6 on the other hand , even lateral pin insertion is not without risks , and a recent meta - analysis29 has shown that the lateral entry pin fixation carries the risk of median nerve injury , indicating that lateral entry pin is not free of nerve injury . this meta - analysis has shown that both lateral and medial entry pin has risk of neuropraxia at a weighted event rate of 3.4% and 4.1% , respectively . a 2% incidence of transient ulnar nerve paresis was recorded in the cross pinning cases ( 1/49 ) in our series , consistent with the published results . zenios et al.30 have expressed technical difficulty in achieving adequate pin placement in lateral entry pin . to get an adequate pin spread and separation at fracture site and at the same time engage both there have been at least two series which have suggested doing a test of stability after lateral pin fixation and , if unstable , an additional third pin is added either on the lateral or medial side.330 this again suggests that it is accepted by these authors that the lateral only pin constructs are susceptible to rotational instability . screening of the elbow in lateral view in internal and external rotation after fixation is recommended to assess the stability intraoperatively . such a screening test is now part of the protocol after fixation of supracondylar fractures in our center and has helped substantially to identify instability often due to faulty pin fixation and bring down the loss of reduction for these cases currently in our center . analysis of our data established the faulty implant placement as the major cause for loss of reduction . all 14 of our patients who lost reduction had at least one contributing technical error . the errors were equally divided between insufficient purchase by the pins in either the proximal or the distal fragment and a convergence of pins in the far cortex in the lateral only entry technique . a fracture treated with technical errors had a 57.63 times higher odds of losing reduction in our series . the most common error in low transverse fracture pattern was poor purchase of one of the pins in either the proximal or the distal fragment , resulting in rotational instability in the postoperative period . the additional analysis looking at total number of cortical holds of the pins and quality of initial reduction did not reach significance in our study . this could be due to the fact that while four cortical holds can be obtained with three pins , there still exists the possibility of having two pins with inadequate purchase in either of the fragments , resulting in an inadequate fixation . our interpretation from this is that it is better to remove a pin which is not technically correct and resite it rather than adding a third pin . if a technically sound pinning is done , then an acceptable reduction which may not be anatomical can go on to union without further loss of reduction . additional pin ( third pin ) analysis also showed no significant benefit , thus supporting our contention . this is biomechanically inferior as it creates a single hold in the proximal fragment and is thus responsible for loss of reduction . technical errors have been found contributory to loss of reduction in other series as well.56911 sankar et al . found three technical errors similar to those seen in our series.6 it is likely that some of the errors in pin placements are induced by imperfect reduction.39 the limitation of this study is that this being a retrospective study , the documentation of the reasons such as instability for choosing a particular kind of pin fixation or the results of test of stability if carried out was not always available . this would have given an insight into other types of instability such as those due to soft tissue disruption which are only recognized intraoperatively and may not be recorded . most children are discharged from the clinic once they regain function and the long term follow up is not available to see the clinical effects on rotational and varus deformity . we would suggest that the role of instability and pin configuration in supracondylar fracture fixation be further validated by a prospective randomized trial . our observations suggest that the loss of reduction following fracture fixation is closely related to technical errors which result in inadequate purchase of the fragments by the pins or errors due to inadequate spread in lateral pinning . cross pinning was found to be superior to an all lateral pin configuration . while the intraoperative understanding of the fracture pattern may help in achieving good fixation , factors such as the unstable fracture patterns , quality of initial reduction , and timing of surgery were not a cause for loss of reduction in our experience .

a balance between energy intake and energy expenditure is responsible for body weight maintenance ; disturbances in this balance may cause individuals to be underweight , anorexic , overweight , or obese [ 1 , 2 ] . signals secreted by peripheral tissues , such as fat or the digestive tract , are active participants in body weight regulation by acting at central level . skeletal muscle has recently been shown to function as a peripheral endocrine organ by releasing peptide signal molecules known as myokines , which are implicated in the regulation of several physiological and metabolic pathways [ 4 , 5 ] . myokines secreted during muscle contraction may exert a protective role against illnesses associated with sedentary lifestyles . the muscle - secreted peptide irisin has been recently described as a cleavage product of the type i membrane protein fibronectin type iii domain containing 5 ( fndc5 ) . in muscle , fndc5 is induced by the peroxisome proliferator - activated receptor ( ppar ) transcriptional coactivator pgc1- , which is described as an essential mediator in the exercise - induced energy expenditure cascade and stimulates several beneficial effects of exercise in muscle tissue [ 7 , 8 ] . after physical activity , fndc5 has been reported to promote white adipose tissue browning by increasing thermogenesis and energy expenditure . thus , augmentation of circulating irisin levels by forced liver expression of fndc5 using adenoviral vectors improves glucose tolerance and reduces obesity of mice fed on a high - fat diet . under this context , irisin was rapidly postulated to be beneficial in the treatment of obesity , diabetes , and a wide range of pathological conditions characterized by a variable imbalance of energy demand and expenditure [ 4 , 913 ] . unexpectedly , some studies have indicated that the circulating irisin levels in humans correlate positively with parameters of adiposity such as bmi and are the highest in obese individuals [ 1417 ] . on the other hand , a recent paper reports that irisin correlates negatively with bmi , waist - hip ratio , and fat mass in men and that circulating irisin is lower in nondiabetic overweight and obese men [ 18 , 19 ] . our group has established a new perspective for fndc5 by describing that fndc5/irisin is also an adipokine expressed and secreted by adipocytes from white adipose tissue ( wat ) in rats and humans . we have observed that short - term endurance exercise increases fndc5/irisin secretion by adipose tissue , while food restriction decreased secretion . accordingly , anorectic animals showed discrete secretion of fndc5/irisin , while fat from obese animals oversecretes fndc5/irisin . thus , we postulated that adipose tissue may contribute to circulating fndc5/irisin and that the contribution may vary depending on the physiological state or the pathological situation . the objective of this study was to evaluate the circulating levels of irisin in conditions of extreme bmi , such as anorexia and obesity , and the correlations of irisin with basal metabolism and daily activity . a total of 145 females participated in this study after being informed about the research and signing informed consent . thirty patients were anorexic ( an ; bmi 17.3 1.8 kg / m ; 28.1 9.4 years ) , 66 were obese ( ob ; bmi 42.8 6.7 kg / m ; 29.0 6.2 years ; 21.2% with type 2 diabetes mellitus ) , and 49 were healthy , normal - weight controls ( nw ; bmi 21.7 1.6 kg / m ; 44.5 11.4 years ) . all consecutive patients were referred to seven centers from six spanish sites ( all involved in the ciberobn spanish research network ) : the eating disorders unit ( department of psychiatry , university hospital of bellvitge - idibell , barcelona ) , the department of endocrinology at the university hospital of santiago ( santiago de compostela ) , the department of diabetes , endocrinology and nutrition ( clinic university hospital virgen de victoria , mlaga ) , the department of endocrinology ( university of navarra , pamplona ) , the diabetes , endocrinology and nutrition department , biomedical research institute of girona ( idibgi - doctor josep trueta hospital , girona ) , the hospital del mar research institute ( imim - hospital del mar , barcelona ) , and the department of basic psychology , clinic and psychobiology ( university jaume i , castelln ) . the an patients were diagnosed by experienced clinicians ( according to dsm - iv - tr diagnostic criteria ) using the structured clinical interview for dsm iv axis i disorders ( scid - i ) . these an patients at extreme bmi were hospitalized , hence showing weaken health and very limited physical activity . the following exclusion criteria were applied to the clinical cases groups ( an and obese patients ) : ( 1 ) male ; ( 2 ) age under 18 or over 60 years , and ( 3 ) a comorbid binge eating disorder in the obese patients ( according to dsm - iv - tr criteria , using scid - i ) . healthy controls were recruited by several methods including word - of - mouth recruitment and advertisements in the local university . prior to assessment , the healthy controls were asked about lifetime or current presence of an endocrine disease or obesity . the lifetime history of health or mental illnesses profile was based on the general health questionnaire ( ghq)-28 . the following exclusion criteria were applied to the healthy control groups ( nw ) : ( 1 ) individuals who have suffered a lifetime eating disorder ( assessed using scid - i ) , ( 2 ) age under 18 and over 60 years , ( 3 ) lifetime obesity ( bmi > 30 ) , and ( 4 ) being male . anthropometric , body composition , and resting energy expenditure ( ree ) measurements were performed by bioelectrical impedance ( tanita mc-180 multifrequency ; tanita corporation of america , inc , arlington heights , il , usa ) , equipped with 8 tactile electrodes with a platform with 2 electrodes for each foot and 2 handgrips with 2 electrodes each . the bioelectrical impedance analysis was chosen since it is able to predict the ree without bias with respect to more costly equipment and invasive procedures needed to directly measure ree . , cambridge neurotechnology , cambridge , uk ) , which is a small ( 39 32 9 ) and light - weight ( 10.5 g ) accelerometer that measures sleep - wake physical activity . the actiwatch is worn on the nondominant wrist for 6 days ( 4 week days and 1 weekend ) , from 00:00 hr on day 1 to 00:00 hr on day 7 . the data were collected over a 1-minute epoch length , and a 6-day average was calculated . the analysis was performed using the actiwatch 7 software ( camntech ltd . and cambridge neurotechnology ltd . , cambridge , uk ) . the actiwatch aw7 originates from the actiwatch aw4 , which has similar reliability to other accelerometers and is an adequate instrument for assessing daily physical activity . the quantitative measurement of irisin in human plasma samples was performed using a commercial enzyme - linked immunosorbent assay ( elisa ) kit directed against amino acids 31143 of the fndc5 protein ( irisin elisa kit ek-067 - 52 ; phoenix pharmaceuticals inc . the absorbance from each sample was measured in duplicate using a spectrophotometric microplate reader at wavelength of 450 nm ( versamax microplate reader ; associates of cape cod incorporated , east falmouth , ma ) . insulin was analysed by an immunoradiometric assay ( biosource international , camarillo , ca , usa ) in a beckman coulter ( fullerton , ca , usa ) , showing 0.3% cross - reaction with proinsulin . glucose was measured using a dimension autoanalyzer ( dade behrng , deerfield , il , usa ) . the homeostatic model assessment index ( homa - ir ) was calculated following the formula ( fasting plasma glucose ( mg / ml ) fasting plasma insulin ( mu / l)/405 ) , as described elsewhere . the human adipose tissue specimen was obtained with written informed consent approved for this particular study by the comit tico de investigacin clnica de galicia ceic de galicia ( spain ) according to the declaration of helsinki . adipose tissue was obtained from nw ( body mass index < 35 ) who underwent cholecystectomy surgery and ob patients ( body mass index > 35 ) who underwent laparoscopic gastrectomy surgery . the visceral fat was located in the hypogastric region around the internal organs , and the subcutaneous fat was located in the mesogastric region . the tissues were transported from the operating room to the laboratory in sterile krh buffer with penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) . secretome and tissues were collected and processed for immunodetection as previously described [ 5 , 16 ] . the sample size of the current trial was estimated by calculating the differences in irisin between the ob and nw patients according to the equation reported by mera et al . . the sample size was established at a minimum of 26 volunteers per group to detect differences according to the adiposity levels . the normal distribution of the variables was tested using the kolmogorov - smirnov and the shapiro - wilk tests . however , based on the sample size of > 60 subjects included in the current study , a one - way anova was used to study the differences between an , nw , and ob subjects adjusted by age . the potential association between anthropometric and biochemical parameters with and irisin levels was evaluated using the pearson coefficient test . multivariate linear regression models were fitted to explain the variations in the irisin plasma levels . data are reported as the mean se , and confidence intervals ( 95% ci ) are used to describe the linear coefficient ( b ) values . as expected , the ob subjects exhibited higher body weight with higher fat mass and fat - free mass compared with the an and nw subjects , with the differences being statistically significant after adjusting by age ( figure 1(a ) ) . the nw subjects exhibited statistically higher body weight compared with the an subjects accompanied by higher fat mass and fat - free mass . considering the body composition as a percentage of the total body weight , the an subjects exhibited higher fat - free mass than the nw and ob subjects ( figure 1(a ) ) . the differences in the anthropometrical parameters were consistent with the significantly increased plasma irisin concentrations in the ob subjects compared with the an and nw groups ( figure 1(b ) ) . the nw subjects exhibited a slight but not statistically significant increase compared with the an subjects ( p > 0.05 ) . further analysis showed that the irisin levels were positively correlated with two parameters of adiposity ( i.e. , body weight ( r = 0.52 ; p < 0.001 ) and bmi ( r = 0.52 ; p < 0.001 ) ) . the irisin plasma levels were not correlated with height ( r = 0.05 ; p > 0.05 ) . the correlations indicated that irisin strongly reflects the body fat mass component , which is further supported by the bioelectrical impedance analysis of body composition in which the irisin plasma levels correlated with the fat mass measured in kg ( r = 0.52 ; p = 0.001 ; figure 2 ) and the percentage of fat mass ( r = 0.48 ; p < 0.001 ) . however , the muscle component may also contribute to irisin circulating levels as shown by the positive correlation between irisin and fat - free mass ( r = 0.43 ; p = 0.001 ; figure 2 ) . an inverse correlation was observed between the irisin levels and the percentage of fat - free mass ( r = 0.53 ; p < 0.001 ) . the associations between irisin and fat mass and fat - free mass were maintained after adjusting for the studied groups ( data not shown ) . in addition , a direct association was observed between plasmatic levels of irisin and glucose ( r = 0.22 ; p = 0.0026 ) , insulin ( r = 0.34 : p < 0.001 ) , and homa - ir ( r = 0.33 ; p = 0.001 ) . this positive association was maintained especially for insulin and homa - ir after adjusting for the studied groups . because irisin secretion was shown to be induced by exercise and energy expenditure , the daily physical activity and resting energy expenditure ( ree ) were evaluated in the three bmi groups . the actiwatch analysis in the ob subjects exhibited statistically lower daily physical activity with higher resting energy expenditure ( ree ) compared with the nw subjects adjusted by age ( figures 3(a ) and 3(b ) , resp . ) . therefore , the ob subjects exhibited higher daily physical activity compared with the extreme an hospitalized subjects displaying very limited activity but lower activity compared with the nw subjects ( p < 0.05 ) . obesity was accompanied by increased ree compared with the an and nw subjects ( p < 0.05 ) . the plasma irisin levels were inversely correlated with daily physical activity ( r = 0.22 ; p = 0.001 ) and directly correlated with ree ( figures 3(c ) and 3(d ) ) . after adjusting by the bmi group , only the association between irisin and ree was maintained ( r = 0.34 ; p = 0.001 ) , and the daily physical activity was not statistically significant ( r = 0.06 ; p = 0.432 ) . to further investigate the potential factors involved in the changes in the irisin plasma levels , a multiple regression model was performed including ree and physical activity , adjusting for age , and bmi group ( table 1 ) . < 0.001 ) of the irisin levels variability , mainly depending on ree , whereas physical activity was not statistically associated . when fat free mass was included in the regression model , the association between ree and irisin plasma levels persisted . however , when the same analysis was performed including fat mass in the multivariate model , ree becomes significantly unassociated with irisin circulating levels , whereas that fat mass was the main factor and explained approximately 30% ( p < 0.05 ) of the irisin concentration variability independently of age , bmi group , fat - free mass , daily physical activity , and ree ( table 1 ) . an increase in 1 kg of fat mass was associated with a 2-fold increase in the irisin plasma levels ( p = 0.003 ) . these results indicate that ree influence the irisin circulating levels but in a lesser extent than fat mass . in order to assay the participation of adipose tissue on irisin circulating levels , the immunodetection of this protein was tested on adipose tissue biopsies from healthy and obese individuals . as shown in figure 4 , fndc5/irisin was detected on visceral and subcutaneous adipose tissue secretion from nw and ob individuals ( figure 4(a ) ) and in ob at both intracellular and secretion level ( figure 4(b ) ) . previous studies are rather controversial , and there is no general agreement about circulating irisin levels and their correlation with bmi [ 14 , 15 , 17 , 18 ] . the current study performed with patients in conditions of extreme bmi confirms the positive correlation of circulating irisin and different parameters of adiposity including body weight , bmi , and fat mass . this work contributes to novel valuable information by showing that circulating irisin levels correlate positively with resting energy expenditure and negatively with daily physical activity . therefore , the inverse correlation of irisin with daily physical activity and the direct relationship between irisin and adiposity indicate that adipose tissue may be a primary inducer of irisin secretion , especially in obesity . the recent discovery of the pgc1--dependent myokine fndc5/irisin , which mediates brown - fat development and thermogenesis in white fat , has opened a new field of research [ 4 , 7 , 913 ] . the soluble form of fndc5 binds to unidentified receptors on the surface of wat to induce the expression of ucp1 and other bat - associated genes partly via increased ppar- expression . as a result , irisin may act as a muscle - derived energy - expenditure signal that directly communicates with adipose tissue and induces browning . this effect may improve the wat metabolic profile and enhance whole - body energy expenditure , making irisin a potential new target for the treatment of metabolic diseases . the possible beneficial effect of irisin on the treatment of obesity has been challenged by recent reports showing unexpected high levels of irisin in obese animals and humans [ 1417 ] as well as its association with the insulin resistance onset in association with weight regain . moreover , it was observed that in obese individuals , body weight and bmi reduction by bariatric surgery or by nutritional intervention significantly reduced irisin circulating levels [ 15 , 17 ] and its depletion after an energy restricted program is associated with the reduction of important lipid and carbohydrate metabolism biomarkers in patients with metabolic syndrome . other reports have described conflicting data indicating a lack of correlation between the circulating irisin concentration and the adiposity parameters . this discrepancy could be due to methodological differences because there is no consensus concerning the identity of the soluble portion of the fndc5 membrane protein , the mechanism of secretion , or the presence of variations on target epitopes among manufacturers . however , most published reports indicate correlations between irisin and adiposity levels . the present work further corroborates these findings ; irisin plasma levels were elevated in obesity , and a strong relationship was found between irisin and several adiposity parameters . in the current work , the obese subjects exhibited higher ree compared with the normal weight and anorexia nervosa subjects and it was directly related to irisin levels , independently of daily physical activity . this association between irisin and energy expenditure was previously reported in a cohort of postmenopausal women and it was proposed that this association could be explained by the irisin action on the brown adipose tissue thermogenesis . however , the association between irisin and ree appears to be masked by the strong association between fat mass and irisin plasma levels . fat mass was revealed as the main factor explaining approximately 30% of the variability in the irisin plasma levels independently of age , fat free mass , daily physical activity , ree , and bmi group . the fact that daily physical activity was found to be independent of plasma irisin levels in our work may seem contrary to what was expected . bostrom 's results have shown the expression and secretion of irisin after endurance exercise , which can not be compared to normal daily activity assayed in our paper . moreover , there are studies that , in contrast to the initial report by bostrom , have shown no variations of fndc5 mrna expression in muscle or at circulating level after aerobic or strength exercise training [ 15 , 33 , 34 ] . on the other hand , an increase of irisin circulating levels was observed after short - term exercise ( sprints ) which suggest that irisin secretion is dependent on acute or chronic sorts of exercise with a possible counter - regulation / adaptation over time . therefore , our results in relation to daily activity are coherent with previous evidences in the bibliography . our group has previously described that fndc5/irisin is a myokine and an adipokine expressed and secreted by wat . interestingly , we observed that explants of adipose tissue from obese animals or humans oversecreted irisin compared with the control subjects . therefore , we suggest that the muscle / adipose secretion ratio may vary and may be affected by the physiological situation ; during exercise , muscle tissue strongly participates in the fndc5 circulating levels , while in atypical bmi cases , such as obesity , the adipose tissue would actively increase the circulating fndc5/irisin . the current work further reinforces this hypothesis , because the main difference between the obesity and normal weight and anorexia nervosa subjects is the higher fat mass ; therefore , adipose tissue could be involved in the circulating irisin concentration differences observed depending on the bmi as reinforced in preliminary experiments assaying fndc5/irisin immunodetection in adipose tissue from nw and ob individuals in figure 4 . the results in the present work with previous reports [ 1417 , 36 , 37 ] might suggest a possible mechanism of resistance to irisin in situations of elevated bmi as previously observed in other adipokines ( e.g. , leptin ) . the elevated concentration of circulating irisin in overweight and obese subjects suggests that a great amount of bat would be expected in these patients . although it is now clear that bat is present and functional in adult humans , significant low amounts of bat have been described in overweight and obesity , which supports the irisin - resistance hypothesis [ 39 , 40 ] . however , the current work demonstrates that irisin circulating levels is associated with energy expenditure independently of daily physical activity but in a lesser degree than fat mass , which was revealed as the main factor influencing the circulating irisin concentration in obese women with extreme bmi from anorexia nervosa to obesity . therefore , these findings reinforce the positive relationship between circulating irisin and adiposity and the relevance of wat - secreted irisin in situations of elevated bmi , such as obesity . further studies are needed to elucidate whether irisin is involved in obesity development or irisin resistance that exist in an obesity state to counteract the metabolic disturbances related to excess adiposity .

a 23-year - old healthy female patient presented with metamorphopsia and photopsia in her left eye . at presentation , her medical history was unremarkable except her smoking habit ( 20/day ) and coke drinking habit ( 2l / day ) . anterior segment examination and intraocular pressures were within normal limits ( 17 mmhg ) in both eyes . dilated fundus examination of the left eye showed blurred optic disc margins with hyperemic disc swelling , venous engorgement , and preretinal hemorrhages in the macula . ( a ) fundus photography of the normal right eye at the initial presentation ( b ) fundus photography of the left eye at the initial presentation . note mildly edematous optic disc , dilated and tortuous retinal veins , and preretinal hemorrhages in the macula extensive laboratory workup including the complete blood count , serum c - reactive protein , erythrocyte sedimentation rate ( esr ) , c - protein , s - protein , d - dimer , lupus test , antinuclear antibody , prothrombin time / partial thromboplastin time , antithrombin iii activity , factor v leiden and prothrombin gene mutation , anticardiolipin antibody , antineutrophil cytoplasmic antibodies , angiotensin converting enzyme , and homocysteine levels were ordered to rule out underlying conditions that might cause papillophlebitis . at the 3 day of follow - up , she noted a sudden visual loss in the left eye . dilated fundus examination revealed crao [ fig . 2 ] and intact cilioretinal artery circulation was determined by fundus fluorescein angiography [ fig . since the macular perfusion was good , there is no proof to accompany of an embolism ; the emergency treatment of crao , including hyperbaric oxygen or anterior chamber lavage , were not done ; only acetylsalicylic acid ( asa ) drug 100 mg / day treatment was started . fundus photography of the left eye on the 3 day of the presentation with central retinal artery occlusion . note retinal edema except foveal region fundus fluorescein angiography of the left eye on the 3 day of the presentation . note intact cilioretinal artery circulation routine laboratory findings only showed a mild elevation of white blood cells , esr , and c - reactive protein . the chest x - ray and magnetic resonance imaging of brain and orbits were normal . all of the tests which were studied to determine the cause of the papillophlebitis were negative except heterozygous ( a1298c ) methylenetetrahydrofolate reductase ( mthfr ) mutation . despite the fact that the heterozygous mutation of this gene is very common and harmless , she was consulted by a cardiologist and a dermatologist who did not reveal any etiological factor . at the 5 day of follow - up , bcva increased but there were cells ( 2 + ) bilaterally in the anterior chamber and serologic study was conducted for iridocyclitis . only the western blot test for since turkey was an endemic region for this infection , the infectious disease specialist suggested starting antibiotheraphy although there was no history of any tick bite . at the 20 day of the examination , left optic disc was pale ; the hemorrhages and retinal edema were decreased , and arteries were attenuated [ fig . 4 ] . on the other hand , bcva was 20/20 in both eyes . since the cardiology department suggested the patient to be on asa , we decided to continue her medication . the left eye with the pale optic disc and attenuated arteries on the 20 day of the presentation papillophlebitis is believed to be a type of crvo in young people ; the exact cause is still not known . it can be isolated or can be seen with retinal artery occlusion , most commonly cilioretinal artery . in the current case , the occluded artery after papillophlebitis was central retinal artery . the increased venous pressure after crvo may have impaired the retinal blood flow , so these two vascular entities may be related . the second possible pathomechanism that is caused to suggest the linkage between them is inflammation of optic disc that caused to disruption of retinal blood flow . the inflammation may be related to smoking , nutritional deficiency , and unhealthy lifestyle of the patient . because of the papillophlebitis has a better natural course compared retinal vein occlusion in older adults and the presence of an intact cilioretinal circulation , our patient gained her visual acuity without any further treatment . the enzyme mthfr has an important role in homocysteine metabolism ; therefore , decreased enzyme activity leads to buildup of homocysteine and can cause thromboembolic events . reported a case of young female with unilateral papillophlebitis who was found to have positive homozygous mutations for mthfr c677 t and a1298c genes . although there was no information about the blood level of homocysteine in that case report , presumed hyperhomocysteinemia was thought as the main cause for that hypercoagulable state . conversely , in our patient , there was a heterozygous mutation of a1298c mthfr and homocysteine level was normal ( = a previous study showed mutation of a1298c mthfr was a risk for early coronary disease without hyperhomocysteinemia . thus , heterozygous a1298c mutations may also present an independent risk factor for thrombosis , especially if combined with unhealthy lifestyle and some risk factors such as smoking , high stress , and toxic exposures . while it is an uncommon cause of papillitis , it can be presented with severe acute anterior uveitis , howbeit previous cases reported unilateral papillitis as the sole significant ocular sign of lyme disease . therefore , due to accompanying bilateral anterior chamber reaction , lyme seropositivity , and being in an endemic area for lyme , she was treated with intravenous ceftriaxone with the recommendation of infectious diseases department , despite the lack of any bite story . performing extensive laboratory workup is very important in such patients to rule out some etiologic factors . furthermore , it should be known that heterozygous mutation of a1298c mthfr gene may cause thromboembolic events even if with the presence of normal homocysteine levels . based on this knowledge , we decided to recommend systemic anticoagulant therapy , other than asa to our patient for prophylaxis . the limitation of our paper is that the exact cause of anterior chamber reaction was not clearly determined , and it might have been a simultaneous self - healing iridocyclitis with false positive lyme serology . although combined crvo ( or papillophlebitis ) and crao entity tend to develop devastating complications such as rubeosis iridis , macular ischemia , and neovascular glaucoma , in the present case , even in the 3 month of vascular occlusion , neovascularization or macular ischemia was not seen and the visual prognosis was excellent , due to good perfusion of cilioretinal artery in the affected eye . in this regard , this patient is one of the rare cases which vision is preserved despite the combined crvo and crao . the most important challenge for this case is to determine the exact etiology . last , heterozygous mutation of a1298c mthfr with an unhealthy lifestyle or lyme disease with an atypical presentation may be the reason for this condition in such a young healthy person .

renal allograft transplantation has been successful in chronic kidney disease ( ckd ) stage v patients with sickle cell disease or trait , although incidence of complications has been higher than other causes of ckd-5d . we report a case of sickle cell trait who had developed acute patchy renal cortical necrosis ( rcn ) following renal transplantation . a 32-year - old woman with sickle cell trait diagnosed on electrophoresis with confirmed sickling phenomenon ( on sodium metabisulfite preparation ) presented in january 2003 with accelerated hypertension , neuroretinopathy , and ckd stage v. she was initiated on regular hemodialysis . her pretransplant journey of 1 year was complicated with recurrent pulmonary edema due to breakthrough accelerated hypertension , acute pancreatitis , and tubercular osteomyelitis . kidney biopsy could not be done to establish the cause of ckd as patient had bilateral small kidneys . the cause appeared to be unrelated to sickle cell trait because of absence of prior episodes of sickling crisis or hematuria and presence of malignant hypertension , all uncommon features in sickle cell trait . triple drug immunosuppression with cyclosporine ( 8 mg / kg ) , prednisolone ( 0.5 mg / kg ) , and azathioprine ( 2 mg / kg ) was used along with induction therapy with basiliximab ( 20 mg on day 1 and day 4 of renal transplant ) . there was brisk post - transplant diuresis ( 10.5 l on first day and 6.5 l on the second day ) and serum creatinine normalized by second day ( 1.1 mg / dl ) as expected in a live renal transplant . she was noted to have spontaneous closure of av fistula ( on day 1 ) with accelerated hypertension which was controlled with nifedipine , clonidine , and atenolol . on day 5 , there was a rise in serum creatinine to 2 mg / dl from a base line of 1 mg / dl , which increased rapidly to 3.8 mg / dl by day 7 . however , her urine output remained above 3 l /day and blood pressure was normal ( 140/80 mmhg ) on three drugs . investigations showed leukocytosis ( wbc count > 13500/cu.mm ) with near normal hematocrit ( pcv 36% ) and normal liver function tests ( total bilirubin , 1 mg / dl ; sgot , 26 u / ml ; sgpt , 32 u / l ; serum proteins , 6.2 gm / dl ; and albumin , 3.5 gm / dl ) . ultrasonogram of graft was normal and radionuclide study of graft showed mild reduction of perfusion with prolonged parenchymal transit . cyclosporine c2 level ( fluorescence polarization immunoassay ) on day 7 was 1932 g / l ( target , 1700 g / l ) . graft biopsy done on day 10 showed 15 glomeruli ( two viable ) with necrosed surrounding tubules , patchy inflammatory infiltrates , and no arteritis [ figure 1a and b ] . acute patchy cortical necrosis was diagnosed and possibility of renal graft rejection or thrombotic microangiopathy as possible alternative diagnosis excluded . the renal function showed an improving trend ( s. creatinine , 3 mg / dl ) . after 4 days , she developed severe pancytopenia ( wbc count , 1000/cu.mm ; platelet count , 30 000/cu.mm ; and hb , 7.5 gm / dl ) . mmf was discontinued and supportive therapy for anemia / leucopenia was given with granulocyte colony stimulating factor , antibiotics , and blood transfusion . later , sirolimus , mmf , and azathioprine were tried one after the other as second agent but were unsuccessful because of recurrence of leucopenia with each of them . hence , patient was continued only on high dose of prednisolone 40 mg / day till 6 weeks when her serum creatinine stabilized at 2.2 mg / dl . she was restarted on low - dose cyclosporine ( 2 mg / kg / day ) which was well tolerated , serum creatinine remaining stable . she was discharged from hospital after 7 weeks post - transplant on prednisolone 30 mg / day and cyclosporine 50 mg twice daily ( 2 mg / kg / day ) with serum creatinine 2.2 mg / dl and hb 9.9 gm / dl . at 72 months follow - up , she is doing well ( serum creatinine , 1.2 mg / dl ; hb , 11.5 gm / dl ; and leukocyte count , 10 200/cu.mm ) and is on low - dose immunosuppression ( prednisolone , 7.5 mg / day and cyclosporine , 1.5 mg / kg / day ) . ( a , b ) renal histology ( pas staining , 400 magnifications ) showing necrotic areas having ghost outlines of glomeruli and tubules with loss of cellular details renal manifestations of sickle cell disease are hematuria , renal infarction and papillary necrosis , diminished concentrating ability , renal tubular acidosis , abnormal proximal tubular function , acute renal failure , progressive renal failure with proteinuria ( sickle cell nephropathy and less commonly mpgn ) , and renal medullary carcinoma . relative low oxygen tension of renal arterial system and hypertonic acidic renal medulla combine to promote local red blood cell sequestration , leading to progressive renal ischemia / infarction and interstitial fibrosis . higher incidence of infection , disease recurrence , and sickling crisis due to improved hematocrit and blood viscosity have contributed to relatively poor long - term clinical outcome in these patients as compared with other etiologies of renal failure after renal transplantation . though 1 year patient and allograft survival rates are similar to that of other categories of renal transplantation , 3 year graft survival rate is reported to be significantly inferior . despite these reported adverse outcomes , long - term survival after renal transplant is still superior to hemodialysis ( median survival , 35 vs 24 months ) in patients of sickle cell disease . in contrast to sickle cell disease , patients of sickle cell trait have usually benign course with sickling crisis not noted except in extremes of metabolic derangement ( e.g. , severe acidosis ) . the possibility of alternative diagnosis of basic disease , specifically compound heterozygote of sickle cell disease ( hb sc and sickle cell - beta ( + ) thalassemia ) , were excluded in our patient based on serum electrophoresis and absence of target cells in peripheral blood smear . absence of anemia , microcytosis , and sickling crisis in the follow - up was consistent with sickle cell trait . our patient had several crises , like acute pancreatitis in pretransplant period and spontaneous closure of arteriovenous fistula and acute patchy cortical necrosis in immediate post - transplant period . such complications could be related or primed by her sickle cell trait . patchy cortical necrosis causing allograft dysfunction is an uncommon complication , more so in sickle cell trait patient . it was related to cyclosporine - basiliximab interaction following renal transplantation , which resulted in high cyclosporine levels . the raised level of cyclosporine was considered to be the primary event rather than related to renal dysfunction because of known drug interaction of cyclosporine with il-2 receptor blocker basiliximab . our success in re - introducing low - dose cyclosporine suggests its long - term safety in this setting . normal graft function even after 72-month follow - up is reassuring despite contrary reports in literature about poor outcome of such histopathological lesion . though cyclosporine nephrotoxicity leading to acute graft dysfunction is well known , the role of cyclosporine in producing patchy cortical necrosis is not reported . cyclosporine upregulates angiotensin-2 receptor and calcium responses in human vascular smooth muscle cells , leading to intrarenal vasoconstriction which could theoretically explain ischemic nephrotoxicity like acute cortical necrosis and hypertension , which was evident in our case . basiliximab which was used as induction therapy potentiates this vasoconstrictive ischemic injury by increasing cyclosporine levels via cytochrome p 450 pathways , especially in first 10 days of post - transplant period . cyclosporine could be reintroduced cautiously in such patients without adverse effects at later date as was done for this patient with good medium - term outcome , though one would logically avoid it should the patient tolerate mmf or sirolimus . this report underlies the importance of being aware of such severe drug interaction in susceptible patients with renal injury risk . rcn after kidney transplantation can result from unusual situations , such as technical surgical problems , graft preservation technique , transplantation across abo incompatible blood groups , positive cytotoxic antibody crossmatch , and any other factor that can generate hyperacute rejection , thrombotic microangiopathy and prothrombotic state ( antiphospholipid syndrome ) , and drugs ( tacrolimus , thymoglobulin ) . such an early vascular complication ( arterial / venous thrombosis ) leading to graft loss / dysfunction remains a constant and devastating complication in 2% of transplantation patients and constitutes a small population of overall acute cortical necrosis ( 5.3% ) . we hypothesize the contribution of sickle cell trait as a co - contributor along with cyclosporine - basiliximab for cortical necrosis . this could be the possible first report of such serious association leading to a complication which could be prevented .

the widely accepted theory is retrograde menstruation and implantation of endometrial cells on peritoneal surface ( 24 ) . endometriotic cells are capable of attaching to the peritoneal mesothelium , break the peritoneal lining , and destroy the extracellular matrix ( ecm ) ; thereby , they invade the surrounding tissue ( 5 ) . ecm is involved in the regulation of these cellular events , and ecm invasion may be on the critical point of endometriosis development . apelin is a new investigated member of adipose tissue peptide ( 6 ) and apelin expression has been shown in different tissues and in the uterus ( 79 ) . apelin and its receptor , also known as apj receptor , make function in vessel formation and cell proliferation ( 10 , 11 ) . being regarded as a new mitogenic peptide for endothelial cells ( 12 ) , apelin can promote cells from g0/g1 to s phase and stimulate epithelial proliferation . apelin and apj have been reported to be expressed in vascular muscle ( 1316 ) . it has been suggested that the apelin / apj system might be involved in the size - sensing mechanism of blood vessels which is an important event for supplying oxygen and nutrients in smaller vessels ( 17 ) . contribution of the apelin / apj system to portosystemic collateralization and splanchnic neovascularization in portal hypertensive rats has been also reported ( 18 ) . reported significant correlation between apelin and tnf - alpha that apelin might play a role in inflammation ( 19 ) . it was reported that apelin - apj signaling was essential for embryonic angiogenesis and it was upregulated during tumor angiogenesis . apelin was non - mitogenic for in vitro culture of primary human endothelial cells but it promoted chemotaxis ( 20 ) . the function of endometrial apelin is not known yet but , apelin may play a role in endometrial regeneration and endometriotic implantation via angiogenesis . we aimed to investigate the expression pattern of apelin in eutopic and ectopic endometrial tissues according to possible angiogenic function of apelin . tissue samples were collected in accordance with the requirements of the firat university hospital ethics committee between november 2008 and september 2009 . eutopic and ectopic endometrial samples were obtained from 45 women aged 18 to 40 years , and a bmi of 18.5 to 24.9 kg / m who were undergoing laparoscopy for benign indications . the participants had a regular 26 to 33 day menstrual cycle and had received no hormonal medication in the preceding 3 months . the women were classified into two groups ; group i : women who did not have any pelvic pathology confirmed by ultrasonography and laparoscopy ( n=15 ) , and group ii : women with endometriosis ( asrm - stage ii and iii ; n=30 ) . samples of endometriotic implants and endometriomas were obtained at laparoscopy , and the disease was confirmed by histological examination of the biopsies . we histologically examined the sections of endometrium and endometriotic tissues taken from these samples and assessed them for proliferative or secretory endometrial morphological features , using the criteria proposed by noyes et al . fortyfive endometrial samples ( 22 proliferative , 23 secretory ) from 15 women with a normal pelvis and 30 women with endometriosis were used for immunohistochemical and enzyme immunoassay analysis . samples of endometriosis ( 16 from women in the proliferative stage and 14 from women in the secretory stage of the menstrual cycle ) were obtained from 15 ovarian endometriomas and 15 peritoneal implants . semi - quantitative estimation of apelin staining was evaluated as an index of staining intensity and distribution . moreover , peripheral venous blood samples ( 5 ml ) were also collected for serum enzyme immunoassay . a written informed consent was obtained from each patient using consent forms . peripheral venous blood samples ( 5 ml ) after centrifugation of blood samples at 4000 rpm at room temperature for 10 min , 2030 l / ml aprotinin ( 400 kallikrein inactivator units , kiu ) and 1/10 volume 1 n hcl were supplemented into serum samples . eutopic and ectopic endometrial tissue samples were divided into two parts : formaldehyde embedded tissues for immunohistochemical analysis and the remaining tissues for elisa . approximately , 15 mg tissue samples were boiled at 100c for 5 min , then were squashed on iron mold and were homogenised in phosphate - buffered saline ( pbs ) containing 5% ( w / v ) dry milk . the homogenised samples were later centrifugated at 4000 rpm at room temperature for 10 min . for accurate quantification of apelin levels in tissue samples , 2030 l / ml aprotinin ( 400 kallikrein inactivator units , kiu ) and 1/10 volume 1 n hcl were supplemented into tissue samples . the extracted plasma and tissue samples were assayed for apelin concentration by human apelin kit ( active ; phoneix pharmaceuticals , germany ) . paraffin - embedded tissue samples were cut into 5 m sections and mounted on poly - l - lysine slides ( lab vision corporation , usa ) . endogenous peroxidase activity was quenched by incubation in 3% h2o2 for 10 min and followed by a rinse with pbs . for antigen retrieval , slides were placed in 10 mmol / l citrate buffer ( ph=6.0 ) and were microwaved twice for 5 min . non - specific binding of the primary antibody was prevented by incubation in ultra v block ( lab vision corporation , usa ) . sections were then incubated with rabbit polyclonal apelin antibody ( ab59469 , abcam inc . , thereafter , sections were washed with pbs and incubated in biotinylated goat antiserum ( lab vision corporation ) for 10 min at 38c . after rinsing in pbs again , the slides were incubated in streptavidin - biotin - peroxidase ( sbp ) ( histomark , usa ) for 10 min . as negative control , the slides were washed with pbs twice for 5 min , and then amino - ethylcarbazole ( aec ) was used as chromogene for 10 minutes . we semiquantitatively evaluated the cytoplasmic intensity for apelin immunoreactivity in eutopic and ectopic endometrial tissues . the stromal and glandular cells positively stained using the following intensity categories : 0 ( no staining ) , 1 + ( weak but detectable staining ) , 2 + ( moderate or distinct staining ) , and 3 + ( intense staining ) . in each slide , five different areas were evaluated under 60 magnification for each intensity within these areas were determined by two investigators blind to the type and source of the tissues . power calculation was performed based on previous studies that involved semiquantification of tenascin expression by immunohistochemistry . a sample size of 15 in each group has a power of 80% to detect a difference between means of 22.07 with a significance level ( alpha ) of 0.05 . statistical analysis was performed by statistical package for social sciences ( spss ) , version 12.0 ( spss , chicago , illinois , usa ) . differences between two groups for continuous variables were analyzed using student 's t - test . differences in two groups for ordinal variables were analyzed using mann - whitney u test . tissue samples were collected in accordance with the requirements of the firat university hospital ethics committee between november 2008 and september 2009 . eutopic and ectopic endometrial samples were obtained from 45 women aged 18 to 40 years , and a bmi of 18.5 to 24.9 kg / m who were undergoing laparoscopy for benign indications . the participants had a regular 26 to 33 day menstrual cycle and had received no hormonal medication in the preceding 3 months . the women were classified into two groups ; group i : women who did not have any pelvic pathology confirmed by ultrasonography and laparoscopy ( n=15 ) , and group ii : women with endometriosis ( asrm - stage ii and iii ; n=30 ) . samples of endometriotic implants and endometriomas were obtained at laparoscopy , and the disease was confirmed by histological examination of the biopsies . we histologically examined the sections of endometrium and endometriotic tissues taken from these samples and assessed them for proliferative or secretory endometrial morphological features , using the criteria proposed by noyes et al . fortyfive endometrial samples ( 22 proliferative , 23 secretory ) from 15 women with a normal pelvis and 30 women with endometriosis were used for immunohistochemical and enzyme immunoassay analysis . samples of endometriosis ( 16 from women in the proliferative stage and 14 from women in the secretory stage of the menstrual cycle ) were obtained from 15 ovarian endometriomas and 15 peritoneal implants . semi - quantitative estimation of apelin staining was evaluated as an index of staining intensity and distribution . moreover , peripheral venous blood samples ( 5 ml ) were also collected for serum enzyme immunoassay . after centrifugation of blood samples at 4000 rpm at room temperature for 10 min , 2030 l / ml aprotinin ( 400 kallikrein inactivator units , kiu ) and 1/10 volume 1 n hcl were supplemented into serum samples . eutopic and ectopic endometrial tissue samples were divided into two parts : formaldehyde embedded tissues for immunohistochemical analysis and the remaining tissues for elisa . approximately , 15 mg tissue samples were boiled at 100c for 5 min , then were squashed on iron mold and were homogenised in phosphate - buffered saline ( pbs ) containing 5% ( w / v ) dry milk . the homogenised samples were later centrifugated at 4000 rpm at room temperature for 10 min . for accurate quantification of apelin levels in tissue samples , 2030 l / ml aprotinin ( 400 kallikrein inactivator units , kiu ) and 1/10 volume 1 n hcl were supplemented into tissue samples . were assayed for apelin concentration by human apelin kit ( active ; phoneix pharmaceuticals , germany ) . paraffin - embedded tissue samples were cut into 5 m sections and mounted on poly - l - lysine slides ( lab vision corporation , usa ) . endogenous peroxidase activity was quenched by incubation in 3% h2o2 for 10 min and followed by a rinse with pbs . for antigen retrieval , slides were placed in 10 mmol / l citrate buffer ( ph=6.0 ) and were microwaved twice for 5 min . non - specific binding of the primary antibody was prevented by incubation in ultra v block ( lab vision corporation , usa ) . sections were then incubated with rabbit polyclonal apelin antibody ( ab59469 , abcam inc . , thereafter , sections were washed with pbs and incubated in biotinylated goat antiserum ( lab vision corporation ) for 10 min at 38c . after rinsing in pbs again , the slides were incubated in streptavidin - biotin - peroxidase ( sbp ) ( histomark , usa ) for 10 min . as negative control , the slides were washed with pbs twice for 5 min , and then amino - ethylcarbazole ( aec ) was used as chromogene for 10 minutes . we semiquantitatively evaluated the cytoplasmic intensity for apelin immunoreactivity in eutopic and ectopic endometrial tissues . the stromal and glandular cells positively stained using the following intensity categories : 0 ( no staining ) , 1 + ( weak but detectable staining ) , 2 + ( moderate or distinct staining ) , and 3 + ( intense staining ) . in each slide , five different areas were evaluated under 60 magnification for each intensity within these areas were determined by two investigators blind to the type and source of the tissues . power calculation was performed based on previous studies that involved semiquantification of tenascin expression by immunohistochemistry . a sample size of 15 in each group has a power of 80% to detect a difference between means of 22.07 with a significance level ( alpha ) of 0.05 . statistical analysis was performed by statistical package for social sciences ( spss ) , version 12.0 ( spss , chicago , illinois , usa ) . differences between two groups for continuous variables were analyzed using student 's t - test . differences in two groups for ordinal variables were analyzed using mann - whitney u test . the age , bmi and waist / hip ratio of the participants , respectively , were as follows : 30.25.5 years , 22.11.8 kg / m , and 0.720.06 with no significant differences between two groups . the serum and tissue concentrations of apelin in both groups are presented in table 1 . there were no significant differences between serum and eutopic tissue levels of apelin in both groups . serum apelin concentrations did not show cyclic changes , but both eutopic and ectopic endometrial tissue apelin concentrations increased during secretory , and decreased during proliferative phases . eutopic endometrial tissue concentration of apelin did not differ between the two groups ( p=0.43 ) . spp = serum proliferative phase ; ssp = serum secretory phase ; eupp = eutopic proliferative phase ; ecpp = ectopic proliferative phase ; eusp = eutopic secretory phase ; ecsp = ectopic secretory phase . immunohistochemical staining of apelin in eutopic endometrium of both groups were dominant in secretory phase ( p<0.01 ) , but comparison of eutopic endometrial tissue apelin staining did not show a significant difference between two groups ( p=0.71 ) . upon immunohistochemical staining , intense immunoreactivity of apelin was observed in glandular cells of eutopic endometrial tissue of both groups during secretory phase ( figure 1 ) . moreover , endometriotic tissue showed intense apelin immunoreactivity in glandular cells during secretory phase ( p<0.01 ) . stromal cells of ectopic and eutopic endometrial tissues showed weak staining for apelin ; therefore , cyclic variation could not be analyzed in their expression levels . during secretory phase of menstrual cycle , apelin staining was intense in eutopic and ectopic endometrial tissue of women with endometriosis and in endometrium of non - endometriosis participants . immunostaining of apelin in eutopic and ectopic endometria . in eutopic and ectopic endometrial glands , stromal staining intensity of apelin was very low in both the eutopic and ectopic endometria ( 200 ) . a : eutopic proliferative endometrium ; b : eutopic secretory endometrium ; c : ectopic proliferative endometrium ; d : ectopic secretory endometrium the present study most probably was the first to evaluate the expression of apelin in eutopic and ectopic endometrial tissues . immunohistochemical and immunoassay results showed increased apelin expression during the secretory phase in glandular cells of eutopic and ectopic endometrium . however , the changes in stromal cells were less dynamic than those in glandular cells of both eutopic and ectopic endometrium . increased local apelin concentration may indicate a paracrine function on endometrium . the limitations of our study were as follows : our small study population limited the statistical power of the analysis and immunohistochemical analysis of apelin could have been supported with apj . new vessel formation is involved in the pathogenesis of endometriosis ( 22 ) , and it is thought that epithelial - mesenchymal interaction with the influence of sex steroids play a critical role in endometriosis ( 2326 ) . it is likely that the growth regulation of endometriotic cells is controlled by a complex interaction of ecm proteins and paracrine factors . we hypothesized that apelin might play a role in the endometrial regeneration via angiogenesis process . apelin mrna and immunoreactive apelin were detected in various tissues and organs such as epithelial cells of the gastric mucosa , myocardium and endocardial endothelium , lungs , kidneys , adrenals , and the endothelium of large and small blood vessels ( 9 , 2729 ) . apelin has also been shown to be involved in the regulation of vessel formation and cell proliferation ( 11 ) . apelin causes endothelium dependent vasorelaxation by triggering the release of nitric oxide ( 3032 ) . moreover , there are reports that apelin is a potent angiogenic factor inducing endothelial cell ( ec ) proliferation , ec migration , and the development of blood vessels in vivo ( 7 , 33 ) . suggested that apelin might act as a chemo - attractant for sprouting endothelial cells ( 34 ) . apelin and apj in the granulosa cells of bovine ovary may be involved in induction of cell apoptosis ( 35 ) . apelin has an important effect on activated t lymphocytes by supressing ifn- , il-2 and il-4 production ( 27 ) . our study is most probably the first for describing apelin expression in ectopic and eutopic endometrium . expression level of apelin was higher in secretory than proliferative endometrium and glandular cells more evidently showed intense expression than stromal cells . similar expression patterns of apelin in eutopic and ectopic endometrium shows that endometriotic lesions share some of the characteristics of the cellular processes involved in the regeneration of endometrium . we observed that expression of apelin was similar in the eutopic endometrium of women with or without endometriosis . presence of apelin in the ectopic endometrium may point to the expression of apelin by ectopic epithelium in the peritoneal environment of women susceptible to the disease . apelin may also play a spatio - temporal role for the maturation of blood vessels by transient expression in ectopic endometrium where angiogenesis is taking place . in addition , genetically engineered mouse models indicate that apelin / apj regulates vascular stabilization and maturation in physiological and pathological angiogenesis ( 36 ) . thus , it is difficult to merely explain the proliferative potential of endometriotic epithelia in terms of quality or quantity of apelin . in conclusion , our results point out secretory phase glandular cell dominancy of apelin expression in both of the eutopic and ectopic endometrium . further studies are needed to investigate the possible effects of apelin on the formation of endometriotic lesions via their paracrine effects on vessel formation and ecm invasion .

according to the world health organization , injuries are still a major cause of severe disability and death . there are approximately 3 - 9 million injuries recorded annually in developed countries . it is predicted that in 2020 they will be a major cause of death . according to statistical data , within an hour in the world around 2000 people the number of deaths due to injuries has doubled in the last 30 years in poland and other european countries . the most common cause of death due to injury is damage to the head , chest , and abdomen and associated haemorrhages . injuries more frequently affect men than women and individuals aged 20 - 60 years [ 46 ] . as shown by statistical studies , in 2008 in poland there were 49,054 traffic accidents , in which 92% of people were injured and 8% died . this was a significant increase in the number of accidents compared to the study conducted in 1985 , which recorded 36,100 accidents with a slightly higher mortality ( 10% ) . the post - traumatic mortality rate in our country is 75/100,000 people a year , which is much higher than in western europe or the united states . in poland , traumatic disability affects approximately 25% of hospitalised patients , and thus 100% more than in the usa and western europe [ 8 , 9 ] . severe infections ( sepsis ) and multiple organ failure ( multiple organ dysfunction syndrome mods ) are among the most severe traumatic complications burdened with high mortality [ 2 , 9 , 10 ] . according to the scarce data from intensive care units ( icu ) , in poland injuries are still a major cause of severe infections [ 11 , 12 ] . according to these analyses , severe infections are diagnosed in 50,000 patients a year , including 16% of patients hospitalised in icus , approximately half of which die . the largest group are surgical patients ( 58.9% ) , and the most common complications are respiratory ( 88.7% ) and cardiovascular failure ( 82.2% ) . this is confirmed by studies from other centres elsewhere in the world . sepsis caused by various factors is the cause of death of every fourth patient [ 11 , 13 ] . in the united states , 750,000 new cases of sepsis are recorded each year , of which 210,000 die . in turn , in canada there are about 30,000 cases of sepsis recorded each year , and 35% of these patients die . in summary , 40% of icu patients are admitted because of sepsis , or they develop its symptoms in a short period of time ; of these , 36.7% are patients from the emergency room . as demonstrated by the study conducted in the united states , severe infections are now the cause of more mortalities than stroke , lung cancer , and breast cancer combined . studies in polish icus showed that severe sepsis is the leading cause of death in 30 - 60% of patients . as shown in a previous study conducted by e. faist in a group of 433 young patients with multiple traffic injuries , mortality in the course of multiple organ failure ( mof ) was 56% . the authors of this study emphasised that in order to improve the results of treatment in these patients , the proper functioning of the respiratory and circulatory systems should be ensured as soon as possible ( preferably still at the scene ) . the results of this study indicated a significant proportion of infections as one of the causes of mof . the resulting disorders of the immune response after trauma are primarily associated with increased production of inflammatory response mediators ( e.g. cytokines ) and the dysfunction of gut - associated lymphoid tissue ( galt ) . according to this hypothesis , translocation of microorganisms from the intestine into the lymphatic system and 1 ) [ 19 , 20 ] . the hypothetical model of pro - inflammatory ( sirs ) and anti - inflammatory response ( cars ) to trauma and infection , which can lead to multiple organ failure ( according to bone et al . ) according to this theory , the immune response to tissue injury and associated infection starts at the site of the injury and is related to the production of pro- and antiinflammatory mediators , primarily by cells of the innate immune response ( e.g. neutrophils ) . this part of the immune response is directed at restoring the homeostasis , and may be considered as an early physiological inflammatory reaction . the local inflammatory response and it is known , however , that persistent infection in the wound or the existence of any additional source of infection ( e.g. in the respiratory tract ) increases the local and systemic immune disorders , which can lead to sepsis and multiorgan failure . according to this concept , exacerbation of immune disorders through increased activation of the immune system can lead to immune paralysis syndrome ( chaos ) , which increases the number of complications and mortality [ 19 , 21 ] . an important element of this hypothesis was the attempt to link immune disorders with clinical signs of disease severity . the authors stressed the importance of apoptosis of the immune system cells ( macrophages , neutrophils , lymphocytes ) , which may exacerbate immunosuppression and is an important element of response to injury / infection , and they indicated the importance of mediators involved in the inflammatory response . a better understanding of the impact of pathological response of sirs / cars type ( compensatory anti - inflammatory response syndrome ) on the occurrence of mods was hindered by poorly understood cooperation mechanisms of immune cells involved in direct organ damage and the influence of other effects ( e.g. the size of the injury , previous comorbidity , type of infection ) . attempts to simplify the classical molecular model have had an adverse impact on the diagnostics and have given a better understanding of the immune response to injury and infection . the above - presented concept of immune disorders has not been widely adopted by clinicians because of the complicated course of sirs / cars response and difficulties in the interpretation of the results of immunological studies in severely ill patients with infections . nevertheless , a simple clinical model of sirs , sepsis , and severe sepsis diagnostics has been introduced ( table 1 ) [ 22 , 23 ] . clinical diagnosis of sirs , sepsis and severe sepsis a lack of conviction of clinicians to perform immunological tests among the most seriously ill patients had a significant impact on reducing early molecular diagnostics in the icu , although numerous studies have indicated that the immune dysfunction observed in these patients preceded the occurrence of organ complications . thus , a routinely applied scheme of sirs , sepsis , and severe sepsis diagnosis still does not include the evaluation of selected immunity parameters ( e.g. testing the levels of cytokines , chemokines , or alarmins ) that could give information about the dynamics of the immune response to trauma and infection , and potential complications . this scheme does not include immune disorders caused by injury and infection , which are a direct cause of multiple organ failure . clinical signs of mods usually occur late , and routine laboratory diagnostics markers ( e.g. levels of crp , pct , lactates , d - dimers , albumins ) are still of little help in the early diagnosis of threatening complications . the increase in the concentration of these indicators is most often observed at the onset of clinical symptoms . as numerous studies have shown , the levels of immune mediators of pro- and anti - inflammatory responses are significantly elevated in patients with mods and usually precede the occurrence of clinical symptoms by dozens of hours . cascade inflammatory response to trauma and infection , including immunological indicators and diagnostic window previous studies have highlighted the disorders of cellular immunity and immunosuppression associated with trauma , stressing the importance of cytokines ( il-2 , il-13 , tgf- ) , prostaglandins , thymus hormones , complement , nk cells , and granulocytes . the relationship has been found between the severity of sirs / cars and mof and impaired apoptosis of cells involved in pro- and anti - inflammatory response ( mainly neutrophils ) , which increases the production of cytokines and free radicals . it has been suggested that uncontrolled immune response to trauma or infection can lead to the induction of self - destruction of tissues and organ dysfunction distant from the site of injury ( e.g. in the lungs ) . it was necessary to modulate immune cell function in order to improve the resistance in severely ill patients . the proposed treatment with antibodies neutralising the action of mediators of the inflammatory response ( anti- tnf- , anti - endotoxin , il-1antagonist , no inhibitor , eicosanoids ) , thymus hormones , indomethacin , thymopentin in combination with indomethacin , stimulation of il-2 production by lymphocytes [ 32 , 33 ] , and regulation of macrophage activity ( including blocking the migration of macrophages ) . in addition , modulation of the immune response was attempted by endotoxin removal ( hemofiltration ) or the blocking action of the complement system activity and neutralisation of active oxygen species [ 10 , 35 ] . as demonstrated by numerous clinical studies , these methods have not caused a significant decrease in mortality in the most severely ill patients . although preliminary experimental results were promising , they have not been confirmed in randomised clinical trials . some of these failures resulted from improper use of animal models or from difficulties in precise classification of clinically septic patients . additionally , there was no correlation between the inflammatory response and the severity of the clinical status of a heterogeneous group of septic patients . it should be assumed that the more precise linking of the inflammatory response with the severity of the disease could be helpful to give a better understanding of the contribution of immune disorders in severe infections . it has been found that the immune response in sepsis is one of the causes of multiple organ failure , but changes in the inflammatory response do not always correlate with the clinical status of the patient . probably , monitoring changes in immunity during treatment could provide an answer to the question of to what extent a given immunological indicator can be used in practice as an early marker of impending organ failure . a division into early and late multiple organ failure has been introduced , depending on the dynamics of changes in the inflammatory response . early organ failure was caused by pathological immune disorders occurring during the sirs response , while late organ failure was caused by pathologic response of the cars type ( fig . 3 ) . a concept of physiological sirs and cars response was also presented , which may develop in patients with extensive trauma and does not lead to severe septic complications and multiple organ failure . an unquestionable problem of this research was the determination of specific numerical values for inflammatory response markers that allow clinicians to distinguish early physiological sirs reaction from pathological response . similar problems were related to cars , in which the increased production of il-4 was assumed to be an early indicator of immunosuppression . dynamics of changes in sirs and cars response to trauma and infection , including the physiological and pathological reactions ( according to moore et al . ) according to a newer concept developed by murphy et al . 4 ) , cars occurs in parallel sirs - compensating response , mainly associated with increased production of anti - inflammatory cytokines ( e.g. il-4 , il-10 ) and the development of immunosuppression . changes in the inflammatory response to trauma and infection , including the parallel course of sirs and cars response ( according to murphy et al . ) according to these authors , an additional factor ( e.g. infection , re - injury ) may be the cause of another sirs type reaction ( second hit sirs ) , and a pathological increase in this response can lead to mods . thus , a new element of this scheme is made up of parallel reactions of sirs and cars and disorders of the innate and acquired immune response , which increase the risk of severe septic complications . despite the simplification , the scheme presented indicates a complex mechanism of immune disorders that occur as a result of severe trauma and infection . according to the hypothesis of the authors , the largest immune disorders , as a result of extensive trauma , occur during the first seven days after injury , and then physiological extinction of inflammatory response a certain novelty was the introduction of the second hit concept of sirs response , which can also lead to mods . in addition , the key role of the immune response self - adaptation to trauma was stressed . this scheme did not include the important parameters of innate antimicrobial response , particularly the participation of toll - like receptors ( tlr2 , tlr4 ) , although the authors of this study pointed out the significant role of these receptors in response to trauma and infection . in patients after injury with infection , increased expression was emphasised of tlrs located on macrophages and neutrophils , as a defence mechanism enhancing the innate immune response . undoubtedly , discrimination between normal and pathological inflammatory response is a difficult element of diagnosis [ 38 , 39 ] . according to the current concept of the inflammatory response to trauma and infection concomitant to sirs , in which the cells of the innate immune response are primarily involved , cars inflammatory response develops , which may lead to immunosuppression . this part of the immune response involves adaptive immune cells including lymphocytes producing anti - inflammatory cytokines , immunosuppressive treg cells ( cd4/ cd25 ) , and antigen - presenting dendritic cells . more recent studies have confirmed that cars can lead to a reduction in the severity of sirs proinflammatory response in order to achieve balance , but in some patients it led to increased immunosuppression . it should be assumed that the adoption of the hypothesis of a parallel course of sirs and cars was another factor that hindered the designation of appropriate immunological markers that would allow the differentiation of physiological reaction and pathologic post - traumatic response . it should be recalled that based on an extensive review of the literature , the control of the dynamics of inflammatory response in severe infection , on the basis of selected sirs and cars parameters ( e.g. tnf- , il-1 , il-6 ) , has repeatedly been proposed . the authors have also suggested other prognostic factors , including genetic predisposition , the type and source of infection , and the degree of multiple organ failure , and formulated on this basis a classification system for sepsis ( piro scoring system predisposition , the insult infection , the response of the host system and organ dysfunction ) ( table 2 ) [ 10 , 42 ] . this system attempted to link the assessment of selected genetic , immunological , and bacteriological markers with clinical signs of infection . multiple organ dysfunction syndrome , sofa sequential organ failure assessment , lods logistic organ dysfunction system , pemod paediatric multiple organ dysfunction , pelod paediatric logistic organ dysfunction the most recent studies , conducted in the netherlands , portugal , and the united states on a large group of patients ( about 3,500 ) , compared the piro system with the scales routinely applied in the evaluation of the severity of the patients status ( apache acute physiology and chronic health evaluation , sofa sequential organ failure assessment , meds , it was found that this system had a higher prognostic value in the assessment of severity of infection , mods and mortality risk . moreover , it was found that the point value in this system correlated with clinical symptoms [ 4345 ] . the next scheme ( fig . 5 ) , which illustrates the dynamics of pro- and anti - inflammatory response to trauma and infection , emphasised the influence of other factors on the inflammatory sirs response ( mechanism of injury , ischaemia , reperfusion , medical procedures , atls advanced trauma life support , type of surgery , anaesthesia , additional infection , and possible reoperation ) . an example of changes in the pro- and anti - inflammatory response to injury and infection ( according to brochner et al . ) , the presented scheme should be reflected in the treatment of post - traumatic patients , which should be supported by monitoring the levels of il-6 in peripheral blood , coagulation markers , lactates , changes in ph , temperature , and diuresis . in the first hours of the occurrence of trauma , the authors of this study emphasise that every surgical procedure may be another stimulus that increases the immune disorders , therefore , subsequent re - operations should be performed only after normalisation of the indicators examined . in the latest studies , the results of these studies have shown that the concept of the socalled second hit sirs response to trauma and infection is unlikely ( fig . based on the expression of genes ( mediators of the immune response ) , it was confirmed that sirs and cars occur simultaneously ( fig . dynamics of changes in sirs and cars response in trauma patients based on genetic studies ( according to xiao et al . ) the following genes have been studied : tlrs ( except for tlr3 and tlr7 ) , haptoglobins , collagenases , cytokines ( il-1ra , il-4 , il-6 , il-8 , il-10 ) expressed in t and b cells , leukocytes , neutrophils , and genes encoding proteins responsible for apoptosis . attention has been drawn to the dynamics of the selected signalling pathways activation or their suppression in conjunction with the degree of the severity of multiple organ failure . the authors introduced the term gene storm stemming from the involvement of virtually all genes in response to external and internal stimuli . it was found that the trauma can induce the production of various inflammatory mediators and activate the receptor protein genes involved in the recognition of molecular patterns of microorganisms ( prr ) , suppressing at the same time receptor genes responsible for antigen presentation , proliferation of t cells , apoptosis , receptor function or activity of nk cells . gene storm is a highly coordinated and repeatable response , so that the immune system undergoes rapid adaptive changes in response to trauma . in this work , it was found that the quantitative and not qualitative changes in genes expression are the cause of post - traumatic complications . in patients without complications , silencing of gene transcription occurs within 7 - 14 days , whereas gene activation in patients with complications lasts significantly longer ( over 28 days ) . thus , the previous concepts assuming that sirs is short term and transient were wrong . the expression of genes involved in the immune response can be prolonged in severely ill patients after the injury , resulting in the production of immature bone marrow cells ( mdscs myeloid derived suppressor cells ) , which exhibit a potent immunosuppressive action and do not differentiate into the cells of the early immune response . the main mediators of myeloid cell activation include cytokines and prostaglandins , which are involved in both pro- and anti- inflammatory response [ 47 , 48 ] . appropriate myeloid stem cells with the ability of self - renewal differentiate successively into cmp cells ( common myeloid progenitor ) and mdscs , which are important in the formation of early immune response cells ( granulocytes , macrophages , dendritic cells ) . trauma excessively increases the production of mdscs , the result of which is that the immature cells can not activate subsequent immune response pathways . it is known that in severely ill patients after trauma , treated long - term in the icu , the cells of the immune response to injury and infection that produce nitric oxide ( no ) , reactive oxygen species ( ros ) , and other mediators of immunosuppressive action consume large amounts of energy ( e.g. arginine ) . rapid depletion of energy resources may give rise to so - called persistent inflammation and immune suppression syndrome catabolism ( pics ) ( fig . 7 ) . a new concept of the immune response to trauma and infection , involving pics and disorders of cell - mediated immunity ( according to gentile et al . ) there is a need to change the therapeutic approach directed at balancing the multi - level defects of immune response and reducing protein catabolism , which in turn requires appropriate monitoring of immunity changes . a wider introduction of testing the concentrations of selected cytokines of pro- and anti - inflammatory action ( il-6 , il-10 , il-1ra , or stnfri ) to the routine diagnostics of inflammatory response to trauma and infection raises some hopes . these indicators give earlier information about the pathological changes in the innate immune response to injury and infection , and their concentration can be routinely assayed using a simple elisa . on the other hand , the routine use of flow cytometry allows rapid evaluation of disorders of cell - mediated immunity , including the examination of the current activity of monocytes involved in the later stage of the immune response to trauma and infection ( analysis of hla - dr or cd80/cd86 expression in cd14 cells ) . more advanced techniques enable evaluation of the expression of selected genes in the mdsc responsible for persistent immunosuppression , but this part of diagnostics is still in the experimental phase . an important element in the treatment of these patients is the need to rapidly provide energy substances required for the proper functioning of the immune cells in a suitable nutritional therapy . this applies in particular to patients with pics requiring longer treatment in the icu [ 47 , 48 ] . these patients are diagnosed with difficult to balance catabolism , exacerbating malnutrition , impaired wound healing , and recurrent infections . pics is characterised by simple parameters assessing the nutritional status : icu stay > 10 days , weight loss > 10% during hospitalisation , or bmi < 18% , crp level > 150 mg / dl , total lymphocyte count ( tlc ) < 0.8 109/l , albumin < 3.0 g / dl , prealbumin < 10 mg / dl , retinol binding protein ( rbp ) < 10 mg / dl . the authors emphasise that the clinical image is accompanied by chronic inflammation , increasing immunosuppression with macrophage function paralysis , and a decreased count and activity of lymphocytes . this group of patients usually requires long - term treatment in the icu and is subject to the highest risk of secondary complications ( respiratory failure , traumatic coagulopathy syndrome , hypothermia , hypocalcaemia , acidosis , infection ) and death [ 49 , 52 ] . previous therapy attempts using antibodies , factors stimulating the activity of immune cells , or immunomodulatory preparations did not yield any significant breakthrough in reducing the high mortality of patients with severe sepsis treated in the icu . some hope for improving the results of treatment is associated with a more accurate assessment of nutritional status and with the wider introduction of new immunomodulatory preparations , e.g. an appropriate nutritional treatment of immunonutrition type agents blocking immune response by affecting the tlrs ( e.g. eritoran ) [ 53 , 54 ] . the clinical usefulness of novel survival biomarkers and severity of infection has also been tested ( e.g. endocan ) . patients in metabolic stress after major trauma , surgery , or sepsis often require supply of a modified diet composition enriched with specific immune- modulating nutrients such as glutamine , arginine , omega-3 fatty acids , or nucleotides ( impact , reconvan , intestamin ) [ 5458 ] . opinions on the use of immunonutrition in severely ill patients with post - traumatic sepsis , treated in the icu , are still controversial . as recent studies have shown , the use of glutamine in patients with multiple organ failure may increase mortality [ 59 , 60 ] . characteristic in these studies are very high doses of glutamine ( 0.35 g / kg bw / d i.v . + 30 g / d enterally ) that may exacerbate organ failure ( according to espen , glutamine dose should not exceed 0.5 g / kg bw / d ) . it should be emphasised that these studies were performed in a heterogeneous group of patients , which could also have influenced the obtained results . in conclusion , one of the important factors that increase mortality in posttraumatic patients is pathological pro- and anti - inflammatory response occurring in the first hours after extensive trauma with massive infection , which is still difficult to control and to discriminate from physiological immune response . interpretation of the results of studies is hindered by the parallel course of sirs and cars response , and the significant heterogeneity of the patient groups investigated . complementing the sirs and cars concept with genetic research partly explains this problem . as demonstrated by the most recent studies , during the so - called gene storm , which occurs immediately after the injury and infection , a large number of genes are induced , which are ultimately responsible for the production of many inflammatory response mediators and the expression of receptors involved in the recognition of molecular patterns associated with pathogens . in parallel , the expression of genes responsible for the proper presentation of antigens , lymphocyte proliferation , and apoptosis is decreasing . these are primarily qualitative rather than quantitative changes in the expression of genes that are one of the causes of severe post - traumatic complications . changes in gene expression detected early after injury precede the occurrence of later complications with a typical clinical picture . furthermore , rapid depletion of energy resources increases the immunosuppression , burdened with the highest mortality rate . early diagnosis of pics and appropriate treatment , nutritional therapy in particular , can reduce the length of stay of patients in the icu and decrease mortality .

sciatica , is characterized by radiating pain that typically follows the dermatome of the affected nerve - root . when strict criteria are used , the prevalence rate is approximately 2% [ 1 , 2 ] . roughly 90% of the lumbar radicular pain cases are caused by intervertebral disc herniation with nerve - root compression . although a spontaneous recovery is seen in most of these patients , only 30% are completely pain - free one year after onset . at present , there are no measurable molecular markers for patient prognosis , choice of treatment , or development of new compounds for persistent pain after disc herniation . earlier studies suggest that leakage of nucleus pulposus into the spinal canal after disc herniation may initiate immunological and inflammatory responses close to the nerve - roots [ 5 , 6 ] that increase the activity in nociceptive pathways [ 711 ] . this inflammatory influence has been attributed to an upregulation of interleukins ( ils ) , tumor necrosis factor ( tnf ) , matrix metalloproteinases ( mmps ) , nitric oxide ( no ) , and prostaglandins ( pgs ) in or around the herniated disc [ 1214 ] . in addition , recent data point to increased levels of circulating cytokines in patients with chronic pain . for example , higher systemic il-2 and tnf levels have been observed in patients with painful neuropathies compared to painless neuropathies . moreover , il-6 and il-8 appear to be elevated in serum of patients with radicular pain due to disc herniation [ 16 , 17 ] . thus , earlier observations suggest that disc herniation may be associated with an upregulation of several inflammatory molecules . in these previous studies , however , the methodological hindrance has limited the number of markers to a few proteins . in the present study , in contrast , we take advantage of the new multiplex proximity extension assay ( pea ) technology to simultaneously analyze the levels of 92 inflammatory proteins . to adjust for multiplicity , we used the false discovery rate ( fdr ) approach in the statistical analysis of the data . the aim of the present study was to investigate if it is possible to define an inflammatory fingerprint in the serum of patients with persistent lumbar radicular pain after disc herniation . our results indicate that the pea technology may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment . patients with lumbar radicular pain were recruited from the outpatient clinic at oslo university hospital , ullevl , norway , during the period 20072009 . the inclusion criteria were age between 18 and 60 years , lumbar disc herniation confirmed by magnetic resonance imaging ( mri ) with corresponding radicular pain , and positive straight leg raising ( slr ) . the exclusion criteria were lumbar spinal stenosis , previous surgery for herniated disc at the same level or fusion at any level in lumbar spine , generalized musculoskeletal pain , inflammatory rheumatic disease , diabetic polyneuropathy , cardiovascular disease ( nyha iii and iv ) , cancer , psychiatric disease , cauda equine syndrome , alcohol or drug abuse , recent surgery ( within one month ) , pregnancy , poor norwegian language , or non - european - caucasian ethnicity . in total , 122 patients ( 82% ) of the 148 patients who met the eligibility requirement were included in the intended follow - up assessment . surgical treatment was given to patients with persistent radicular pain lasting for more than 8 weeks , neurological deficits ( sensory changes , muscle weakness , and depressed or absent deep tendon reflexes ) , and corresponding magnetic resonance imaging findings in the anticipated location . patients who did not clearly fulfill these criteria were managed conservatively by a treatment comprised of a brief cognitive intervention , activity guidance during the acute phase of disc herniation , and , for the majority of patients , physiotherapy . at inclusion , at 6 weeks and at 12-month follow - up , the patients were asked to rate their pain intensity in activity during the last week on a 10 cm visual analog scale ( vas ) with endpoints no pain and worst possible pain . hopkins symptom checklist ( hscl ) , a 25-item questionnaire , was used to register psychological distress , that is , anxiety and depression . the drop - out rate was 8% and ultimately , 112 patients were assessed at 12-month follow - up . among these 112 patients , patients with vas > 6 at 12-month follow - up were defined as a high pain group ( n = 23 ) . moreover , a gender and age matched sample from the same 112 patients with vas < 1 at 12-month follow - up was defined as a low pain group ( n = 22 ) . the serum levels in inflammatory - related proteins in the high pain group versus the low pain group were analyzed . the study was approved by the norwegian regional committee for medical research ethics and the norwegian social science data services . at 12-month follow - up , after centrifugation at 2,000 g for 10 min at 4c , the supernatant serum was collected and stored in aliquots at 80c until further analysis . serum was used to analyze the biomarkers for the levels of 92 inflammatory proteins . in the present study we took advantage of the multiplex proximity extension assay ( pea ) technology in which a panel of 92 proteins ( see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/3874964 ) is simultaneously analyzed . the serum samples were assessed with proseek multiplex inflammation i ( olink bioscience , uppsala , sweden ) using the pea according to the manufacturer 's instructions . briefly , 3 l incubation mix containing two pea probes , that is , antibodies equipped with single strand dna oligonucleotide , against each protein was mixed with 1 l serum and the mixture was incubated at 8c overnight . the mixture was then mixed with 96 l extension mix containing pea enzyme and pcr reagents and incubated for 5 min at room temperature before the plate was transferred to a thermal cycler for 17 cycles of dna amplification . a 96.96 dynamic array ifc ( fluidigm , ca , usa ) was prepared and primed according to the manufacturer 's instructions . 2.8 l of sample mixture was mixed with 7.2 l detection mix in a new 96-well plate and 5 l was loaded into the right side of the primed 96.96 dynamic array ifc . the unique primer pairs for each cytokine were loaded into the left side of the 96.96 dynamic array ifc , and the protein expression program was run in fluidigm biomark reader according to the instructions for proseek . for the baseline characteristics , unpaired student 's t - test was chosen to compare mean values for normally distributed data , whereas for nonnormally distributed data the mean values were compared by a nonparametric two - sided mann - whitney u test . pearson chi - square test was used to analyze the frequency of baseline categorical variables between the high and low pain group . in order to identify differentially expressed serum proteins between the two groups of patients , protein - level analyses for each individual biomarker were performed . for a group of patients , the protein expression levels of some proteins were below the laboratory 's limit of detection ( lod ) . therefore , we used a multiple linear regression model with lod imputed values in place of below - lod values to test the differences between the groups . because of the difficulties associated with analyzing data with a high proportion of below - lod values [ 2022 ] , only proteins for which the proportion of below - lod values was less than 20% were included in the analyses . the false discovery rate ( fdr ) was controlled using the procedure of benjamini and hochberg ( 1995 ) . the difference in expression levels was estimated by computing the ratio of the group medians on the original non - log scale . the linear discriminant analysis [ 23 , 24 ] was performed to visualize the differences between the two groups . using vector notation and letting xij denote the vector of protein measurements of patient j in group i ( with i = 1 corresponding to high pain and i = 2 corresponding to low pain ) , x - i denote the sample mean vector in group i , and ni denote the number of patients in group i , the discriminant score for each individual was computed as(1)x1x2i=12j=1nixijxixijxi1xij12x1+x2.this resulted in a score , essentially based on a weighted average of the differentially expressed proteins , such that positive values indicated high levels of inflammatory proteins and negative values indicated low levels of inflammatory proteins . the weighted protein score was visualized in a simple dot plot where each patient was represented by one dot . a positive score indicated a high inflammatory activity , whereas a negative score indicated a low inflammatory activity . the high pain group had more pain when they arrived at the hospital , at 6 weeks and 12-month follow - up , than the low pain group ( figure 1 ) . more smoking and higher hscl score were also observed in the high pain group ( table 1 ) . therefore , smoking and hscl were corrected for in the further analyses , that is , in the multiple linear regression model to test the differences between the high and low pain group . the analyses of the levels of 92 proteins showed 76 proteins for which at most 20% of the patients had expression levels below the lod . among these 76 proteins , we identified 41 with fdr < 0.10 and 13 with fdr < 0.05 , which were considered upregulated in the patients with severe pain one year after disc herniation ( table 2 ) . the analyses of the inflammation - related protein levels showed increased expression of many proteins in the high pain group compared to the low pain group . on the top of the list ranked by estimated increase we found c - x - c motif chemokine 5 ( cxcm5 ; 217% increase ) , epidermal growth factor ( egf ; 142% increase ) , and monocyte chemotactic protein 4 ( mcp-4 ; 70% increase ) . for each patient , an inflammation score , that is , weighted average of the 41 protein levels , was also constructed . notably , the two groups were completely separated ; all high pain patients had positive scores , whereas all low pain patients had negative scores ( figure 2 ) . thus , an inflammatory fingerprint in the serum of the patients with persistent lumbar radicular pain after disc herniation was demonstrated . the data showed a clear increase in cytokine levels in the patients that developed chronic pain ( figure 3 ) . in the present study , we examined the serum levels of 92 inflammatory proteins in patients one year after lumbar disc herniation . among these , 41 proteins with fdr < 0.10 , including il-6 and il-8 , seemed to be upregulated in the chronic pain patients compared to the patients that fully recovered within one year . hence , in accordance with our earlier elisa findings from the same cohort where il-6 and il-8 in almost all patients were increased the first weeks after disc herniation [ 17 , 25 ] , the present data showed that patients that develop long lasting radicular pain may be different from patients that recover with regard to measurable cytokines in serum . moreover , a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated . many of the upregulated proteins with fdr < 0.05 were chemokines and other molecules associated with inflammation , activation of macrophages , and/or immune responses including regulators of glial cells . c - x - c motif chemokine 5 ( cxcl5 ) is a neutrophil chemoattractant which increases the expression of several proinflammatory mediators . moreover , monocyte chemotactic proteins 2 , 3 , and 4 ( mcp-2 , mcp-3/ccl7 , and mcp-4 ) recruit macrophages , whereas c - x - c motif chemokine 10 ( cxcl10/ip10 ) activates t - cells and microglia . macrophages and microglia cells may also be activated by macrophage colony stimulating factor ( m - csf / csf-1 ) thought to be upregulated in nucleus pulposus tissue after disc herniation [ 10 , 30 ] . several of the upregulated proteins with fdr < 0.05 have also previously been linked to healing and tissue regeneration . c - c motif chemokine 4 ( ccl4 ) that reduces the expression of tnf and il-1 may be involved in nucleus pulposus resorption after disc herniation . vascular endothelial growth factor a ( vegf - a ) supports healing , regeneration , and revascularization [ 3335 ] and facilitates opening of the blood - nerve - barrier . interleukin-15 receptor- ( il-15r ) is expressed in monocytes , nk cells , and t - cells . in addition , some of the upregulated proteins with fdr < 0.05 may induce cell differentiation , apoptosis , and neuronal loss and influence cell cycle regulation . moreover , transforming growth factor ( tgf ) has important immune - regulative role and may reverse inflammation , whereas caspase-8 ( casp-8 ) is involved in fas mediated apoptosis [ 40 , 41 ] and cleavage of cytoskeletal proteins . stam - binding protein ( stambp ) is a zinc metalloprotease involved in cell cycle regulation recently linked to the neuronal loss underlying microcephaly - capillary malformation syndrome . in pea , each target molecule is recognized by a pair of affinity binders in which both have to bind the same target molecule prior to signal amplification via qpcr . similar to other proximity assays [ 43 , 44 ] , the pea technology also has an increased assay specificity . the specificity of the assay has been validated using a complete pool of pea probes to analyze either all or submixes of eight antigens , for which significant signal over background was obtained only when the cognate antigen was present . still , as some of the protein expressions were below the lod , the actual expression levels of these proteins remain unknown . as high proportions of below - lod measurements lead to tests with low power , we opted to only analyze the proteins for which the proportion of below - lod measurements was at most 20% . moreover , the two groups were balanced in terms of age , sex , and treatment but differed in smoking habits and hscl scores ( table 1 ) . our linear regression model was therefore adjusted for smoking and hscl scores to exclude the possibility that any differences found actually were due to differences in smoking and hscl . in the present study thus , the analyses of the data include multiple testing , that is , associated with many challenges . clearly , we can not control the family wise error rate by bonferroni correction as such corrections tend to be far too conservative , resulting in statistical procedures with very low power . however , since this is a study where the focus is on evaluation of the methodology ( proof of concept ) , a limited rate of false positives is acceptable . given a fdr of 0.10 and 0.05 , up to 1/10 and 1/20 of our findings are likely to be false positives . moreover , although a few false positive markers may be present , our study clearly demonstrated that inflammatory profiling can be used to characterize chronic sciatica patients . the present data also showed that the pea is a reliable and sensitive method to study proteins in serum associated with clinical measures . despite the above mentioned challenges , the linear discriminant analysis demonstrated a clear difference between the high pain group and the low pain group . notably , the two groups were completely separated ; all high pain patients had positive scores , whereas all low pain patients had negative scores . thus , even though there might be one or two false positive markers , an inflammatory fingerprint in the serum of the patients that developed persistent lumbar radicular pain after disc herniation was defined by the pea technology . although the present study had a prospective design when it comes to the pain ratings , the serum protein levels of the patients were only measured at one time point , that is , 12 months after disc herniation . thus , it is hard to tell whether the observed differences in serum proteins are a trait or a state of the patients that develop chronic pain . moreover , medication and other life style factors may also influence the serum protein levels . however , the analgesics and anti - inflammatory - drugs taken by the patients were higher in the high pain group than in the low pain group . therefore , it seems likely that the effect of medication reduces rather than increases the difference in inflammatory serum proteins . nevertheless , the results demonstrated that it is possible to detect differences between persistent and nonpersistent pain patients . the fact that this protein pattern can be detected in serum suggests that the pea technology may have a clinical potential . in summary , the present data demonstrated that sciatica patients with a chronic outcome have increased levels of inflammatory proteins in serum one year after disc herniation . this is to our knowledge the first protein serum profiling study of patients with chronic lumbar radicular pain . we conclude that serum proteins may be measurable molecular markers of patients that develop persistent pain after disc herniation .

to improve treatment results , repeat transurethral resection ( retur ) has been recommended for non - muscle - invasive bladder cancer ( nmibc ) , especially in high - grade / t1 disease . several reports have documented occult t2 disease in up to 10% of second turs . meanwhile , high - grade , t1 or multifocal disease is included as the risk of disease remaining.[35 ] indeed , retur is considered to improve recurrence - free survival ( rfs ) and progression - free survival ( pfs ) as compared to a single tur . moreover , at the second tur , absence of residual disease ( t0 ) showed a better correlation with a low recurrence or progression rate at follow - up as compared to pta , tis , or t1 residual disease . in some cases , residual disease was found even after the third tur . however , the number of times retur should be performed in order to reach t0 is unclear , and the predicting factor for the same is also unidentified . retur is not only a treatment for nmibc ; it also has an important role in providing stratification of patients based on its results . the stratification is directly related to the selection for subsequent treatment such as intravesical prophylaxis or cystectomy . based on the stratification , therefore , the end point of retur should be considered in a treatment algorithm . in this study a total of 117 patients underwent a second tur between july 2006 and february 2010 at yokohama minato red cross hospital and yokohama citizens municipal hospital . six patients were excluded from this evaluation : 2 had macroscopically incomplete resections and 4 had inadequate muscular samples for a diagnosis at the first tur . tur of all visible tumors was extended to the lamina propria , and several deep muscular samples were also taken from the tumor base . the location , number , and size of bladder tumors were recorded on a cystoscopy diagram . a third tur was recommended when t1/a/ is residual disease was observed during the second tur . here , we used retur as a general term indicating the second and third turs . if stage migration to muscle - invasive disease was observed at retur , radical cystectomy retur was performed 2 - 8 weeks after the last tur ( a mean of 3.9 weeks ) . retur consisted of wide resection of the margins and depth of each tumor site as well as complete resection and fulguration of all suspected residual tumors . deep muscle specimens were taken , especially from the previous tumor areas and the margins . tumors were classified according to the tnm system of the union internationale contre le cancer and were graded according to the world health organization classification . the definitions classify nmibc into 3 different levels : low- ( solitary , primary low - grade ta ) , intermediate- ( multiple or recurrent low - grade ) , and high- ( any t1 , high - grade , or cis ) risk disease . chi - square or fisher 's exact test was used to determine whether the incidence of residual disease significantly correlates with the risk levels informed consent for the treatment strategy was obtained from each patient , and approval from a local ethics committee was obtained . risk levels and residual disease at the second tur ( n ) the clinicopathological characteristics of the 111 patients at initial tur are shown in table 2 . of the 111 patients who underwent a second tur , 57 ( 51% ) had residual disease , as determined by histopathological analysis . multivariate logistic regression analysis identified tumor grade ( p = 0.006 ) and multifocality ( p = 0.02 ) as the predicting factors of residual tumor [ table 3 ] . residual tumor was observed in 33% ( 17/52 ) and 68% ( 40/59 ) of low- and high - grade tumors , respectively . residual tumor was observed in 40% ( 21/53 ) and 62% ( 36/58 ) of single and multifocal tumors , respectively . table 2 shows the number of patients and the incidence of residual disease at the second tur at each of the risk levels . a statistically significant difference was observed by comparison of the 3 groups ( p = 0.01 , x test ) . of the 57 patients who had residual disease at the second tur , of these 26 patients , 25 were recommended a third tur , which they declined . of the 26 patients , 1 with stage migration to muscle - invasive disease at the second tur underwent cystectomy . of the 31 patients who underwent a third tur , multivariate logistic regression analysis did not identify any predicting factors for the incidence of residual tumor at the third tur ( data not shown ) . table 4 shows the number of patients and the incidence of residual disease at the third tur at each risk level . in the high - risk disease group , there was a tendency for a higher incidence of residual disease at the third tur ( p = 0.06 , fischer 's exact test ) . the incidences of not reaching t0 were 17% ( 3/18 ) , 23% ( 7/31 ) , and 52% ( 32/62 ) in the low- , intermediate- , and high - risk groups , respectively . a statistically significant difference was observed by comparison of the 3 groups ( p = 0.01 , x test ) . patient characteristics multivariate logistic regression analysis to determine independent variables of residual disease at the second tur risk levels and residual disease at the third tur ( n ) next , we evaluated the change in the t category by retur [ figure 1 ] . of the 50 patients with high - grade / t1 at the first tur , 1 ( 2% ) , 8 ( 16% ) , 25 ( 50% ) , and 16 ( 32% ) showed changes in the t category to t2 , t1 , ta / is , and t0 at the second tur , respectively . at the third tur , t1 or worse disease was not found . in the 9 patients with high - grade / ta at the first tur , neither t2 nor t1 were found at the retur . of the 49 patients with low - grade / ta at the first tur , 17 ( 35% ) had residual tumors at the second tur , and all were ta / is . two patients had residual tumors at the third tur , and the both were ta / is . changes in t category no patient had major complications , such as bladder wall perforation or massive bleeding . divrik et al . showed incidences of residual tumor a t the second tur of 39% and 63% in grade 2 and 3 tumors , respectively ; the risk of residual tumor directly correlated with the grade of the initial tumor . the incidence of understaging at the first tur was only 1% , which was relatively lower than that reported in other studies . we showed that the incidence of residual tumor correlates with the risk levels for nmibc . our results are as follows : ( i ) in the high - risk disease group , a high incidence of residual disease was identified even after the third tur . ( ii ) in the intermediate - risk disease group , nearly complete resection was reached after the third tur . ( iii ) in the low - risk disease group , nearly complete resection was reached after the second tur . in the high - risk disease group indeed , retur is considered to improve recurrence - free survival ( rfs ) and progression - free survival ( pfs ) as compared to a single tur . moreover , at the second tur , absence of residual disease ( t0 ) showed a better correlation with a low recurrence or progression rate at follow - up as compared to pta , tis , or t1 residual disease . an algorithm for the treatment of high - grade / t1 bladder cancer has been proposed . in the algorithm , bladder - sparing treatment by bcg induction is recommended in the case of ta / is/0 at the second tur . our data showed that ta / is residual tumor was found even after the third tur , especially in cases of the high - risk disease . therefore , doubt regarding whether the presence of ta / is residual disease after the third tur can cause disease recurrence or progression could not be clarified completely . actually , a higher progression rate was observed in ta / is residual disease than in t0 residual disease at the second tur . hereafter , the necessity of constructing a new treatment algorithm in which a third tur is included might arise for high - risk disease . intermediate - risk disease has a low potential to progress to muscle - invasive disease or distant metastasis . therefore , a cystectomy as a result of muscle - invasive progression would be relatively rare . instead , two or more repetitive recurrences of non - muscle - invasive disease must be prevented . an algorithm for the treatment of intermediate - risk disease has also been proposed ; however , retur has not been included in this algorithm . on the other hand , our data showed that residual disease was found in about 40% of patients with intermediate - risk disease after the second tur . therefore , the doubt that the presence of residual disease after the second tur may cause disease recurrence or progression could not be clarified fully . hereafter , if the incidence of repetitive recurrence is considerably high , the necessity for construction of a new treatment algorithm in which a second tur is included might arise for intermediate - risk disease . in low - risk disease , second tur can be omitted because the incidence of residual tumor after the second tur was relatively low in our study . retur is considered to improve rfs and pfs as compared to single tur . however , retur is not only a treatment for nmibc , but it also plays a crucial role in providing stratification of nmibc patients based on the results . our data will provide the essential guidance required to establish a treatment algorithm that functions as a system in toto .

mirs are a family of short non - coding transcripts involved in the regulation of at least a third of all translated genes [ 13 ] . they are expressed as primary transcripts , which are subsequently processed by the drosha rnase , thus generating the hairpin - shaped precursor mirs ( pre - mirs ) . pre - mirs are then cleaved by another rnase iii ( dicer ) , which leads to the formation of mature duplexes ( 1924-nucleotide long ) . one of the two strands is selectively transferred to the rna - induced silencing complex ( risc ) , which interferes with gene expression at post - transcriptional level . the classic view of mir action was that in mammalian cells mirs act predominantly , if not exclusively , by blocking the translation of mrna targets . however , more recently , it has become apparent that the action is more complex , with mrna degradation being quite frequent , in a similar fashion to sirnas [ 5 , 6 ] . to further increase the intricate nature of mir action mirs continue to be at the centrestage of the non - coding rna revolution , currently being suspected to regulate virtually all known cellular mechanisms , such as cell differentiation , proliferation , death and metabolism [ 811 ] . on the one hand , specific mir expression profiles have been associated with human cancers , and in some cases correlated with clinico - pathological features [ 1318 ] . on the other hand , mechanistically individual mirs can function as bona fide oncogenes ( such as mir-1792 , mir-10b , mir-21 [ 21 , 22 ] , ) or tumour suppressor genes ( such as mir-1516 clusters ) . recent studies have shed significant light into the regulation of mir expression and information is accumulating on the impact of specific stresses on the mir - nome. arguably , the best - documented cellular stress is oxygen deprivation ( hypoxia ) , which is of relevance for a variety of diseases of major impact . tumours with extensive low oxygen tension tend to exhibit poor prognosis and resistance to conventional therapy . moreover , hypoxia is also a crucial pathogenic component of major cardiovascular diseases , such as myocardial infarction and stroke . the molecular mechanisms of response to oxygen deprivation are extremely complex , a key role being played by hypoxia - inducible factor ( hif ) , which orchestrates an expression program involving in excess of 100 genes . while gene induction by low oxygen has arguably dominated hypoxia research , more recently the study of gene repression has received increasing attention [ 2730 ] . thus , a large percentage of genes continue to be expressed at quasi - normoxic levels , while the translation(transcription of others is significantly suppressed . studies from several groups identified a variety of hypoxia - regulated mirs , providing a link between a tumour - specific stress factor and gene expression control [ 3139 ] . for example , one group identified a wide set of hypoxia - induced mirs in breast- and colon - cancer cells , which in addition to mir-210 included mir-21 , 23a , 23b , 24 , 26a , 26b , 27a , 30b , 93 , 103 , 106a , 107 , 125b , 181a , 181b , 181c , 192 , 195 and 213 . of these , only mir-30b , 93 and 181b were independently confirmed by a separate study . a more recent set of data identified only three mirs ( mir-210 , ambi - mir-7105 and mmu - mir-3223p ) , which showed at least 2-fold induction in response to hypoxia . in non - cancer cells , mir-210 was identified as a key player of endothelial cells response to low oxygen tension , and therefore this mir is emerging as a universal responder to hypoxia , with likely deep biological impact in the response to this type of stress . finally , according to two studies , hypoxia can also lead to mir down - regulation , including : mir-15b , 16 , 19a , 20a , 20b , 29b and 197 [ 38 , 39 ] . whether these represent specific targets of hif , or this process is simply the result of cell cycle arrest , is not known at this point . as results from above , beyond general agreement with regards to mir-210 , there has been rather limited overlap in mirs regulated by hypoxia . these discrepancies are not necessarily surprising , and could be explained by the differences in cells examined , technology employed , differences in the thresholds and time investigated , as well as oxygen concentrations [ 35 , 36 ] . in most tumours , the expression of mir-210 is significantly up - regulated compared to the corresponding non - malignant tissue [ 14 , 16 , 18 , 33 ] . the exception , according to one study , is represented by the ovarian carcinomas that tend to lose the locus encoding for mir-210 , and as consequence exhibit decreased expression of this mir . more significantly than altered expression in cancers , mir-210 has been shown to correlate with a hypoxia signature score in human breast cancers and strongly associate with an adverse clinical outcome . classic genes are now known to regulate the expression of specific mirs , therefore mirs may be rather common targets of transcription factors . from a historic perspective , the first transcription factors shown to involve mirs are c - myc and e2f , which activate the mir-1792 oncogenic cluster [ 4042 ] . recently , mir-34a joined the targets of the transcription factor ( and tumour suppressor gene product ) p53 , and has been shown to contribute to its function [ 4345 ] . it is not surprising that the hif was interrogated first with regards to a role in mir regulation . at least for mir-210 , hif is clearly a critical factor for the hypoxic induction , as determined in several studies [ 31 , 33 , 34 ] by multiple strategies : transfection of active hifs , chromatin immunoprecipitation and luciferase - based reporters driven by fragments of select hrm promoters . additionally , mir-210 hypoxic induction was shown to be dependent on the von hippel - lindau ( vhl ) tumour suppressor gene in rcc4 renal carcinoma cells . inactivating mutations of the vhl gene block proteolytic degradation of hif , leading to constitutive activation of hypoxia pathways , therefore further confirming the central role of hif in mir-210 induction ( fig . 1 ) . finally , sirna - mediated suppression of hif-1 or hif-2 in mcf7 cells led to abrogation of mir-210 induction under hypoxia . in primary endothelial cells , the situation seems more complex : the knock - down of hif2 , the main hif species in this cell type , does not affect mir-210 , while sirnas to hif1 effectively prevents mir-210 induction , suggesting a potential hif isoform specificity , at least in certain cell systems . moreover , the dynamic of mir-210 induction exhibits a notable difference compared to hif , as it continues to increase beyond the peak of hif protein level [ 31 , 34 ] . the dual aspect of the hypoxic response . in addition to the well - documented hypoxia - inducible genes that are direct transcriptional targets of hif , the response to low oxygen triggers expression of select mirs , which in turn down - regulate select genes . several issues remain to be elucidated with respect to mir-210 regulation , for example the existence of other possible mechanisms . growth factor deprivation , osmotic stress , acidosis and oxidative stress did not elicit mir-210 increase , but additional conserved candidate transcription factor sites are present in the proximity of mir-210 : oct - c , ap2 , ppar and e2f [ 35 , 36 ] . these could potentially regulate its expression as part of the hypoxia response in the case of oct-4 , itself a hypoxia regulate gene or as part of unrelated pathways . while mir-210 was initially thought to be intergenic , a more recent study showed that it is in fact contained within the sequence of a transcript with virtually unknown function ( ak123483 ) . this transcript is also hypoxia inducible , consistent with published observations of coordinated expression of mirs and the corresponding host genes . whether this transcript encodes for a protein or plays any biological role in the hypoxia response an increasing number of prediction programs for target identification are currently available , such as pictar , targetscan and miranda [ 4749 ] . when more than one search program is used , the in silico predictions often exceed one hundred genes , thus posing significant challenges to the effort to identify the biologically relevant targets . the first clues about genes(pathways that may be targeted by mir-210 were provided by a computational analysis . over 70 targets of mir-210 were predicted in drosophila , with a significant overrepresentation of genes involved in female gamete generation ( according to gene ontology ) , including : cut , egghead , germ cell - less , gurken , lozenge , par-1 , rhomboid-4 , rna - binding protein 9 , singed and slalom . biochemically , these genes belong to receptor tyrosine kinases , notch , wingless , or hedgehog signalling , and also function at more advanced stages during embryogenesis . based on the available prediction programs , most of these targets are not conserved in more complex organisms , such as mammals . for human mir-210 , in silico searches reveal a highly complex spectrum of candidate targets , including genes involved in proliferation , dna repair , chromatin remodelling , metabolism and cell migration [ 31 , 3336 ] . this diversity , combined with the salient discrepancies between prediction programs , raises experimental challenges , one prediction being that manipulation of any individual target will fail to fully capture the phenotypic impact of the corresponding mir in low oxygen . despite the above challenges , biologically relevant mir-210 targets have started to emerge [ 32 , 34 ] ( fig . one study focusing on the response of endothelial cells to hypoxia identified ephrina3 as target of mir-210 in hypoxic conditions . mir-210 was shown to play an important role in this system , as its inactivation decreases the ability of huvec cells to form capillary - like structures and migrate in response to vegf . ephrin ligands and their corresponding receptors are known to be involved in the development of the cardiovascular system , and at least for the efna1/epha2 system there is evidence for involvement in vegf signalling / angiogenesis [ 5153 ] . the finding that efna3 inhibition is necessary for mir-210-mediated stimulation of tubulogenesis suggests that this particular ephrin may also be part of angiogenic regulation . whether this mechanism functions only in the cardiovascular system , or its significance can be extended to neoplastic angiogenesis , remains to beelucidated . predicted wide impact of mir-210 in the hypoxic response . a selection of potential targets is shown ( identified using targetscan 4.1 and pictar programs ) . standard gene i d nomenclature was used and experimentally confirmed targets are shown in blue . another mir-210 target derived from computational analysis and subsequently backed by experimental studies is the e2f transcription factor 3 ( e2f3 ) . this protein plays a pivotal role in the control of the cell cycle , therefore the study potentially reveals a novel link between hypoxia and cell proliferation . however , there are limited data about a possible biological impact of this connection . the recent study by camps et al . provided the first evidence that mir-210 represents not only a marker of tumour hypoxia in vivo , but is also a prognostic indicator of adverse prognosis in breast cancer , and may represent a valuable drug target . although proteins represent the overwhelming majority of therapeutic targets , recent developments of mir derivatives such as anti - mir oligonucleotides ( amos ) and locked nucleic acids ( lnas ) are regarded as important steps toward clinical applications [ 5559 ] . while classic gene therapy strategies have failed to fulfil expectations in most cases , the small size of mirs and their high rate of transduction in eukaryotic cells represent distinctive advantages . indeed , the feasibility of oligonucleotide - based therapeutics such as micrornas and sirnas is already supported by ongoing clinical trials . to list only a few , sirna , inc . developed sirna-027 , which targets vegfr-1 and is now in a phase ii clinical trial for age - related macular degeneration . additionally , alnylam has initiated a phase i clinical trial of an inhaled sirna - based drug for the treatment of respiratory syncytial virus ( rsv ) infections , and santaris pharma has developed spc3649 , which specifically targets mir-122 , which is important for hepatitis c virus replication . in cancer , one can hypothesize that inactivation of an mir critically important for the response to lox oxygen could have a beneficial effect against a tumour compartment notoriously resistant to therapy [ 2426 ] . mir-210 analysis also serves as proof of principle for a novel class of non - invasive cancer diagnostic tools , as it is readily detectable in the serum from patients with diffuse large b - cell lymphoma ( dlbcl ) versus healthy controls . the impact of mir-210 ( and by extension , of other less thoroughly validated hypoxia - regulated mirs ) may not be limited to cancer . hypoxia represents a central component of other clinical conditions with major impact on morbidity and mortality , such as cardiac ischaemia and cerebrovascular diseases . although to date there are no formal data to substantiate a mechanistic role in such disorders , mir-210 was recently found up - regulated in a mouse model of cardiac hypertrophy / cardiac failure and in response to brain transient focal ischaemia in rats [ 6466 ] . another example of disease with a hypoxic component is preeclampsia , which is associated with accumulation of hif - alpha proteins at placental level . consistent with its status of hif target , mir-210 was found up - regulated in placentas from patients with preeclampsia , compared to normal pregnant women . again , whether mir-210 contributes to the clinical manifestations of this condition is yet to be determined .

the heart is responsible for supplying the organs and tissues of the body with blood , and the kidneys , amongst other functions , play an integral role in fluid balance and salt homeostasis . it should therefore come as little surprise that renal dysfunction frequently accompanies cardiac failure and that cardiac dysfunction frequently accompanies renal failure . this phrase has been in use since 2004 , but despite generating a plethora of papers in the literature and being discussed at length in dedicated conferences , crs has until very recently lacked a universally accepted definition , and numerous key questions remain unanswered . we shall cover the epidemiology , pathophysiology , and current management of crs in this paper , but we will begin with brief case histories which help demonstrate the heterogeneity of patients who fall under the umbrella term of crs . case 1a 63-year - old patient with known severe heart failure and chronic renal impairment ( baseline creatinine 190 mmol / l , estimated glomerular filtration rate ( egfr ) 23 mls / min ) was admitted with acute decompensated heart failure ( adhf ) . creatinine on admission was similar to baseline , but over the next week renal function deteriorated significantly ( urea 51.1 mmol / l , creatinine 503 mmol / l , egfr 8) requiring inotropic support and then haemofiltration . her inpatient stay lasted 7 weeks , of which over half was spent on high dependency or intensive care units . a 63-year - old patient with known severe heart failure and chronic renal impairment ( baseline creatinine 190 mmol / l , estimated glomerular filtration rate ( egfr ) 23 mls / min ) was admitted with acute decompensated heart failure ( adhf ) . creatinine on admission was similar to baseline , but over the next week renal function deteriorated significantly ( urea 51.1 mmol / l , creatinine 503 mmol / l , egfr 8) requiring inotropic support and then haemofiltration . her inpatient stay lasted 7 weeks , of which over half was spent on high dependency or intensive care units . case 2a 31-year - old previously fit and well indian man was admitted with a two - week history of malaise and a 2-day history of hemoptysis . admission blood tests revealed urea level of 20 mmol / l and creatinine level of 1100 after his first three sessions of hemodialysis , echocardiography was repeated and revealed normal systolic function . a 31-year - old previously fit and well indian man was admitted with a two - week history of malaise and a 2-day history of hemoptysis . admission blood tests revealed urea level of 20 mmol / l and creatinine level of 1100 after his first three sessions of hemodialysis , echocardiography was repeated and revealed normal systolic function . case 3a 32-year - old lady developed end - stage renal failure secondary to type 1 diabetes mellitus . she commenced hemodialysis in 2007 , and just prior to this , transthoracic echocardiography revealed concentric ventricular hypertrophy and severely impaired systolic function . 6 months after she had been started on hemodialysis , repeat echocardiography revealed marked improvement in systolic function , with lv dysfunction now only mild rather than severe . a 32-year - old lady developed end - stage renal failure secondary to type 1 diabetes mellitus . she commenced hemodialysis in 2007 , and just prior to this , transthoracic echocardiography revealed concentric ventricular hypertrophy and severely impaired systolic function . 6 months after she had been started on hemodialysis , repeat echocardiography revealed marked improvement in systolic function , with lv dysfunction now only mild rather than severe . case 4a 28-year - old fit gentleman , with no past medical history , was admitted feeling unwell for the past 3 days . he was extremely ill when first seen : temperature 40c , bp 70/35 mmhg , and pulse rate 130 . initial blood tests revealed marked leukocytosis ( white cell count 41.5 10/l , neutrophil count 38.5 10/l ) and acute renal failure ( urea 6.2 he was diagnosed with septic shock and treated with fluids and broad - spectrum intravenous antibiotics . in less than 72 hours , he was feeling significantly better and renal function had returned to normal . repeat echocardiography one week later revealed normal systolic function . a 28-year - old fit gentleman , with no past medical history , he was extremely ill when first seen : temperature 40c , bp 70/35 mmhg , and pulse rate 130 . initial blood tests revealed marked leukocytosis ( white cell count 41.5 10/l , neutrophil count 38.5 10/l ) and acute renal failure ( urea 6.2 mmol / l and creatinine 184 mmol / l ) . he was diagnosed with septic shock and treated with fluids and broad - spectrum intravenous antibiotics . in less than 72 hours , he was feeling significantly better and renal function had returned to normal . all of these patients had coexistent cardiac and renal dysfunction but clearly with grossly different underlying pathology and , therefore , prognoses . renal dysfunction is unfortunately extremely prevalent in patients with congestive cardiac failure ( ccf ) , and the associated statistics make sombre reading . data from the acute decompensated heart failure national registry ( adhere ) of over 100,000 patients ( admitted with adhf ) revealed that almost one third of patients have a history of renal dysfunction . another study found that , in a survey of outpatients with congestive cardiac failure , 39% patients in new york heart association ( nyha ) class 4 and 31% of patients in nyha class 3 had severely impaired renal function ( creatinine clearance < 30 mls / minute ) . baseline renal function is as important an adverse prognostic marker as ejection fraction and nyha functional class . elevated serum creatinine on admission to hospital with adhf and worsening renal function during admission for adhf have both been shown to predict prolonged hospitalisation , increased need for intensive care facilities , and increased mortality [ 7 , 8 ] . almost 44% of deaths in patients with end - stage renal failure ( esrf ) are due to cardiovascular diseases , and a 2006 meta - analysis indicated that patients with esrf are more likely to die from cardiovascular causes than from renal failure itself . death from cardiovascular causes is 1020 times more common in patients with chronic renal failure than in matched segments of the general population . half of patients commencing hemodialysis will suffer a myocardial infarction within the following two years , and mortality in this patient population is high . increased myocardial mass ( i.e. , left ventricular hypertrophy)which increases myocardial oxygen demand is increased in mild - to - moderate as well as more advanced stages of renal failure . on the other hand , treatment of renal dysfunction can improve cardiac function , although the majority of this evidence comes from esrf patients receiving kidney transplants . a study of over 100 dialysis patients with known heart failure who underwent renal transplantation showed an improvement of ejection fraction from 32% to 52% and over two thirds of patients had complete normalisation of cardiac function . there are a few other such reports , albeit , all in the transplant population [ 1517 ] . crs has , in the absence of a generally accepted definition , usually been perceived as renal dysfunction secondary to chronic cardiac dysfunction ( i.e. , heart failure ) . however , this clearly failed to address the numerous other instances in which cardiac and renal dysfunction coexist . first proposed a five - part classification scheme for the cardiorenal syndromes in 2008 , and this has since been incorporated into the report from a consensus conference held in the same year . the classification system is outlined in table 1 but essentially recognises the multiple ways in which cardiorenal dysfunction occurs and defines the primary and secondary organ dysfunction in each case . disorders of the heart and kidneys whereby acute or chronic dysfunction in one organ may induce acute or chronic dysfunction of the other . this phraseology was chosen as it helps explain the bi - directional nature of the various syndromes . cardiorenal connection as an addition to the haemodynamic framework ( on the control of extracellular fluid volume ( ecfv ) ) developed by the late physiologist arthur guyton and termed this the severe cardiorenal syndrome ( scrs ) . they stated that scrs is a syndrome with accelerated and extensive cardiovascular disease that has distinct properties not occurring in conditions that affect either organ alone . they proposed the renin - angiotensin - aldosterone system ( raas ) , balance between nitric oxide ( no ) and reactive oxygen species ( ros ) , inflammation , and sympathetic nervous system ( sns ) as circuits within the cardiorenal connection . derangement of any connector was thought to initiate a vicious downward spiral culminating in disturbance in the other connectors and culminating in cardiac and renal dysfunction via common final pathophysiological pathways . as our knowledge of crs expands , it is becoming increasingly clear how complex the interaction between heart and kidneys is once one organ becomes diseased . conventional thinking for decades held that the progressive deterioration in renal function in heart failure patients was primarily as a result of reduced renal blood flow secondary to reduced cardiac output . inadequate renal afferent flow was said to activate the renin - angiotensin - aldosterone system ( raas ) leading to fluid retention , increased preload , and thus worsening pump failure . however , recent work suggests that , though correct , this is a very narrow and incomplete picture . the evaluation study of congestive heart failure and pulmonary artery catheterization effectiveness ( escape ) trial assessed pulmonary artery catheter - guided management of over 400 patients admitted with adhf . it found no correlation between baseline renal function and cardiac index , and improvement of the latter did not result in improved renal function . others have also found that improved cardiac index or reduced wedge pressure during pulmonary artery catheter - guided management failed to predict improvement in renal function [ 23 , 24 ] . additionally , worsening renal function has been demonstrated in adhf patients despite normal systolic function ( ejection fraction ) , and thus , presumably , renal blood flow . in combination , these data suggest much more than simply reduced renal blood flow as an explanation for crs . activation of the raas by reduced perfusion pressure is a protective mechanism against potentially dangerous conditions like haemorrhage . unfortunately , when chronically stimulated as in both heart and renal failure the pathophysiological consequences are severe and deleteriously affect function of both organ systems . renin is produced in the juxtaglomerular apparatus of the kidneys and catalyses the conversion of angiotensinogen i to angiotensinogen ii , which is subsequently turned into angiotensin ii ( ang ii ) by angiotensin - converting enzyme ( ace ) . ang ii has numerous negative effects upon the cardiovascular system in heart failure patients , increasing both preload and afterload and thus myocardial oxygen demands . the main changes induced by ang ii are illustrated in figure 1 , but one of the most important recent advances has been recognition of the promotion of vascular inflammation . ang ii activates the enzyme nadph oxidase in endothelial cells , vascular smooth muscle cells , renal tubular cells , and cardiomyocytes . a growing body of evidence suggests that ros are responsible for the processes of aging , inflammation , and progressive organ dysfunction . nitric oxide ( no ) is responsible for vasodilation and natriuresis and assists in renal control of ecfv . oxidative stress damages dna , proteins , carbohydrates , and lipids and also shifts cytokine production towards proinflammatory mediators such as interleukin-1 , interleukin-6 , and tumour necrosis factor alpha . sns activation is initially a protective mechanism in ccf patients , akin to raas activation . the aim is to maintain cardiac output by positive chronotropic and inotropic effects on the myocardium . unfortunately , chronic sns activation also results in numerous negative effects upon the cardiovascular system and kidneys . sns overactivity leads to reduction in beta - adrenoceptor density within myocardium and also reduced adrenoceptor sensitivity in both renal and cardiac failure . sns activation leads to increased cardiomyocyte apoptosis and increases the release of the neurohormone neuropeptide y ( npy ) . npy is a vascular growth promoter leading to neointimal formation ( and thus atherosclerosis ) , induces vasoconstriction , and also interferes with normal immune system function . renal sympathetic denervation in patients with resistant hypertension significantly improved renal function in one quarter of patients , and bilateral renal nerve ablation has been shown to reduce blood pressure at one - year followup . however , these therapies have not been tested in the heart failure population and thus still requires further evaluation . heart failure is marked by an elevation in central venous pressure which reduces the perfusion gradient across the renal capillary bed . studies performed in the early part of the last century demonstrated that rising renal venous pressures could reduce or even abolish urine production , and rising renal venous pressure was more important than falling arterial ( perfusion ) pressure in this setting . intraabdominal pressure ( iap ) is said to be elevated when > 8 mmhg , and intraabdominal hypertension has been defined as a pressure > 12 mmhg . a study of 40 patients admitted with adhf found that 24 had an iap > 8 mmhg though none had abdominal symptoms . the degree of reduction of iap with diuretic treatment correlated with an improvement in renal function . the escape trial found that baseline right atrial pressure , but not arterial blood flow , correlated with baseline serum creatinine . patients with baseline renal dysfunction or worsening renal function after admission have significantly elevated central venous pressure compared to those with less or no renal dysfunction . additionally , elevated jugular venous pressure on physical examination is associated with higher baseline serum creatinine and increased risk of hospitalisation due to adhf and death due to pump failure . cras was first described almost a decade ago by silverberg et al . as a vicious cycle of deterioration that leads to poor outcomes , including faster progression to esrf and further progression of congestive heart failure . their simple model suggested anaemia as a condition induced by dysfunction of either organ but also exacerbating dysfunction of either organ . the candesartan in heart failure : assessment of reduction in morbidity and mortality ( charm ) study suggested that anaemia was an independent adverse prognostic factor in ccf patients . there has , however , like for crs , been a lack of consensus over the true definition , significance , and management strategy for patients with cras ( if even such a syndrome exists ) . this has mainly stemmed from a lack of large - scale randomised controlled trials to guide management . anaemia is widely thought to have a multifactorial aetiology in patients with ckd or ccf , but iron deficiency is thought to play a prominent role in both [ 54 , 55 ] . the ferinject assessment in patients with iron deficiency and chronic heart failure ( fair - hf ) study assessed intravenous ( iv ) iron therapy in 459 symptomatic ccf patients with iron deficiency . it demonstrated that the treatment group had a significant improvement in heart failure symptoms , exercise capacity , and quality of life irrespective of whether they actually had underlying anaemia or not . long - term safety data on the newer dextrans - free iv preparations are still awaited , but iv iron does appear to be emerging as an important therapy in patients with cras . however , current european guidelines for the management of heart failure ( published before results of trials such as fair - hf were available ) describe correction of anaemia in ccf patients as unproven and not established as routine therapy . the role of erythropoietin stimulating agents ( esas ) is also controversial due to conflicting evidence . erythropoietin is a cytokine produced in the kidneys that is essential for red blood cell production . scientific studies have shown that erythropoietin protects cardiomyocytes from apoptosis [ 58 , 59 ] and that the mechanism appears to be upregulation of endothelial nitric oxide synthase . a study of 26 heart failure patients with anaemia who received esa found significantly improved exercise capacity which appeared to be principally due to increased oxygen delivery due to higher haemoglobin concentration . however , studies have shown that patients with ccf have elevated endogenous erythropoietin levels and that this is associated with poorer survival independent of haemoglobin level [ 62 , 63 ] . an additional study showed that an erythropoietin level higher than expected was an independent predictor of increased mortality even after adjustment for possible confounding variables . although initial small studies suggested benefit in correcting anaemia due to ckd with esas , three large multicentre phase iii trials all had negative outcomes and put a severe question mark over the future of these agents . the cardiovascular risk reduction by early anaemia treatment with epoetin beta ( create ) trial found that correcting anaemia early in patients with renal failure does not reduce their risk of cardiovascular complications . the trial to reduce cardiovascular events with aranesp therapy ( treat ) study found that diabetic patients with renal failure and moderate anaemia had no benefit from receiving esa and in fact had a statistically higher risk of stroke . finally , the correction of hemoglobin and outcomes in renal insufficiency ( choir ) trial found that aiming for a higher haemoglobin level in ckd patients with anaemia was associated with a higher risk of adverse outcome including death , hospitalisation for heart failure , or myocardial infarction . a randomised trial of two dosing regimens of the esa darbepoetin alfa in patients with heart failure and anaemia showed no improvement in nyha class , lv ejection fraction , or minnesota living with heart failure questionnaire score . consequently , the routine use of esa therapy to increase haemoglobin levels in anaemic ccf patients does not have a sound evidence base . medical management of patients with concomitant cardiac and renal dysfunction remains tremendously challenging , and this is exacerbated by the fact that the vast majority of trials providing evidence for treatments in heart failure excluded patients with significant renal impairment . the heterogeneous nature of patients with crs also poses unique challenges with no single success - guaranteed therapy . there is limited trial data proving mortality benefit for diuretics in crs , but they have long been deemed an essential management strategy in these patients . data from the adhere registry suggests that 81% of patients were using chronic diuretic therapy at the time of admission with adhf . studies have shown , however , that furosemide decreases gfr in many patients , and higher doses of diuretics are independently associated with sudden cardiac death or death from pump failure [ 71 , 72 ] . furosemide also stimulates the raas and can thus increase fibrosis . a systematic review and meta - analysis on the use of loop diuretics in management of patients with acute kidney injury found no mortality benefit , though there was a shorter required duration of renal replacement therapy . a large observational cohort study examining the use of diuretics in intensive care patients with acute renal failure found a significantly increased risk of death or nonrecovery of baseline renal function in the patients receiving diuretics . however , the two papers mentioned above looked at all mechanisms of renal dysfunction , not just the heart failure population . there is unfortunately a dearth of high - quality randomised controlled evidence to support or refute the use of diuretics in patients with cardiac and renal dysfunction . therefore , in the absence of definitive data proving harm in heart failure population , diuretics should not be withheld from volume - overloaded patients . diuretic resistance is frequently used as a surrogate marker of poor prognosis in ccf patients . the most probable culpable mechanisms are inadequate diuretic dose , excessive sodium intake , delayed intestinal absorption due to gut mucosal oedema , decreased diuretic excretion into urine , and increased sodium reabsorption from other parts of the nephron not blocked by loop diuretics ( e.g. , distal convoluted tubule ) [ 75 , 76 ] . concomitant use of nonsteroidal anti - inflammatory drugs can also contribute to diuretic resistance by diminishing synthesis of vasodilator and natriuretic prostaglandins . firstly , one should bear in mind that furosemide does not have a smooth dose - response curve , meaning that no natriuresis would occur until a threshold rate of drug excretion is reached . consequently , a patient not responding to 40 mg furosemide should have the dose doubled to 80 mg rather than the frequency doubled to twice daily . secondly , patients should be instructed to restrict their salt intake to help achieve net fluid loss . thirdly , the patient may require iv diuretic therapy to avoid the poor bioavailability frequently encountered due to reduced gastrointestinal blood flow , reduced intestinal peristalsis , and intestinal mucosal oedema . a cochrane review has confirmed that continuous iv furosemide infusion achieves a greater diuresis than bolus iv doses and this is associated with reduced mortality and shorter hospital stay . other treatment options include adding in a thiazide diuretic to block distal sodium reabsorption , a potassium - sparing diuretic such as spironolactone , or adding salt - poor albumin . salt - poor albumin is thought to enhance delivery of furosemide to the kidney , and one small study suggested adding salt - poor albumin to a furosemide infusion significantly increased sodium excretion . ace inhibitors are known to reduce mortality in patients with heart failure , though the majority of these studies excluded patients with significant renal impairment . the cooperative north scandinavian enalapril survival ( consensus ) study revealed that patients with the most severe ccf had a substantial increase in creatinine on initiation of an ace inhibitor irrespective of baseline creatinine . however , it is comforting to note that in the consensus trial the outcomes were better in the treatment arm even though mean creatinine increased . indeed , some have proposed that the rise in creatinine after initiation of an ace inhibitor actually may identify the subgroup of patients who derive the most benefit . ace inhibitors should be used with caution in patients with crs and renal functional monitored closely during initiation and uptitration . studies have shown that patients with first presentation of pulmonary oedema are frequently discharged without initiation of ace inhibitor therapy for fear of worsening renal function . however , as mentioned above , patients who derive prognostic benefit over the longer term from these drugs may experience slight deterioration of renal function in the short term . a concomitant reduction in diuretic dosage may be required ( especially once the patient is euvolemic ) to facilitate safe uptitration of the ace inhibitor . the chances of deterioration of renal function after starting ace inhibitor therapy can also be minimised by avoiding simultaneous use of nsaids and ensuring the patient is not hypovolemic at onset of treatment . patients with crs are often hypotensive , and admissions due to adhf frequently result in severe hypotensive episodes or frank cardiogenic shock . this may be accompanied by oligo - anuria , and inotropes are frequently used in this setting with the aim of improving cardiac output and thus renal blood flow . renal or low - dose dopamine is known to increase renal blood flow though there is conflicting evidence regarding its effect upon gfr [ 85 , 86 ] . one study of 13 patients suggested that dopamine reduces renovascular resistance , though the baseline renal function of these patients is not stated . however , another larger study in which 75% had acute renal failure showed an increase in renovascular resistance in these patients with a fall in resistance in those with normal renal function . trials of dobutamine and milrinone have shown improvement of cardiac index and , in proportion , renal blood flow however , this has not translated into mortality benefit . the outcomes of a prospective trial of intravenous milrinone for exacerbations of a chronic heart failure ( optime - hf ) trial clearly rejected the hypothesis that milrinone would improve renal function and overall survival in adhf patients . the patient population who requires inotropic support for adhf or cardiogenic shock is inherently complex , and thus designing adequately powered and well - conducted randomised trials poses clear challenges . however , it seems likely that short - term inotropic support for such patients in a low - output state is likely to continue . current esc heart failure guidelines state the evidence for using dobutamine as class iia level b , dopamine class iia level c , milrinone class iib level b , and levosimendan class iia level b ( i.e. , none has a class i or level of evidence a recommendation ) . certain pharmacological agents which held much promise during development have failed to make the expected impact following results of phase iii clinical trials . nesiritide is an analogue of brain natriuretic peptide ( bnp ) and known to induce vasodilation and reduce filling pressures as well as augment cardiac output . the first large randomised trial of nesiritide in patients with crs demonstrated no difference in gfr , renal plasma flow , urine output , sodium excretion , or mortality between treatment and placebo groups . a meta - analysis of seven large randomised trials of nesiritide also showed a lack of mortality benefit at 30-day and 180-day followup . a pooled analysis of three trials showed a strong trend ( p value .057 ) towards increased early mortality with nesiritide . the results of the acute study of clinical effectiveness of nesiritide in decompensated heart failure trial ( ascend ) are currently awaited and may help clarify if this agent has a future in the management of crs ( type 2 ) patients . the ability to make a diagnosis of crs early in a patient 's assessment may allow early introduction of management strategies which would hopefully prevent further clinical and biochemical deterioration . therefore , the development of novel biomarkers of acute kidney injury is a promising step . neutrophil gelatinase - associated lipocalin , cystatin c , kidney injury molecule-1 , n - acetyl--(d)glucosaminidase , and interleukin-18 have all been shown to act as markers of renal injury in a variety of different clinical scenarios , and further work is ongoing to help define their role in diagnosis and management . this extracorporeal treatment permits removal of large fluid volumes more speedily than diuretics and without inducing profound hypotension . the unload trial showed that , 48 hours after treatment , uf safely produced greater weight and net fluid loss than conventional iv diuretic therapy and at 90 days the uf group had fewer repeat admissions to hospital for fluid reaccumulation . however , another trial demonstrated that uf did not improve renal haemodynamics ( as judged by urine output , egfr , and renal plasma flow ) . arginine vasopressin ( avp ) is released from the posterior pituitary gland and mediates water retention via the v2 receptor in the renal collecting ducts . avp levels are elevated in heart failure patients and avp antagonists ( the vaptans ) have thus been developed . tolvaptan was initially shown to reduce body weight and help normalize serum sodium in adhf patients without adverse effect on blood pressure , heart rate , or renal function . later studies also demonstrated that tolvaptan , when compared to placebo , significantly reduced pulmonary arterial pressure , pulmonary capillary wedge pressure , and right atrial pressure as well as increasing urine output without adverse effect on renal function . the multicentre international phase iii efficacy of vasopressin antagonism in heart failure outcome study with tolvaptan ( everest ) trial randomised patients admitted with adhf within 48 hours to receive either tolvaptan 30 mg once daily or placebo for a minimum time duration of 60 days . tolvaptan was associated with more weight loss and less dyspnoea on days 1 and 7 and without adverse effect on renal function . however , the key end points of all - cause mortality , cardiovascular mortality , cardiovascular death , or hospitalization , and worsening heart failure were not different between the two groups . adenosine - a1 receptors are found in the kidney and thought to mediate urine output . adenosine levels are increased in heart failure and thus adenosine - a1 receptor antagonists were conceived ; unfortunately , again , the vital randomised trial failed to show any benefit in adhf patients . ccf patients characteristically have an expanded extracellular fluid volume and contracted arterial blood volume with resultant regional perfusion abnormalities . this results in a series of complex neurohormonal changes leading to peripheral and central congestion and reduced renal blood flow . hypertonic saline solution ( hss ) has been proposed as a useful adjunct to iv furosemide in adhf patients with crs with several postulated mechanisms of action : mobilisation of fluid from the extravascular space to the intravascular compartment by the osmotic effects of hss and an increase in renal blood flow which can thus help overcome diuretic resistance . small studies have demonstrated the ability of hss to augment renal blood flow and a larger trial which randomised nyha class 4 patients to iv furosemide plus hss or iv furosemide bolus alone revealed a greater degree of diuresis and natriuresis , lower rehospitalisation rate , and lower mortality rate in the hss group versus placebo group . however , routine use of hss in adhf patients remains rare , and its role in this patient population is yet to be defined . as our review has hopefully demonstrated , crs is an ominent development in many patients . however , prognosis is not uniform across all five subtypes and highly dependent upon the nature of the underlying disease process(es ) . crs has generally been used so far to describe patients with renal dysfunction secondary to chronic heart failure ; this group of patients have a particularly high morbidity and mortality . fortunately , however , cardiologists and nephrologists are now acutely aware of the scale of the problem posed by crs , and this awakening will hopefully translate into greater research into this fascinating yet challenging clinical conundrum .

chronic bronchitis ( cb ) is a progressive disease characterized by chronic increase in the production of mucous . it is defined as the presence of a chronic cough with sputum production for at least three months , in each of two consecutive years , after other causes of cough , such as tuberculosis and lung cancer , have been eliminated.1,2 acute exacerbations of chronic bronchitis ( aecb ) are defined as recurrent attacks of worsening bronchial inflammation that occur on average 1.53 times a year , and are superimposed on baseline cb.1 these exacerbations are marked by an increase in the volume of daily sputum produced ; a change in color of the expectorated sputum ( ie , darker , more yellow , more green ) ; and worsening dyspnea . it also induces the synthesis of surfactant in lung alveolar type ii cells . by this mechanism , it has also been proved to have antioxidant and anti - inflammatory activities.3 it became evident that theophylline can exert anti - inflammatory and immunomodulating actions at lower plasma concentrations than those required for bronchodilation.4 theophylline inhibits the production and release of pro - inflammatory cytokines , including il-1 , tnf- , and ifn-. the production of the anti - inflammatory cytokine il-10 is increased . the guidelines of the global strategy for the diagnosis , management , and prevention of chronic obstructive lung disease ( gold ) currently recommend consideration of the addition of an oral or intravenous methylxanthine to aerosolized bronchodilators , for management of severe exacerbations of chronic obstructive pulmonary disease ( copd).5 the effect of theophylline and ambroxol , when combined together , has been proved to give more than additive effects in improving the mucociliary clearance and increasing the release of respiratory surfactants.6,7 guaiphenesin is a commonly used over - the - counter drug for productive cough . an us food and drug administration ( fda ) review of preparations available over - the - counter concluded that guaiphenesin was an effective expectorant.8 farcosolvin ( pharco pharmaceuticals , alexandria , egypt ) is a cough mixture of ambroxol ( 15 mg ) ; theophylline ( 50 mg ) ; guaiphenesin ( 30 mg ) , per 5 ml syrup . the purpose of this study is to test the clinical efficacy of farcosolvin in the treatment of acute exacerbation of chronic bronchitis . a pragmatic , randomized , single - blinded , comparative study , designed to determine the efficacy of farcosolvin syrup in reducing clinical symptom scores under conditions that reflect the usual medical care for aecb ( defined as worsening of the baseline symptoms [ cough , sputum , and breathlessness ] of cb ) , was conducted . to detect a difference in the mean severity of cough and expectoration score between the two treatment groups of 4 points with a standard deviation ( sd ) of 5 ; a p value < 0.05 ; and an approximate power of 90% , we calculated the minimal needed sample size to be 68 patients . one hundred eligible patients were included in the study analysis according to the following criteria defined below . patients were eligible for inclusion in the study if they were a male or female aged 45 years , with a history of smoking and a physician s diagnosis of aecb . the diagnosis of cb was based on the history of cough and expectoration on most days during a period of at least three consecutive months for two consecutive years.1 aecb was classified as type i according to anthonisen et al 9 when it met all three of the following criteria : increases in amount of sputum ; purulence of sputum ; and increasing dyspnea . aecb was classified as type ii if it met two of the above three criteria and type iii if it met only one criterion.9 patients were excluded if they had any of the following conditions : heart failure or multiple organ dysfunctions : bronchiectasis ; bronchial carcinoma ; interstitial lung disease ; pneumonia ; inability to comply with the study procedures because of dementia ; or if they had any other medical problems that in the opinion of the investigator would interfere with the conduct of study . the study was conducted at mabarah hospital and green clinics and was approved by the ethics committee and all participants provided written informed consent . all consecutive patients presenting to both centers who met the eligibility criteria between november 2005 and october 2006 were allocated sequential numbers as they entered the study . patients were randomized into an experimental group , who were treated with farcosolvin ( 5 ml syrup three times daily ) ; and a control group , who were treated with a generic guaiphenesin syrup ( 100 mg per 5 ml three times daily ) . a block randomization technique was used to ensure that equal numbers were allocated to each group . in addition to farcosolvin or guaiphenesin , all patients received standard treatment for their exacerbation as specified by their condition and the guidelines . this was antibiotic ( ciprofloxacin 500 mg , twice daily , for 7 days ) ; inhaled or nebulized bronchodilators in the form of salbutamol ( two inhaler puffs or 5 mg in 2 ml saline nebulized ) ; and/or ipratropium 0.5 mg as needed , or up to four times daily . mucolytics , theophylline and expectorants were not permitted during the trial , except as in the protocol . severity of cough and sputum clearance was measured using the modified questionnaire for ease of cough and sputum clearance.10 at the end of one week treatment , all patients were asked to grade their impression of improvement or change in their condition using the clinical global impression of improvement or change scale ( cgic),11 which is a 7-point rated scale where 0 = not assessed ; 1 = very much improved ; 2 = much improved ; 3 = minimally improved ; 4 = no change ; 5 = minimally worsened ; 6 = much worsened ; and 7 = very much worsened . the two treatment groups were compared at baseline and at 3 and 7 days , using tests for nonparametric data ( friedman , kruskal all tests were two - tailed and a 5% significance level was maintained throughout these analyses . a pragmatic , randomized , single - blinded , comparative study , designed to determine the efficacy of farcosolvin syrup in reducing clinical symptom scores under conditions that reflect the usual medical care for aecb ( defined as worsening of the baseline symptoms [ cough , sputum , and breathlessness ] of cb ) , was conducted . to detect a difference in the mean severity of cough and expectoration score between the two treatment groups of 4 points with a standard deviation ( sd ) of 5 ; a p value < 0.05 ; and an approximate power of 90% , we calculated the minimal needed sample size to be 68 patients . one hundred eligible patients were included in the study analysis according to the following criteria defined below . patients were eligible for inclusion in the study if they were a male or female aged 45 years , with a history of smoking and a physician s diagnosis of aecb . the diagnosis of cb was based on the history of cough and expectoration on most days during a period of at least three consecutive months for two consecutive years.1 aecb was classified as type i according to anthonisen et al 9 when it met all three of the following criteria : increases in amount of sputum ; purulence of sputum ; and increasing dyspnea . aecb was classified as type ii if it met two of the above three criteria and type iii if it met only one criterion.9 patients were excluded if they had any of the following conditions : heart failure or multiple organ dysfunctions : bronchiectasis ; bronchial carcinoma ; interstitial lung disease ; pneumonia ; inability to comply with the study procedures because of dementia ; or if they had any other medical problems that in the opinion of the investigator would interfere with the conduct of study . patients were eligible for inclusion in the study if they were a male or female aged 45 years , with a history of smoking and a physician s diagnosis of aecb . the diagnosis of cb was based on the history of cough and expectoration on most days during a period of at least three consecutive months for two consecutive years.1 aecb was classified as type i according to anthonisen et al 9 when it met all three of the following criteria : increases in amount of sputum ; purulence of sputum ; and increasing dyspnea . aecb was classified as type ii if it met two of the above three criteria and type iii if it met only one criterion.9 patients were excluded if they had any of the following conditions : heart failure or multiple organ dysfunctions : bronchiectasis ; bronchial carcinoma ; interstitial lung disease ; pneumonia ; inability to comply with the study procedures because of dementia ; or if they had any other medical problems that in the opinion of the investigator would interfere with the conduct of study . the study was conducted at mabarah hospital and green clinics and was approved by the ethics committee and all participants provided written informed consent . all consecutive patients presenting to both centers who met the eligibility criteria between november 2005 and october 2006 were allocated sequential numbers as they entered the study . patients were randomized into an experimental group , who were treated with farcosolvin ( 5 ml syrup three times daily ) ; and a control group , who were treated with a generic guaiphenesin syrup ( 100 mg per 5 ml three times daily ) . a block randomization technique was used to ensure that equal numbers were allocated to each group . in addition to farcosolvin or guaiphenesin , all patients received standard treatment for their exacerbation as specified by their condition and the guidelines . this was antibiotic ( ciprofloxacin 500 mg , twice daily , for 7 days ) ; inhaled or nebulized bronchodilators in the form of salbutamol ( two inhaler puffs or 5 mg in 2 ml saline nebulized ) ; and/or ipratropium 0.5 mg as needed , or up to four times daily . mucolytics , theophylline and expectorants were not permitted during the trial , except as in the protocol . severity of cough and sputum clearance was measured using the modified questionnaire for ease of cough and sputum clearance.10 at the end of one week treatment , all patients were asked to grade their impression of improvement or change in their condition using the clinical global impression of improvement or change scale ( cgic),11 which is a 7-point rated scale where 0 = not assessed ; 1 = very much improved ; 2 = much improved ; 3 = minimally improved ; 4 = no change ; 5 = minimally worsened ; 6 = much worsened ; and 7 = very much worsened . finally , all subjects were interrogated about any possible adverse effects . the two treatment groups were compared at baseline and at 3 and 7 days , using tests for nonparametric data ( friedman , kruskal all tests were two - tailed and a 5% significance level was maintained throughout these analyses . the analysis included 100 patients with aecb grouped into two equal sized groups : an experimental group ( 50 patients ) , who were treated with farcosolvin 5 ml syrup three times daily ; and a control group ( the remaining 50 ) , who were given guaiphenesin syrup 3 times daily . the two groups were almost matched at baseline in terms of age , smoking history , lung function , oxygen saturation , severity of cough , and breathlessness ( table 1 ) . also , there was no significant difference between both groups for the concurrent medications they received during the study . all patients needed to be treated with antibiotic and bronchodilators were given on an as - needed basis , except for four patients in the farcosolvin group and 6 in the guaiphenesin control group who required regular inhaler use . one patient needed to be given a budesonide dry powder inhaler at doses of 200 g twice daily at day 3 due to worsening dyspnea . the effects of treatment on breathlessness scores and cough scores were analyzed by nonparametric statistical methods as the distribution was found by kolmogorov smirnov to be not normal ( p < 0.001 ) . there was a statistically significant improvement of breathlessness and cough scoring in both groups when compared before and after treatment at day 3 and at day 7 ( tables 2 and 3 ) , using a nonparametric related samples friedman test ( p < 0.05 ) . there was a highly statistically significant difference in improvement in breathlessness score , as well as cough score , in favor of farcosolvin treatment when the two groups were compared using an independent samples kruskal wallis test ( tables 4 and 5 ) . out of 50 patients , 48 ( 96% ) in the farcosolvin treated group rated their improvement on the cgic scale as much and very much improved , while only 41 patients ( 82% ) reported the same degree of improvement in the control group . five of the subjects treated with farcosolvin reported nausea compared with four of the subjects treated with guaiphenesin . to our knowledge , this is the first study to address the effects of a mixture of theophylline , ambroxol and guaiphenesin in relation to clinical manifestations and severity of symptoms of aecb . this study showed significant improvement in breathlessness and severity of cough scores in patients treated with the farcosolvin mixture than those treated with guaiphenesin alone . ambroxol has been tested in many large multicenter , randomized , double blind controlled studies in cases of cb . the results confirm the previously seen improvement in symptomatology and the significant reduction in number and severity of exacerbations in groups treated with ambroxol versus placebo.1214 in an open , long - term multicenter study including 5635 patients , ambroxol was effective and well - tolerated for the prophylaxis of exacerbations of chronic bronchitis.2 positive effects have been seen with the use of ambroxol in patients with early hypersecretory cb , including improvement in coughing ; dyspnea ; color and consistency of sputum ; and ease of expectoration , when compared to a control ( p < 0.05).13 theophylline has also been proven effective in cases of cb and copd.4 the accumulated evidence has led theophylline to be recommended as a treatment in addition to aerosolised bronchodilators for severe exacerbations of copd , in the gold guidelines.5 the combination of theophylline plus ambroxol has been proven to give more than additive effects in mucociliary clearance and increasing the release of respiratory surfactants.6,7 this was the basis of our study for the efficacy of the triple mixture of a theophylline , ambroxol , plus an expectorant , in cases with aecb , when sputum amount , content and viscosity changes added to compromised mucociliary clearance and surfactant release are among the factors leading to worsening of symptoms and general condition of patients . our results , to some extent , can support our assumption that this mixture can be helpful in such patients through working against these factors . we concluded from our study that farcosolvin syrup might be safe and effective in improving symptoms in cases of aecb .

with the recent widespread introduction of cone beam computed tomography ( cbct ) , dentists and otolaryngologists are better able to identify anatomical abnormalities and pathological states within the structures of the nasal cavity and the surrounding paranasal sinuses . mucosal inflammation can be easily identified in computed tomography ( ct ) scans , arguably making this radiographic modality the standard for accurately evaluating the nasal cavity and paranasal sinuses . on each side of the nasal cavity , it is widely believed that osteomeatal obstructions may impede ventilation and mucociliary clearance from the sinuses , predisposing affected patients to sinus disease . less is understood about the role of a deviated septum or pneumatization of the conchae as potential contributors to the development of sinusitis . while some studies suggest that deviations of the nasal septum or the presence of concha bullosa may interfere with proper airflow , potentially predisposing to sinus disease , other studies have produced contradictory findings [ 1 , 3 , 4 ] . the purpose of this study was to determine the prevalence of concha bullosa and nasal septal deviation and to examine their possible relationship to maxillary sinus disease . a retrospective study was conducted of 883 cbct scans taken between september 2005 and june 2008 at creighton university school of dentistry ( omaha , ne ) . all scans were taken using an icat cbct scanner ( imaging sciences international ) at a 0.3 mm voxel size . scans were reconstructed using osirix software and evaluated in the axial , sagittal , and coronal planes . two trained investigators , well versed on the anatomy of the region , independently reviewed the scans . the gender and age of the patient were the only patient - specific variables included in this study . scans were reviewed for any nasal cavity and/or paranasal anatomical abnormalities , with specific evaluation on the presence of concha bullosa , deviated septa , and sinusitis of the maxillary sinuses . concha bullosa was defined as the presence of pneumatization of any size within in the superior , middle , or inferior conchae . septal deviation was defined as a deviation of greater than 4 mm from the midline . the presence of any radiographic mucosal thickening above the bony floor of the maxillary antrum was defined as abnormal [ 1 , 4 ] . the mean age of the patients was 44.2 years of age , with a range of 4 to 99 years . of the 883 scans evaluated , 43.6% were from male patients and 56.3% were female patients . from the 883 scans , 12.3% were located in one of the right conchae , 13.0% involving the left conchae , and 43.2% bilaterally distributed . the majority of concha bullosa were located in the middle concha ; 7.8% on the right side , 8.3% left ( figure 1 ) , and 20.8% bilateral . in the concha bullosa group , 56.3% were female and 43.7% were males ( p = .856 ) . the mean age of patients with concha bullosa ( 45.6 years of age ) was similar to the overall study population ( table 2 ) . there was no statistical difference between gender and the presence of nasal septal deviation ( 19.9% female ; 18.9% were male ; p = .703 , table 2 ) . there was a statistically significant higher prevalence of maxillary sinusitis in males ( 61.8% ) compared to females ( 41.8% ; p < .0001 ) . 12.1% had right maxillary sinusitis , 15.6% had left - sided involvement , and 21.0% had bilateral sinus disease ( figure 3 ) . there was no statistical significance when comparing the relationship of patients with concha bullosa ( 67.6% ) and those with sinusitis ( 41.8% ) . 49.3% of patients had a combination of both ( figures 4 , 5 , and 6 ) , 50.7% had concha bullosa without evidence of sinusitis , and 33.5% had sinusitis in the absence of concha bullosa ( p = .533 , table 3 ) . the relationship between unilateral or bilateral concha bullosa and ipsilateral sinusitis was not statistically significant . of the 109 patients with right concha bullosa , only 12.8% also had right maxillary sinusitis ( p = .804 ) . of the 115 patients with left concha bullosa , only 18.3% of patients also demonstrated left maxillary sinusitis ( p = .426 ) . of the 381 patients with bilateral concha bullosa , only 21.3% of patients had maxillary sinusitis ( p = .559 , table 4 ) . the relationship between the presence of concha bullosa and nasal septal deviation was not statistically significant . of the 596 patients with concha bullosa , 19.5% also had deviation of the nasal septum ( figure 7 ) . 32.2% of the 171 patients with a deviated septum had no evidence of concha bullosa ( p = .916 ; table 5 ) . examining the potential relationship between sinusitis and nasal septal deviation , there was no statistical significance . 87 ( 19.7% ) of the 442 patients with maxillary sinusitis also had nasal septal deviation ( figure 8) . 84 ( 49.1% ) of 171 patients with deviated septum had no evidence of maxillary sinus disease ( p = .811 ; table 6 ) . in our study , 67.5% of patients had concha bullosa , which is somewhat higher than other studies , in which the prevalence of concha bullosa varied from 35% to 53% [ 14 ] . this variation may be due to differing criteria used to define concha bullosa . in our study , we defined any degree of pneumatization , regardless of size or location , as consistent with concha bullosa . other studies restricted concha bullosa to specific locations on the turbinates and/or to a minimum size of pneumatization [ 1 , 3 , 4 ] . in subramanian 's study , there was a higher incidence of concha bullosa in females ( 58.9% ) compared to males . 19.4% of patients in our study had nasal septal deviation , which is significantly lower than stallman 's 65% and sazgar 's 62.9% prevalences . the reason for this difference is most likely due to our stricter criteria for classification as deviated septum , which we defined as a deviation of greater than four millimeters from the midline . subjectively categorized deviations as mild , moderate , or severe , and sazgar et al . defined septal deviation as any asymmetric curvature of the septum . sinusitis , which was defined in our study as any evident thickening of the mucosa in the maxillary sinus , occurred in 50.0% of our patient population . bolger 's study noted mucosal thickening of the sinus floor in 83.2% of patients . while the difference may be the result of referral bias ( our patients were primarily referred for radiographic assessment prior to dental implant placement and not evaluation of suspected sinus disease ) , other potential variations such as seasonal bias , in which a small consecutive patient sample is chosen during a season that may predispose patients to higher incidence of allergies , may have contributed to this discrepancy . one significant finding in our study was the relationship between sinusitis and gender , with males having a 20.0% higher incidence of sinusitis . such a difference may be due to anatomical variations or mucosal secretion differences between the sexes . while it has been suggested that abnormalities of the concha can predispose patients to obstruction of the sinuses , leading to chronic sinusitis [ 46 ] , other studies with findings similar to those in the current study concluded that there was no correlation between the presence of concha bullosa and sinusitis [ 3 , 5 , 7 ] . previous studies that supported the validity of a relationship have typically included a majority of patients with pre - existing chronic sinusitis . while studies have suggested an association between septal deviation and the presence of concha bullosa [ 2 , 3 ] , the presence of septal deviations was usually associated with the presence of dominant or large concha bullosa [ 2 , 3 ] . however , in our study , only 19.5% of patients with septal deviation had concha bullosa , suggesting that in many cases there is no relationship . . found that there was an association between the degree of deviation and the presence of sinusitis . a meta - analysis conducted by collet et al . failed to confirm a definite relationship between these 2 factors , which is in agreement with the current study . we found no definitive relationship between the presence of concha bullosa or nasal septal deviation and the development of maxillary sinusitis .

the term responsibility has become an integral but diverse element of bioethical and public health debates . thus , the promotion of responsible health behavior constitutes a major effort of health care policies in europe and the united states . in the name of personal responsibility for health , the us health care reform proposes cost - sharing options that are supposed to reflect mainstream thinking ( center for medicare and medicaid services 2006 : 4 ) . furthermore , there are heated debates about the responsibility of the physician , for instance in the case of assisted suicide . recently , the german medical association declared in its revised guidelines on end - of - life treatment that assisted suicide is not a medical task ( bundesrztekammer 2011 : 346)but there still exists the physician s own responsibility in a concrete situation ( ibid . ) . it states a social responsibility of societies and governments regarding the promotion of health and social development for their people ( unesco 2005 : article 14 ) . these examples elucidate that responsibility is a term in bioethics that appears everywhere and at the same time nowhere . this might be a reason for some scholars to avoid its systematic use and not invest much normative scrutiny . one of the most common conceptions in applied medical ethics , the principlist approach developed by tom beauchamp and james childress ( 2009 ) , provides an excellent example . the whole work has no particular chapter or paragraph dealing with responsibility as a normative principle or framework . 1997 ) , another standard work in medical ethics , lacks an entry on responsibility . this lacuna points to a serious problem : responsibility is a key term in so many contexts related to medicine , health care , and biotechnology that we can not afford to ignore or delude its meaning . despite the broad use of responsibility in empirical practice , assuming that it is mainly a however , rather than of offering one single model of responsibility as a substantial normative solution for all bioethical problems , we suggest to deal with responsibilities ( in plural ) . this means that there are different models of responsibility , each of them appropriate in particular contexts . thus , instead of dismissing responsibility as a term non grata for bioethics , we consider its relational composition as well as the plurality in models of responsibility to have an important analytical value for ethical reflection . by acknowledging responsibility as a leading concept for applied ethics , a more concrete approach and more social relevance will be gained and the individual dimension of the doctor patient relationship can easily be linked to the institutional as well as societal level and the public health dimension . by pursuing this line of argument , we aim at the following : first , we provide a formal classification of various conceptions of responsibility , given that there exists a lot of unsystematic or even rhetorical use of the word . this summary is based on a systematic analysis of different theoretical and applied works in philosophy and includes an analysis of conditions as well as of contexts of application . responsibility as a normative concept is on a meta - ethical level distinguished from and at the same time related to other formal ethical principles , such as autonomy , justice , or non - maleficence . secondly , we analyze the use of different models of responsibility in bioethics in a descriptive perspective . with models , we identify three stages of the use of responsibility within western bioethics since the late 1960s . these stages are based on the following : a holistic model of global responsibility , a model of professional responsibility , and a model that focuses on the relation between the individual and the community . we do not argue for one particular model , but for the normative importance of an explication and differentiation of models , preserving their diversity . the juxtaposition of these models allows us to detect moral problems related to power relations and normative inconsistencies and to analyze limitations and one - sided usage of specific models . in this sense , the ethical consideration of responsibility can overcome limits of other common approaches in applied ethics . we demonstrate the hermeneutic value of such a broader understanding of responsibility and show how ethical analysis can benefit from taking different models into account those which are stated explicitly , and those which are implicit or still neglected . once one has accepted that there are different possible models , a discussion on the underlying subjects , norms , instances , and normative consequences has to start . the following analysis divides into three dimensions : first , the basic normative structure of all responsibility models is disentangled by introducing a general concept . this concept is a relational one : it is described in a formula comprising a set of relations between different positions ( relata ) . third , the fact is considered that different norms and instances used as relata imply different moral presuppositions and commitments . in philosophy , various authors have made attempts to develop a typology of responsibility ( e.g. , baier 1991 ; french 1991a , b , c , d ; ropohl 1996 ) . moreover , the dominant use of the term in the legal sphere has shaped all attempts to differentiate types of responsibility ( holl et al . still is often used in the sense of being blameworthy or being guilty . however , the explanation itself provides an important hint at the formal structure of responsibility : in the legal context , it means a person is answerable the person has to respond to accusations raised in front of a court or in parliament ( holl et al . , responsibility turns out to be a relational concept : a person is being held responsible by a court / authority regarding the blameworthy outcomes of his or her actions and must await a judgment that implies legal or social consequences . in other contexts , positive outcomes and rewards can occur , too . with this formal conception , we refer to an analytical , meta - ethical understanding , but not to a particular normative approach of responsibility . at least , the concept of responsibility requires three relata , a subject , an object , and an instance : someone ( the subject , as we will argue the moral agent ) is responsible for someone or something ( the object ) against someone ( the instance ) . however , on closer inspection , it turns out that more five , six , or even seven relata are necessary to reconstruct and analyze the use of the concept responsibility in an adequate manner ( werner 2002 ) . for the subsequent analysis of the role of responsibility in the bioethical debate , we suggest a conception involving seven relata : someone ( subject ) is in a particular time frame ( time ) retrospectively / prospectively ( temporal direction ) responsible for something / someone ( object ) against someone ( norm - proofing instance ) on the basis of certain normative standards ( standard ) with certain sanctions or rewards ( consequences ) . we argue that this seven - relata formula covers the most important normative aspects of different models of responsibility in the bioethical context . with regard to this relational conception , the relata can be interpreted as variables : one can theoretically fill in the different categories with a particular , possibly infinite number of items . the number of possible relations in the model is vast ; they can be expressed by the formula ( n = number of items ) : n(a ) * n(b ) * n(c ) * n(d ) * n(e ) . with regard to the explanation of this formula , the temporal direction and distinction between retrospective / prospective is fundamental for the understanding of responsibility . in the legal context the concept of guilt is used to ascribe a certain course of harmful past events to a particular person who can be identified as their author . this retrospective concept is therefore also called causal responsibility ( bayertz 1995 : 119 ; french 1991c : 113 ) or liability model ( young 2011 : 97 ) . in the rise of modern society , however , a second , prospective meaning became prominent ( bayertz 1995 ) . according to this meaning , having responsibility for an object , a person , or a group means being in charge of or taking care of future events.prospective ( future- or forward - oriented ) responsibility became important in the context of modern functional differentiation and work division which led to an increasingly complex network of social roles and functions . furthermore , technological progress made the consequences of decisions ever harder to anticipate ( e.g. , by the decoupling of place , time , and effects , see strydom 1999 ; bayertz 1995 ) . responsibility is therefore associated with competence and/or power to decide ( ibid . ) . with this temporal shift , the whole normative meaning changes ( young 2011 : 7693 ) . it allows expressions and justifications of socio - political engagements as a forward - oriented model of taking responsibility ( ibid . some are object to controversy with regard to moral status , moral commitment , and justification of norms ( see below ) . when it comes to bioethics , some items are more accepted , or at least more common , than others . so with regard to the category subject , all items that can be seen as moral agents are useful . note that agency includes active performance of actions as well as omissions ( e.g. , omitting of help for a person in a life - threatening case ) ( feinberg 1991 ; held 1991 ) . of course , in everyday language we also tend to say the virus is responsible for the infection or however , this way , the ideas of causality and responsibility are confounded . while causality is an important part of responsibility - assignments and judgments , it is problematic to equalize both in the moral context . there is consensus that any moral meaning of responsibility can only be assigned to moral agents ( yoder 2002 : 24 ) . between different moral theories exists , however , dissent about whether only persons or also collectives ( e.g. , families , states / nations , political groups , random groups , companies ) can be classified as moral agents ( see below ) . with regard to the category objects , all moral agents can be seen as moral objects , but there can be even more entities . for example , in bioethics , we discuss whether human embryos , brain - dead patients , next generations , animals , plants , or nature as a whole ( warren 2000 ) can be according to their moral status and underlying anthropologies considered as objects of responsibility . the relatum object also includes sub - aspects of moral objects , such as the health or body of a person , or entities understood as property of a moral agent ( e.g. , the donated organ ) . with regard to the category instance , we already saw that the court ( or similar legal authorities ) is a well - accepted instance . but in moral terms , we can ( and must ) think of other authorities , as well , such as social peer groups , individual conscience , or god . an instance obviously has a norm - proofing ( and even norm - enforcing ) function . it is the authority that decides whether a norm has been met or violated and thus the corresponding responsibility has been fulfilled or not . some instances also can be norm - founding instances , depending on assumptions based on different ethical theories . the category standard refers to normative standards , such as moral principles or legal norms ( e.g. , to respect others , avoid harm , save life , dispense justice ) . these principles can be very general or very concrete and context - specific ( e.g. , such professional virtues as confidentiality , trustworthiness , honesty but also such personal values as efficiency or austerity ) . the standards depend , however , on their understanding as duty / obligation , right , or virtue . the level of moral commitment can differ according to the standards bindingness and acceptable excuses . finally , the category consequences refers to a list of actions or judgments that are supposed to take place if a subject has or has not acted in a responsible way . some consequences are external ( e.g. , legal punishment , economic disadvantage , social exclusion , social power ) and some internal ( feelings of guilt or shame , a bad conscience , or increased moral identity ) . moral philosophy and applied ethics ( in contrast to legal studies ) have an ambiguous relationship to sanctions . while moral philosophy lengthily debates whether an action is morally wrong or right , only a few precepts discuss the consequences associated with a moral subject that does something morally wrong ( tonry 2011 ) . the sphere of consequences is left to the social and legal domain . however , this is no reason to dismiss the whole concept of responsibility ; our aim is to point out that it is possible to morally assess the actual social practice of sanctions regarding whether they are appropriate in kind and degree . the question of who counts as a moral agent is central to the need to clarify the plausibility of a particular constellation of responsibility . this debate can be traced back to francis herbert bradley ( 1991 : 6274 ) . responsibility implies a capacity for acting rationally , to act so that your actions can be counted on he also states that a man is only responsible for what issues from an act of volition ( 72 ) . for bradley , without personal identity responsibility is sheer nonsense harry frankfurt and john l. mackie contributed to this debate by discussing the important role of choice and intention . an agent is responsible for all and only ( its ) intentional actions ) is later modified by peter french into responsibility based on being willing to do it ( french 1991c : 129ff ) . for example , a physician involved in the tuskegee syphilis case states that he did not have the intention to harm a patient by this experiment ; his intention was to increase helpful knowledge for further vaccination research . nevertheless , he can be held responsible for the effect of his action on the patient . it therefore seems plausible to choose being willing as the crucial criterion , rather than intention . this concept includes all cases where a person is willing to take some risks ( e.g. , harm others ) and has the necessary knowledge ( or could have it ) . furthermore , this reformulation links retrospective and prospective meanings of responsibility . if individuals have to fulfill specific conditions ( such as being willing to commit an action and being aware of possible consequences ) for ascribing responsibility , what does this imply for collectives ? , collectives must be seen as aggregations of individual agents ( e.g. , ladd 1991 ) . hannah arendt insisted that the liability model of responsibility should not be applied to collectives : where all are guilty , nobody is guilty . guilt , unlike responsibility , always singles out ; it is strictly personal ( cited in young 2011 : 76 ) . her statement has to be seen in the context of her analysis of the eichmann lawsuit . if we accept that eichmann and other nazis can excuse their actions by referring to collective orders they had to obey , we lose all measures to ascribe liability and force individuals to take responsibility.2 however , whether this fear justifies a general rejection of the concept of collective responsibility is questionable . under the impression of various ecological , medical , and economical catastrophes such as the bhopal disaster in 1984 or the bp deepwater horizon oil spill in 2010 , the idea is implausible to ascribe responsibility for wrongdoings only to the single worker , a captain , or a ceo , while they must be explained by the interplay of various people in a system , such as a corporative or organized group . one important argument that supports the idea of collectives as moral agents points to our intuition that sometimes the whole ( a nation , a corporation ) is more than just the sum of its single parts . those social groups , parties , a state , or a corporation can be seen as moral agents if their actions are based upon an espirit de corps ( cooper 1991 : 258259 ) . this has to be understood as a joint commitment , and its effect can not simply be explained by the aggregation of individual actions ( french 1991b , 296 ) . the argument gets even stronger where the members of organized groups3 have deliberatively chosen a collective action ( e.g. , by voting or being a voluntary member of a social group , such as a party ) . this group membership should be regarded as active commitment as young stresses ( 2011 : 137).4 french calls it the corporation s internal decision structure as requisite redescription device that licenses the predication of corporate intentionality ( french 1991b : 298 ) . french s definition applies not only to economical corporations but also to organized social groups , such as political parties , nongovernmental groups , and professions . if we have good reasons to believe that an action was based on the condition of a joint commitment or a system effect that goes beyond the impact of single actors , we can identify a collective as agent and therefore also claim collective responsibility . this is even more so if collectives possess economical , structural , or political power . with the concept of active commitment and identification and the process of decision - making within a group there are cases where both the individual members and the collective can be held responsible or only single members or only the whole . without a model of collective responsibility , important moral and political considerations in the whole debate on solidary health care or the role of professional associations , institutions such as hospitals or patient organizations , can not be fully articulated . an intermediary concept that relates individual and collective responsibility is the so - called task responsibility that was introduced by hart ( 1968 ) . it relies on a socially ascribed role or relationship ( e.g. , a mother , a director of a company ) . hence , it is important whether the agents have actively dedicated themselves to the task ( e.g. , as professional or in a position of an institution ) or whether they find themselves in charge involuntarily ( e.g. , some family members ) . within collectives , the latter is related to the mental and practical abilities to perform an action ; diminished capacities can be seen as an excuse or justification for being less responsible . however , the distinction between task responsibility and capacity responsibility is not very helpful in cases where tasks are defined upon capacities ( e.g. , in cases of professional responsibilities of doctors ) . instead , we suggest that by analyzing the agent , the instance , and the norms , one will also identify and differentiate the respective capacities and tasks . an instance is an authority that has to oversee , control , and judge the responsibility of the agents . this point is important with regard to social - practical questions : if an instance has no agency power ( can not practically judge ) or can not impose sanctions , its value as a moral instance is questionable . thus , while individual conscience plays a crucial role for our internal process of moral deliberation and judgment , there might be cases where referring to the psychological state of a bad conscience is insufficient . for example , the phrase doctors are responsible ( only ) towards their own consciences ( as a central statement in the german professional guidelines for doctors indicates ) is very unspecific . on the one hand , it points to the fundamental requirement of moral responsibility that physicians should reflect upon their own values and can not solely rely on social norms . on the other hand and that would be problematic it can be interpreted in the sense that it is sufficient if someone feels shame because something went wrong . related to the question whether a moral agent counts as responsible is the issue of how moral responsibility is ascribed in terms of the underlying moral standards . this question refers to the normative complex between moral standards ( norms ) and sanctions . in the following , this relationship is defined in terms of proportionality : the more binding and important an underlying moral standard is the more demanding or powerful is the sanction that we would expect . furthermore , moral bindingness and consequences differ with regard to the temporal direction of an action : we can distinguish guilt , liability , or blame ( or excuse ) in retrospective cases , and precaution , prevention , or power and authority in prospective cases . in this sense , retrospective and prospective responsibility are categorically different ( see above ) . retrospective responsibility requires that something went bad or a consequence is assessed as morally wrong ( french 1991c : 135 ) , while prospective responsibility focuses on doing morally right or at least avoiding doing wrongs . nevertheless , moral actions must be understood in a temporal continuum , in which backward and future - oriented views often complement each other . as discussed later , prospective responsibility of preventive health behavior can result over time in retrospective liability models if someone claims that person x did not sufficiently prevent a disease . the second aspect refers to the nature ( or binding character ) of moral standards . are the underlying ethical principles for instance to respect autonomy or justice seen as strict moral obligations , as prima facie norms , or even as virtues ? if this is the case , it should also have consequences for our understanding of responsibility : the sanctions for violating strict rules should be seen as more demanding than for failing virtues . by this standard , a core problem of the various models of responsibilities can be addressed : how well founded and binding are the underlying norms ? and if they are well founded and binding , is the assumed instance the correct one to proof and implement sanctions ? in the case of the liability model of responsibility , we often assume that a court is the instance to clarify guilt . but on closer examination , various instances can be considered : the conscience , the profession , the family , or the broader society . this conceptual variety illustrates one aspect of responsibility in comparison with other moral approaches prominent in applied ethics that we understand as a strength and advantage : the analysis of explicit notions of responsibility allows and requires a stringent analysis of the relationship between actors and their tasks and capacities , the underlying moral standards and their validity and binding character , as well as the instance and its power . hence , being responsible can be defined in terms of duty , obligation , or right or virtue depending on the context , but always in relation to an agent , an instance , and the sanctions we have in mind . while responsibility as such is a relational term , other moral concepts , such as duty or rights , can be seen as entitlements to treat or to be treated in a particular way . however , especially in modern , complex societies , they can not define the whole social , relational context of application and consequences . therefore , we argue that the proper use of responsibility always requires a thorough definition of the relata . in philosophy , various authors have made attempts to develop a typology of responsibility ( e.g. , baier 1991 ; french 1991a , b , c , d ; ropohl 1996 ) . moreover , the dominant use of the term in the legal sphere has shaped all attempts to differentiate types of responsibility ( holl et al . still is often used in the sense of being blameworthy or being guilty . however , the explanation itself provides an important hint at the formal structure of responsibility : in the legal context , it means a person is answerable the person has to respond to accusations raised in front of a court or in parliament ( holl et al . 2001 ) . already in this context , responsibility turns out to be a relational concept : a person is being held responsible by a court / authority regarding the blameworthy outcomes of his or her actions and must await a judgment that implies legal or social consequences . in other contexts , positive outcomes and rewards can occur , too . with this formal conception , we refer to an analytical , meta - ethical understanding , but not to a particular normative approach of responsibility . at least , the concept of responsibility requires three relata , a subject , an object , and an instance : someone ( the subject , as we will argue the moral agent ) is responsible for someone or something ( the object ) against someone ( the instance ) . however , on closer inspection , it turns out that more five , six , or even seven relata are necessary to reconstruct and analyze the use of the concept responsibility in an adequate manner ( werner 2002 ) . for the subsequent analysis of the role of responsibility in the bioethical debate , we suggest a conception involving seven relata : someone ( subject ) is in a particular time frame ( time ) retrospectively / prospectively ( temporal direction ) responsible for something / someone ( object ) against someone ( norm - proofing instance ) on the basis of certain normative standards ( standard ) with certain sanctions or rewards ( consequences ) . we argue that this seven - relata formula covers the most important normative aspects of different models of responsibility in the bioethical context . with regard to this relational conception , the relata can be interpreted as variables : one can theoretically fill in the different categories with a particular , possibly infinite number of items . the number of possible relations in the model is vast ; they can be expressed by the formula ( n = number of items ) : n(a ) * n(b ) * n(c ) * n(d ) * n(e ) . with regard to the explanation of this formula , the temporal direction and distinction between retrospective / prospective is fundamental for the understanding of responsibility . in the legal context the concept of guilt is used to ascribe a certain course of harmful past events to a particular person who can be identified as their author . this retrospective concept is therefore also called causal responsibility ( bayertz 1995 : 119 ; french 1991c : 113 ) or liability model ( young 2011 : 97 ) . in the rise of modern society , however , a second , prospective meaning became prominent ( bayertz 1995 ) . according to this meaning , having responsibility for an object , a person , or a group means being in charge of or taking care of future events.prospective ( future- or forward - oriented ) responsibility became important in the context of modern functional differentiation and work division which led to an increasingly complex network of social roles and functions . furthermore , technological progress made the consequences of decisions ever harder to anticipate ( e.g. , by the decoupling of place , time , and effects , see strydom 1999 ; bayertz 1995 ) . responsibility is therefore associated with competence and/or power to decide ( ibid . ) . with this temporal shift , the whole normative meaning changes ( young 2011 : 7693 ) . it allows expressions and justifications of socio - political engagements as a forward - oriented model of taking responsibility ( ibid . . some are object to controversy with regard to moral status , moral commitment , and justification of norms ( see below ) . when it comes to bioethics , some items are more accepted , or at least more common , than others . so with regard to the category subject , all items that can be seen as moral agents are useful note that agency includes active performance of actions as well as omissions ( e.g. , omitting of help for a person in a life - threatening case ) ( feinberg 1991 ; held 1991 ) . of course , in everyday language we also tend to say the virus is responsible for the infection or alcohol is responsible for hepatic damage . however , this way , the ideas of causality and responsibility are confounded . while causality is an important part of responsibility - assignments and judgments , it is problematic to equalize both in the moral context . there is consensus that any moral meaning of responsibility can only be assigned to moral agents ( yoder 2002 : 24 ) . between different moral theories exists , however , dissent about whether only persons or also collectives ( e.g. , families , states / nations , political groups , random groups , companies ) can be classified as moral agents ( see below ) . with regard to the category objects , all moral agents can be seen as moral objects , but there can be even more entities . for example , in bioethics , we discuss whether human embryos , brain - dead patients , next generations , animals , plants , or nature as a whole ( warren 2000 ) can be according to their moral status and underlying anthropologies considered as objects of responsibility . the relatum object also includes sub - aspects of moral objects , such as the health or body of a person , or entities understood as property of a moral agent ( e.g. , the donated organ ) . with regard to the category instance , we already saw that the court ( or similar legal authorities ) is a well - accepted instance . but in moral terms , we can ( and must ) think of other authorities , as well , such as social peer groups , individual conscience , or god . an instance obviously has a norm - proofing ( and even norm - enforcing ) function . it is the authority that decides whether a norm has been met or violated and thus the corresponding responsibility has been fulfilled or not . some instances also can be norm - founding instances , depending on assumptions based on different ethical theories . the category standard refers to normative standards , such as moral principles or legal norms ( e.g. , to respect others , avoid harm , save life , dispense justice ) . these principles can be very general or very concrete and context - specific ( e.g. , such professional virtues as confidentiality , trustworthiness , honesty but also such personal values as efficiency or austerity ) . the standards depend , however , on their understanding as duty / obligation , right , or virtue . the level of moral commitment can differ according to the standards bindingness and acceptable excuses . finally , the category consequences refers to a list of actions or judgments that are supposed to take place if a subject has or has not acted in a responsible way . some consequences are external ( e.g. , legal punishment , economic disadvantage , social exclusion , social power ) and some internal ( feelings of guilt or shame , a bad conscience , or increased moral identity ) . moral philosophy and applied ethics ( in contrast to legal studies ) have an ambiguous relationship to sanctions . while moral philosophy lengthily debates whether an action is morally wrong or right , only a few precepts discuss the consequences associated with a moral subject that does something morally wrong ( tonry 2011 ) . , this is no reason to dismiss the whole concept of responsibility ; our aim is to point out that it is possible to morally assess the actual social practice of sanctions regarding whether they are appropriate in kind and degree . the question of who counts as a moral agent is central to the need to clarify the plausibility of a particular constellation of responsibility . this debate can be traced back to francis herbert bradley ( 1991 : 6274 ) . responsibility implies a capacity for acting rationally , to act so that your actions can be counted on ( 67 ) . is only responsible for what issues from an act of volition ( 72 ) . for bradley , without personal identity responsibility is sheer nonsense ( 73 ) . harry frankfurt and john l. mackie contributed to this debate by discussing the important role of choice and intention . is later modified by peter french into responsibility based on being willing to do it ( french 1991c : 129ff ) . for example , a physician involved in the tuskegee syphilis case states that he did not have the intention to harm a patient by this experiment ; his intention was to increase helpful knowledge for further vaccination research . nevertheless , he can be held responsible for the effect of his action on the patient . it therefore seems plausible to choose being willing as the crucial criterion , rather than intention . this concept includes all cases where a person is willing to take some risks ( e.g. , harm others ) and has the necessary knowledge ( or could have it ) . if individuals have to fulfill specific conditions ( such as being willing to commit an action and being aware of possible consequences ) for ascribing responsibility , what does this imply for collectives ? some philosophers doubt that collectives can ever be seen as moral agents . for them , collectives must be seen as aggregations of individual agents ( e.g. , ladd 1991 ) . hannah arendt insisted that the liability model of responsibility should not be applied to collectives : where all are guilty , nobody is guilty . guilt , unlike responsibility , always singles out ; it is strictly personal ( cited in young 2011 : 76 ) . her statement has to be seen in the context of her analysis of the eichmann lawsuit . if we accept that eichmann and other nazis can excuse their actions by referring to collective orders they had to obey , we lose all measures to ascribe liability and force individuals to take responsibility.2 however , whether this fear justifies a general rejection of the concept of collective responsibility is questionable . under the impression of various ecological , medical , and economical catastrophes such as the bhopal disaster in 1984 or the bp deepwater horizon oil spill in 2010 , the idea is implausible to ascribe responsibility for wrongdoings only to the single worker , a captain , or a ceo , while they must be explained by the interplay of various people in a system , such as a corporative or organized group . one important argument that supports the idea of collectives as moral agents points to our intuition that sometimes the whole ( a nation , a corporation ) is more than just the sum of its single parts . those social groups , parties , a state , or a corporation can be seen as moral agents if their actions are based upon an espirit de corps ( cooper 1991 : 258259 ) . this has to be understood as a joint commitment , and its effect can not simply be explained by the aggregation of individual actions ( french 1991b , 296 ) . the argument gets even stronger where the members of organized groups3 have deliberatively chosen a collective action ( e.g. , by voting or being a voluntary member of a social group , such as a party ) . this group membership should be regarded as active commitment as young stresses ( 2011 : 137).4 french calls it the corporation s internal decision structure as requisite redescription device that licenses the predication of corporate intentionality ( french 1991b : 298 ) . french s definition applies not only to economical corporations but also to organized social groups , such as political parties , nongovernmental groups , and professions . if we have good reasons to believe that an action was based on the condition of a joint commitment or a system effect that goes beyond the impact of single actors , we can identify a collective as agent and therefore also claim collective responsibility . this is even more so if collectives possess economical , structural , or political power . with the concept of active commitment and identification and the process of decision - making within a group there are cases where both the individual members and the collective can be held responsible or only single members or only the whole . without a model of collective responsibility , important moral and political considerations in the whole debate on solidary health care or the role of professional associations , institutions such as hospitals or patient organizations , can not be fully articulated . an intermediary concept that relates individual and collective responsibility is the so - called task responsibility that was introduced by hart ( 1968 ) . it relies on a socially ascribed role or relationship ( e.g. , a mother , a director of a company ) . hence , it is important whether the agents have actively dedicated themselves to the task ( e.g. , as professional or in a position of an institution ) or whether they find themselves in charge involuntarily ( e.g. , some family members ) . within collectives , the latter is related to the mental and practical abilities to perform an action ; diminished capacities can be seen as an excuse or justification for being less responsible . however , the distinction between task responsibility and capacity responsibility is not very helpful in cases where tasks are defined upon capacities ( e.g. , in cases of professional responsibilities of doctors ) . instead , we suggest that by analyzing the agent , the instance , and the norms , one will also identify and differentiate the respective capacities and tasks . an instance is an authority that has to oversee , control , and judge the responsibility of the agents . this point is important with regard to social - practical questions : if an instance has no agency power ( can not practically judge ) or can not impose sanctions , its value as a moral instance is questionable . thus , while individual conscience plays a crucial role for our internal process of moral deliberation and judgment , there might be cases where referring to the psychological state of a bad conscience is insufficient . for example , the phrase doctors are responsible ( only ) towards their own consciences ( as a central statement in the german professional guidelines for doctors indicates ) is very unspecific . on the one hand , it points to the fundamental requirement of moral responsibility that physicians should reflect upon their own values and can not solely rely on social norms . on the other hand and that would be problematic it can be interpreted in the sense that it is sufficient if someone feels shame because something went wrong . related to the question whether a moral agent counts as responsible is the issue of how moral responsibility is ascribed in terms of the underlying moral standards . this question refers to the normative complex between moral standards ( norms ) and sanctions . in the following , this relationship is defined in terms of proportionality : the more binding and important an underlying moral standard is the more demanding or powerful is the sanction that we would expect . furthermore , moral bindingness and consequences differ with regard to the temporal direction of an action : we can distinguish guilt , liability , or blame ( or excuse ) in retrospective cases , and precaution , prevention , or power and authority in prospective cases . in this sense , retrospective responsibility requires that something went bad or a consequence is assessed as morally wrong ( french 1991c : 135 ) , while prospective responsibility focuses on doing morally right or at least avoiding doing wrongs . nevertheless , moral actions must be understood in a temporal continuum , in which backward and future - oriented views often complement each other . as discussed later , prospective responsibility of preventive health behavior can result over time in retrospective liability models if someone claims that person x did not sufficiently prevent a disease . the second aspect refers to the nature ( or binding character ) of moral standards . are the underlying ethical principles for instance to respect autonomy or justice seen as strict moral obligations , as prima facie norms , or even as virtues ? generally , strict or prima facie rules rank higher than virtues . if this is the case , it should also have consequences for our understanding of responsibility : the sanctions for violating strict rules should be seen as more demanding than for failing virtues . by this standard , a core problem of the various models of responsibilities can be addressed : how well founded and binding are the underlying norms ? and if they are well founded and binding , is the assumed instance the correct one to proof and implement sanctions ? in the case of the liability model of responsibility , we often assume that a court is the instance to clarify guilt . but on closer examination , various instances can be considered : the conscience , the profession , the family , or the broader society . this conceptual variety illustrates one aspect of responsibility in comparison with other moral approaches prominent in applied ethics that we understand as a strength and advantage : the analysis of explicit notions of responsibility allows and requires a stringent analysis of the relationship between actors and their tasks and capacities , the underlying moral standards and their validity and binding character , as well as the instance and its power . hence , being responsible can be defined in terms of duty , obligation , or right or virtue depending on the context , but always in relation to an agent , an instance , and the sanctions we have in mind . while responsibility as such is a relational term , other moral concepts , such as duty or rights , can be seen as entitlements to treat or to be treated in a particular way . however , especially in modern , complex societies , they can not define the whole social , relational context of application and consequences . therefore , we argue that the proper use of responsibility always requires a thorough definition of the relata . the question of who counts as a moral agent is central to the need to clarify the plausibility of a particular constellation of responsibility . this debate can be traced back to francis herbert bradley ( 1991 : 6274 ) . responsibility implies a capacity for acting rationally , to act so that your actions can be counted on ( 67 ) . is only responsible for what issues from an act of volition ( 72 ) . for bradley , without personal identity responsibility is sheer nonsense ( 73 ) . harry frankfurt and john l. mackie contributed to this debate by discussing the important role of choice and intention . is later modified by peter french into responsibility based on being willing to do it ( french 1991c : 129ff ) . for example , a physician involved in the tuskegee syphilis case states that he did not have the intention to harm a patient by this experiment ; his intention was to increase helpful knowledge for further vaccination research . nevertheless , he can be held responsible for the effect of his action on the patient . it therefore seems plausible to choose being willing as the crucial criterion , rather than intention . this concept includes all cases where a person is willing to take some risks ( e.g. , harm others ) and has the necessary knowledge ( or could have it ) . if individuals have to fulfill specific conditions ( such as being willing to commit an action and being aware of possible consequences ) for ascribing responsibility , what does this imply for collectives ? some philosophers doubt that collectives can ever be seen as moral agents . for them , collectives must be seen as aggregations of individual agents ( e.g. , ladd 1991 ) . hannah arendt insisted that the liability model of responsibility should not be applied to collectives : where all are guilty , nobody is guilty . guilt , unlike responsibility , always singles out ; it is strictly personal ( cited in young 2011 : 76 ) . her statement has to be seen in the context of her analysis of the eichmann lawsuit . if we accept that eichmann and other nazis can excuse their actions by referring to collective orders they had to obey , we lose all measures to ascribe liability and force individuals to take responsibility.2 however , whether this fear justifies a general rejection of the concept of collective responsibility is questionable . under the impression of various ecological , medical , and economical catastrophes such as the bhopal disaster in 1984 or the bp deepwater horizon oil spill in 2010 , the idea is implausible to ascribe responsibility for wrongdoings only to the single worker , a captain , or a ceo , while they must be explained by the interplay of various people in a system , such as a corporative or organized group . one important argument that supports the idea of collectives as moral agents points to our intuition that sometimes the whole ( a nation , a corporation ) is more than just the sum of its single parts . those social groups , parties , a state , or a corporation can be seen as moral agents if their actions are based upon an espirit de corps ( cooper 1991 : 258259 ) . this has to be understood as a joint commitment , and its effect can not simply be explained by the aggregation of individual actions ( french 1991b , 296 ) . the argument gets even stronger where the members of organized groups3 have deliberatively chosen a collective action ( e.g. , by voting or being a voluntary member of a social group , such as a party ) . this group membership should be regarded as active commitment as young stresses ( 2011 : 137).4 french calls it the corporation s internal decision structure as requisite redescription device that licenses the predication of corporate intentionality ( french 1991b : 298 ) . french s definition applies not only to economical corporations but also to organized social groups , such as political parties , nongovernmental groups , and professions . if we have good reasons to believe that an action was based on the condition of a joint commitment or a system effect that goes beyond the impact of single actors , we can identify a collective as agent and therefore also claim collective responsibility . this is even more so if collectives possess economical , structural , or political power . with the concept of active commitment and identification and the process of decision - making within a group there are cases where both the individual members and the collective can be held responsible or only single members or only the whole . without a model of collective responsibility , important moral and political considerations in the whole debate on solidary health care or the role of professional associations , institutions such as hospitals or patient organizations , can not be fully articulated . an intermediary concept that relates individual and collective responsibility is the so - called task responsibility that was introduced by hart ( 1968 ) . it relies on a socially ascribed role or relationship ( e.g. , a mother , a director of a company ) . hence , it is important whether the agents have actively dedicated themselves to the task ( e.g. , as professional or in a position of an institution ) or whether they find themselves in charge involuntarily ( e.g. , some family members ) . within collectives , the latter is related to the mental and practical abilities to perform an action ; diminished capacities can be seen as an excuse or justification for being less responsible . however , the distinction between task responsibility and capacity responsibility is not very helpful in cases where tasks are defined upon capacities ( e.g. , in cases of professional responsibilities of doctors ) . instead , we suggest that by analyzing the agent , the instance , and the norms , one will also identify and differentiate the respective capacities and tasks . baier points to an interesting aspect : any instance presupposes a task responsibility . an instance is an authority that has to oversee , control , and judge the responsibility of the agents . this point is important with regard to social - practical questions : if an instance has no agency power ( can not practically judge ) or can not impose sanctions , its value as a moral instance is questionable . thus , while individual conscience plays a crucial role for our internal process of moral deliberation and judgment , there might be cases where referring to the psychological state of a bad conscience is insufficient . for example , the phrase doctors are responsible ( only ) towards their own consciences ( as a central statement in the german professional guidelines for doctors indicates ) is very unspecific . on the one hand , it points to the fundamental requirement of moral responsibility that physicians should reflect upon their own values and can not solely rely on social norms . on the other hand and that would be problematic it can be interpreted in the sense that it is sufficient if someone feels shame because something went wrong . related to the question whether a moral agent counts as responsible is the issue of how moral responsibility is ascribed in terms of the underlying moral standards . this question refers to the normative complex between moral standards ( norms ) and sanctions . in the following , this relationship is defined in terms of proportionality : the more binding and important an underlying moral standard is the more demanding or powerful is the sanction that we would expect . furthermore , moral bindingness and consequences differ with regard to the temporal direction of an action : we can distinguish guilt , liability , or blame ( or excuse ) in retrospective cases , and precaution , prevention , or power and authority in prospective cases . in this sense , retrospective responsibility requires that something went bad or a consequence is assessed as morally wrong ( french 1991c : 135 ) , while prospective responsibility focuses on doing morally right or at least avoiding doing wrongs . nevertheless , moral actions must be understood in a temporal continuum , in which backward and future - oriented views often complement each other . as discussed later , prospective responsibility of preventive health behavior can result over time in retrospective liability models if someone claims that person x did not sufficiently prevent a disease . the second aspect refers to the nature ( or binding character ) of moral standards . are the underlying ethical principles for instance to respect autonomy or justice seen as strict moral obligations , as prima facie norms , or even as virtues ? generally , strict or prima facie rules rank higher than virtues . if this is the case , it should also have consequences for our understanding of responsibility : the sanctions for violating strict rules should be seen as more demanding than for failing virtues . by this standard , a core problem of the various models of responsibilities can be addressed : how well founded and binding are the underlying norms ? and if they are well founded and binding , is the assumed instance the correct one to proof and implement sanctions ? in the case of the liability model of responsibility , we often assume that a court is the instance to clarify guilt . but on closer examination , various instances can be considered : the conscience , the profession , the family , or the broader society . this conceptual variety illustrates one aspect of responsibility in comparison with other moral approaches prominent in applied ethics that we understand as a strength and advantage : the analysis of explicit notions of responsibility allows and requires a stringent analysis of the relationship between actors and their tasks and capacities , the underlying moral standards and their validity and binding character , as well as the instance and its power . hence , being responsible can be defined in terms of duty , obligation , or right or virtue depending on the context , but always in relation to an agent , an instance , and the sanctions we have in mind . while responsibility as such is a relational term , other moral concepts , such as duty or rights , can be seen as entitlements to treat or to be treated in a particular way . however , especially in modern , complex societies , they can not define the whole social , relational context of application and consequences . therefore , we argue that the proper use of responsibility always requires a thorough definition of the relata . within bioethics , the term responsibility has its own particular evolution . dating the emergence of this field ( dealing not only with clinical dilemmas but also with societal risks of life science research ) back to the late 1960s since then , its use and meaning(s ) went manifold paths , often overlapping each other . in the following section , we summarize this evolution by pointing to three paradigms.5 we do not claim a strict historical model of distinct stages , but different discursive moments of dominance.6 the first phase was dominated by discourses of collective forward - oriented responsibility , often directed toward next generations , human kind in general , and nature as such . prominent and influential advocates were hans jonas or van rensselaer potter who both expressed their moral concerns about technology development and increasing ecological crisis . in this context , the asilomar conference on recombinant dna molecules and its risks of biohazard ( berg et al . 1975 ) is noteworthy . its impact on the wider public discussion of bioethics beyond the medical - clinical context should not be underestimated . it concerns the collective ( but professional ) responsibility with regard to mankind and nature . interestingly , the model of genetic responsibility , used more recently as a term for biopolitical impacts on individual life style ( lemke 2006 ) , was coined by lipkin and rowley ( 1974 ) in the early 1970s to argue in favor of reproductive and positive - eugenic considerations for collective responsibility . the second stage , in the mid-1970s , sees the intensified discussion of professional responsibility towards individuals . the belmont report of 1976 ( us department of health ) , often considered a new touchstone for informed consent procedures and the legal protection of human research participants , also defined new , very precise responsibilities for researchers.7 the belmont report ( as a reaction to the tuskegee study scandal ) and the success of its theoretical extension by beauchamp and childress in 1979 contributed to an understanding of professional ethos based upon balancing various ethical principles : respecting patients autonomy , beneficence , non - maleficence , and justice . both approaches refer in a subtle and implicit way to the professional ethos of physicians . of course , the authors use the term responsibility in various constellations , such as individual responsibility , financial responsibility , collective responsibility , community responsibility , but never explain the meaning nor reflect the different models and their interrelations ( beauchamp and childress 2009 : 33 , 48 , 83 , 122 , 157 , 249 ) . the perception of professional responsibility still dominates , especially when linked to legal discussions of the dos and donts of doctors and nurses . in our regular teaching of medical ethics , for instance , we notice that for students professional responsibility is synonym for being a good doctor . this popular usage is backed by such influential organizations as the world medical association , which explains in an ethical manual : whereas in traditional medical ethics the sole responsibility of physicians was to their individual patients , nowadays it is generally agreed that physicians should also consider the needs of society , for example , in allocating scarce healthcare resources ( williams 2009 : 22 ) . however , professional responsibility was not only an explicit model in medical contexts , but also in other areas such as technology assessment ( e.g. , ropohl 1996 ; strydom 1999 ) . professional responsibility can be understood as a backward model of liability , but also as a future - oriented model of guidance . with regard to the more reflective , forward character of professional responsibility , suggestions made by turoldo ( 2009 ) can be cited . he understands his account of ethics of responsibility as a more context - sensitive approach for professional moral reasoning , which takes into account charges and potential social consequences in the treatment and communication with patients . a third stadium , starting in the 1990s , deals with the interrelation between social and individual responsibility . this debate can be explained by two factors : first , bioethics reacts to political reforms in which public welfare and health care systems are cut back so that the tasks and burdens of the individual citizen are redefined . there arose an increase of individual responsibility ( as ) an explicit purpose of the introduction of market forces in european health care systems according to the political philosopher iris young ( 2011 ) , a key term helping to propel welfare reform in the united states in the 1990s , as well as in some other reforming states was personal responsibility ( 10 ) . each must self - sufficiently bear the costs of its choices , and has no moral right to expect help from others , even if the individual or the family should suffer harm or disadvantages ( young 2011 : 10 ) . young s conclusion even goes beyond the analysis of some public health scholars . for her , the emergence of personal responsibility has an absolving functionto pin responsibility on one agent and to absolve others ( 11 ) . according to ter meulen and jotterand ( 2008 ) , personal responsibility is more a policy to control costs , but in such mid - european countries as the netherlands , germany , or the united kingdom , the government keeps monitoring accessibility and quality of care . individualization and free choice are limited by the principles of solidarity and equity ( 195 ) . they contrast this interpretation to the health care system in the united states where the strong tradition of individualism prevails ( 196 ) and also argue that personal responsibility does not sufficiently result in free choice and autonomous decisions of the health care consumer . second , internal theoretical critiques concerning the perversion of autonomy ( gaylin and jennings 1996 ) in bioethics arose , introducing moral responsibility as an opponent for too much autonomy . thus , communitarian and feminist scholars criticized the dominant liberal paradigm of autonomy that trumps all other ethical values ( callahan 1994 ; held 2006 ; scully 2008 ) . for instance , gauthier ( 2002 ) suggests the concept of personal responsibility as a virtue that strikes a balance between the individual and the community , between rights and obligations patients should include the consequences for their families and the community in their deliberations about health care choices , and also the question of who bears the costs . gauthier s considerations are , however , very general , being unspecific with regard to different moral objects , possible instances , and even sanctions . in this sense , this approach tends to use the concept of moral responsibility again as a general place holder for being a moral person . such a generalization runs the risk of arbitrariness or ending as rhetoric , leading to an unjustified shallowness in the use of the term ( see above ) . the dilemma between social and individual responsibility added another point of view , especially the controversial character of knowledge and risk assessment . yoder ( 2002 ) summarizes recent ambiguities of individual responsibility , especially with regard to the increasing amount of information about healthy life styles . he concludes : we are told how to improve our health or reduce our risk of illness by eating properly , exercising regularly , or taking aspirin daily . if we can control our health , we can [ be ] blamed for being ill ( 23 ) . yoder thus identifies an existing trend , already described 20 years earlier ( veatch 1980 ) . for veatch , health care choices and technical opportunities bear the risk that disease and disabilities are seen as someone s fault because individuals , parents , or the state have omitted to do something against the risk . for yoder , the crucial and problematic aspect of dealing with responsibility is that information and risks the epistemic part of assessing consequences and agency are too often seen as a matter of objectives facts . instead , he pleads for a more pragmatic perspective : first , we have to acknowledge that there are limits in our knowledge of outcomes and also limits in causal explanations . second , we have to acknowledge power distributions and how they shape our perception and construction of responsibilities . while yoder does not reject the model of individual responsibility in general , he argues that too often the underlying values , as well as the consequences , are assumed without further deliberation . therefore , his pragmatist - oriented suggestion is a move into the public realm for the purpose of having more public deliberations about the contribution and constitution of different kinds of moral responsibility ( yoder 2002 : 30 ) . even the historical sketch of the evolution of responsibility in the bioethical debate already reveals a deep discrepancy : on the one hand , the word is highly relevant in public and academic debates , but on the other hand , its meaning and implications are quite often far from being self - evident , or are even controversial . to provide a solution to this problem , explication means that the use of responsibility needs to specify the norms and instances . as we will show , claims concerning responsibility are often based on unclear or conflicting norms , such as particular professional virtues or universal moral principles . also a wide range of instances is conceivable , but not always explicitly addressed , from individual conscience to state authorities . with pluralization , we demand that the roles and responsibilities of the different agents involved in moral conflicts are taken into consideration . the formula developed above provides a tool to analyze the meaning and role of responsibility in particular practical contexts . the juxtaposition of different forms and levels should bring out what is predominant in the one context and perhaps missing in the other . in the subsequent section , we choose three prominent cases of moral responsibility which illustrate the purpose of our tool . on the one hand , the individual patient is still often perceived as the central object of the physician s prospective responsibility . they require that the doctor dedicates his or her efforts to this single patient s survival and well - being for the time of treatment ( tauber 2005 ) . on the other hand , in the understanding of organs as scarce resources , the physician also increasingly functions as a gate keeper . he or she adopts the prospective responsibility to ensure fair and efficient allocation of organs over a virtually unlimited space of time . the new distributive responsibility is defined by temporal dimensions and public health norms of efficiency and justice that are often not part of doctors traditional professional competence . it also requires doctors to distance themselves from the individual needy person and the concrete setting of treatment . they now have to envisage the collective of potential recipients as the object of their responsibility . this may also involve the task to evaluate , compare , and weigh several persons life expectancies and quality of life against each other from an impartial point of view . such a conflict of responsibilities is , however , easily neglected because the discussion of doctors professional responsibilities is still mainly confined to the individual doctor interestingly , sanctions in the individual and collective area are located on different levels of severity . while failure to meet the traditional responsibility of beneficence primarily results in professional sanctions , arbitrary discrimination regarding access to the waiting list will be tried in front of court and be punished according to criminal law ( see table 1 for summary).table 1summary of 7 major relata for professional responsibility in organ transplantationsubject ( in different roles)object ( seen as morally relevant)instance ( norm - proofing)moral - relevant standardtemporal directionconsequencestime framephysicianpatient ( recipient)law / ethosbeneficenceretrospective_negativeprofessional sanctions?limited to individual treatment until cure or deathprospective_positiverecognition by patientcollective of recipientsjustice / efficiency?retrospective_negativelegal sanctionstheoretically unlimited ( as long as there are not enough donor organs)prospective_positivepower as gate keeper summary of 7 major relata for professional responsibility in organ transplantation the second example is concerned with responsibilities emerging in the context of advance directives ( ads ) . the mere legal possibility to lay down one s preferences , as it is now common in many western countries , aims at ensuring patient s autonomy and self - determined death . at the same time it also creates a responsibility to take care of oneself , thus avoiding the prospective negative consequences of depending on the decisions of others additionally , ads affect close relatives as objects of responsibility . surviving family members are relieved from the insecurity involved in making advocatory decisions over life and death and thus are spared the agony of not knowing what the patient would have wanted ( gauthier 2002 : 279 ) . however , deriving from this a moral responsibility of each member of the community to make their wishes regarding end - of - life treatment known in advance they have acquired the prospective moral ( sometimes even legal ) responsibility of ensuring that the incompetent patient s living will is interpreted and executed correctly . if the relatives fail to meet the norm , they can lose their decision power . therefore , it is inappropriate to understand the individual as the sole subject and the relatives only as the object of responsibility . the responsibilities are mutual and the normative standards the principle of personal autonomy on the one hand , the virtue of being considerate regarding potential psychological distress on the other have to be juxtaposed in order to assess their relative weight . a thorough analysis of responsibilities allows an understanding of the complexity of different moral subjects and norms involved , even within one family ( see table 2 for summary).table 2individual and family responsibility in the context of advance directivessubject ( in different roles)object ( seen as morally relevant)instance ( norm - proofing)moral - relevant standardtemporal directionconsequencestime framecitizens as potential patientselfconsciencevirtue : to be considerateretrospective_negativedependency ( on physician and family ) if no ad , or even blamefor the time of being competent , but disputable how long in advance is validrelativesprospective_positiveself - determined end of lifefamilypatientlaw ( as attorney)respect for autonomy / careprospective_negativeonly in life - ending cases : loss of decision power ( to court)in case of patient s loss of competence individual and family responsibility in the context of advance directives the third case refers to responsibility in the context of genetic testing . in contrast to its initial meaning ( see above ) , it is now used to describe and criticize a process of geneticization of the identity of patients and their families but also of healthy persons ( e.g. , novas and rose 2000 ; lemke 2006 ) . this knowledge of being at genetic risk means that individuals and families have a right , a duty , even a compulsion , to choose in relation to managing the risk of themselves and others the preventing - risk paradigm underlying this model must be primarily understood as a prospective responsibility . reflecting upon the risk to manifest an inheritable disease , different proactive actions appear possible : family planning , or in the case of such a treatable disease as cancer , to adopt a healthy lifestyle ( raz and schicktanz 2009 ) autonomy as a norm would mean that genetic testing allows individuals to choose freely ( to be tested or not , to prevent or not ) . the negative consequence based on a strict autonomy - norm would then be to accept if such a disease occurs . if autonomy is also understood as freedom to know or not to know , no one else has the right to judge or guide individual decisions . therefore , suggestions in the realm of prevention strengthening the causal and practical need for genetic knowledge to shape preventive campaigns and preventive medicine often tacitly replace the norm autonomy by the norm of self - care ( smith et al . 2005 ) . for instance , the user of an internet forum commenting on the direct - to - consumer genetics offered by 23andme states : just got my 23andme results back ! glad i can be proactive on delaying or preventing some of these conditions that i m prone to ( http://twitter.com/#!/23andme/favorites/11.7.2011 ) . this is exactly the ambiguity of individual responsibility with regard to health prevention that yoder pointed out . liability in a strong sense would mean that individuals are blamed for being not preventive or careful enough . this is discussed in cases like obesity , a condition where genetic as well as life style factors interact ( hilbert 2008 ) . moreover , genetic responsibility often does not differentiate between the individual s responsibility toward himself and the one toward his family . ( 2008 ) reveal in their interview study , about genetic testing the majority of individuals as family members practiced nondisclosure : the prospect of disclosure was expressed as a practical moral burden of calculating who to tell and when to tell them . . they justified nondisclosure in terms of their own guilt , the possibility of incurring blame or by pre - empting a relative s right not to know . one of the most common explanations for not disclosing intergenerationally was the strong desire to protect the future autonomy of children and young adults . therefore , the authors argue for a different sense of genetic responsibility , which is neither reducible to transcendental communication nor that of rational autonomy ( 20 ) . they instead suggest that people resist the onerous obligation of managing genetic risk and disclosing bad news to kin to escape the moral / discursive confinement of autonomy ( 19 ) . they interpret this as an intensity of affect ( ibid . ) , a pre - discursive strategy of these family members practicing nondisclosure . however , we take more seriously what their informants gave as reasons . here , norms such as respecting the right not to know , and care for family members to avoid socio - psychological burdens for others and the family as whole were mentioned . according to our analysis , those who disclose genetic knowledge to family members do not subsequently act upon autonomy as the leading norm ; rather , they act on the norm that others have a right to know or in the case of nondisclosure a right not to know . within the ethics of genetics , these norms are intensively discussed and are also part of legal - political debates ( chadwick 2009 ) . blame as well as feelings of guilt are feared by family members , in both liability models . long - lasting distress within a family can result if the persons involved favor different norms . furthermore , positive - prospective consequences vary , too . in the case of following the norm right to know / duty to inform , this choice results in relying on a genetic paradigm of risk prevention and therefore proactive health behavior for many years , even lifelong . in contrast , following the norm not to know will yield prospective consequences to master a harmonic family life , often based on the task responsibility to take the power of decisions , as parents do for their children , often directly after the counseling process ( hildt 2009 ) . as we already see this ambiguity can be avoided by being more precise , detailed , and explicit about who is responsible toward whom on the basis of which norm , proofed by which instance , and with which consequences . as the case of individual and family responsibility also reveals , we have to reflect upon such concepts as conscience and family communication to understand the loci of everyday moral dilemmas of genetic counseling . moreover , the case of genetic testing shows that not only individuals and their families are involved , but also professionals . however , the conflicts and concerns that individuals as well as family members express invite us to question whether the concept of nondirective counseling is always appropriate and justified ( raz and atar 2003 ) ( see table 3 as summary).table 3summary of the major 7 relata for individual , family , professional , and social responsibility in the case of genetic testingsubject ( in different roles)object ( seen as morally relevant)instance ( norm - proofing)moral - relevant standardtemporal directionconsequencestime frameselfselfconscienceautonomyretrospective_negativeaccept possible harmin late onset disease : life long , decadesprospective_positivefree choiceself - careretrospective_negativeguilt / blamein late onset disease : life long , decadesprospective_positivepreventionfamilyconscienceright to know / duty to informretrospective_negativeblame / guilt if not informedclosely after the knowledge is gained , but theoretical for many yearsprospective_positivepreventive ( e.g. , family planning ) , good risk managementright not to know / duty not to tellretrospective_negativefamily distress if toldclosely after the knowledge is gained , but theoretical for many yearsprospective_positiveprotection of families from social - psychological burdensprofessionalpatientsoft law / lawnon - directive counselingretrospective_negativelegal liability for counselorduring the treatmentprospective_positiveprofessional exclusiveness ( power)public healthcare systemcitizens?justice and equalityprospective_negativewaste of health care resources / inefficient allocationtheoretically decades , but changing political systems ? ? summary of the major 7 relata for individual , family , professional , and social responsibility in the case of genetic testing while it is assumed it is the best standard to be consistent to the norm of autonomy , it can easily conflict over time with the norm right to know of family members . if we even assume there are preventive measures that are really easy , harmless , and cheap to apply , nondirective counseling can be questioned from a social responsibility point of view . the latter wants to ensure just and fair allocation of financial resources to all potential patients . however , when dealing with responsibility in a case that involves a collective such as a society or nation we need more clearness about what is the appropriate instance to judge and evaluate norms and consequences . we also need to know how to deal with long - term effects in a political system of changing parties and power relations . hence , political - philosophical considerations that seek to solve the problem by procedural , political , or expertocratic solutions might help ( yoder 2002 ) . the aim of the three cases was to illustrate the application of the responsibility - formula - tool to structure bioethical as well as socio - empirical insights . this approach allows a comparison , detection of inconsistencies , and analysis of implicit or even conflicting normative relata . complexity will increase with the number of relata to be considered . with regard to restriction in each of these areas , it will be necessary to detect and analyze the complexity within each of these fields . responsibility is a concept commonly and prominently used in academic , political , and public discourses on bioethical questions , but mostly in an underdetermined , ambiguous , or even ideologically instrumentalized way . therefore , its analysis constitutes an important step toward a clarification of these discourses and a critical reflection of the claims made . in this sense , bioethics does not only deal with moral problems in medical - clinical settings , it also constantly includes reflection about the tools and conceptions scholars use . our considerations want to contribute to this reflection , taking real - world needs into account . various concepts of responsibility can be found in empirical contexts and even lay notions , sometimes in a coherent , sometimes in an incoherent way . these real - world problems need an ethical language suitable to face , understand , and deal with social connectivity and complexity . in the perspective of responsibility , the use of other meta - ethical conceptions of duties , rights , or virtues can be integrated into a broad action - theoretical framework that forces us to consider the subject object relationship , time , instances , and consequences . this perspective also allows us to cross - section differences between deontological and utilitarian approaches that sometimes dominate moral philosophical debates . furthermore , the proposed formula of responsibility also bridges deductive ( top - down ) and inductive ( bottom - up ) approaches as it invites into a discourse about the identification , determination , and interrelation between subjects and objects . responsibility is not a normative concept that replaces ethical principles such as autonomy , beneficence , justice , nor does it supersede them . without denying the importance of personal autonomy for bioethics and clinical practice , however , responsibility can help to complement the bioethical debate by highlighting the normative points of view of other actors , social groups , and society as a whole . it should have become clear that responsibility goes beyond the debate of middle - range principles in which many dilemmas are only understood and conceptualized as conflicts between competing ethical principles . this might be still true for many cases . however , our analysis of responsibility as a tool illustrates that the structure and context of moral dilemmas in particular fields can ( sometimes additionally or also exclusively ) rely on many variables , including competing subjects , objects , consequences aimed for , and instances . further research has to show that the suggested tool can contribute systematically to the resolution of conflicts and dilemmas based on insufficient , implicit , or inappropriate ascriptions of responsibility . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

the first reported case was in a dog near a large stable ; the dog became inappetant and lethargic and had had a slight nasal discharge and a persistent cough for several days . over the next 23 weeks , dogs in or near stables with infected horses , including dogs whose owners were handling infected horses or dogs ( n = 6 ) that were only housed with infected dogs , were examined . samples were also collected from dogs kept with horses at 5 other locations 2060 km from the first case . of the 40 dogs , examined , 10 had clinical signs consistent with influenza ( anorexia , lethargy , and , for some , a harsh cough that persisted for several weeks ) . nasal swabs and serum were collected from each of the 40 dogs ; 23 were seropositive according to influenza type a blocking elisa ( 5 ) and hemagglutinin inhibition ( hi ) assay ( 5 ) using a / equine / sydney/2007 virus as antigen ( table ) . results were discordant for 5 dogs : for 2 , hi titer was high but elisa results were negative ; for 3 , elisa results were positive but hi titer was negative . convalescent - phase serum samples were collected 1416 days later from 26 of the dogs ; seroconversion was noted for 4 of the 5 dogs with discordant elisa and hi results . testing of 19 dogs 2 years later showed no change in hi titer , although elisa results were negative for each . each seropositive dog had been in close proximity to eiv - infected horses but not always in direct contact . no evidence of lateral transmission was found for dogs that did not have contact with horses . * hi , hemagglutination inhibition ; x , cross - breed ; ns , not sampled ; neg , negative ; na , not applicable ; pos , positive ; uk , unknown ; mn , male neutered ; fn , female neutered . nasal swabs from 1 clinically healthy dog had a positive result in an influenza a real - time reverse transcription pcr assay ( 5 ) on 2 consecutive days . the dog remained clinically healthy and was seropositive ( titer 64 ) on day 16 after the first positive swab was collected . nucleic acid sequencing was conducted for the ha , neuraminidase ( na ) , and matrix ( m ) genes amplified by pcr from the rna purified from 2 samples from this dog ( a / canine / sydney/6525/2007 and a / canine / sydney/6692/2007 ) and from a nasal swab from an infected horse ( a / equine / sydney/6085/2007 ) in the same stable ( genbank accession nos . sequences were aligned with representative sequences from genbank by using clustal w ( www.clustal.org/ ) before phylogenetic trees with bootstrapping were generated ( n = 1,000 ; random seed n = 111 ) with megalign ( lasergene ; dnastar , madison , wi , usa ) . complete nucleotide homology was found for each of the ha , na , and m gene sequences from the 2 dogs and the sequence from the infected horse in the same stable ( a / equine / sydney/6085/2007 ) . when influenza subtype h3n8 sequences from horses and dogs were compared with other subtype h3n8 sequences in genbank , the ha , na , and m sequences were most similar to strains a / equine / kanazawa/1/2007 and a / equine / ibaraki/1/2007 , which were isolated during the 2007 equine influenza outbreak in japan ( figure ) . the ha , na , and m gene sequences from the dogs in australia were positioned on separate clades of the phylogenetic trees , as opposed to those from subtype h3n8 viruses from dogs in the united states , which all grouped closely together ( figure ) . phylogenetic trees of influenza subtype h3n8 viruses showing analyses conducted on a ) hemagglutinin genes , b ) neuraminidase genes , and c ) matrix genes . boldface indicates viruses identified in dogs and horses in australia , 2007 , asterisks ( * ) indicate viruses from horses in japan , and daggers ( ) indicate viruses from dogs in the united states . researchers in japan have described transmission of eiv from 3 experimentally infected horses to 3 dogs individually housed with each horse ( 6 ) . both studies showed direct linkage between active influenza virus infection in dogs and horses . because some naturally infected dogs were only in the vicinity of stables and not in direct contact with horses , we believe that eiv may be readily transmitted from horses to dogs in close proximity . the mechanism of spread remains unclear , although in the united kingdom aerosol transmission was believed to be a major means of spread to dogs ( 4 ) . studies conducted during the equine influenza outbreak in australia indicate that the levels of virus excretion from horses not previously exposed to the virus can be extremely high ( a.j . data ) . although humans readily spread virus from horse to horse , either directly during handling or by fomite transmission , human transmission of eiv to dogs that were not in the immediate vicinity of infected horses was not found . similarly , dog - to - dog transmission was not found when infected dogs were transported and kept with other dogs in urban locations where there was no opportunity for contact with horses . although clinical signs were not observed for any of the dogs in japan , > 35% of the naturally infected dogs in australia exhibited clinical signs , some quite severe and protracted . nevertheless , virus was rarely detected in nasal secretions of the dogs in australia , and there was no evidence of horizontal transmission to other dogs . the lack of clinical signs in experimentally infected dogs may be because of the small numbers of dogs or because of inoculum attenuation after passage in embryonated chicken eggs . that the experimentally infected dogs in japan also had lower hi titers than did naturally infected dogs may be relevant . finally , when 19 of the dogs in australia were tested 2 years after infection and without opportunity for reexposure , with only 1 exception , the hi antibody titers had not changed . this finding supports the interpretation that antibodies detected in dogs in the united kingdom ( 3,4 ) had been acquired during the equine influenza outbreak several years earlier . the nucleotide gene sequences encoding the 2 surface proteins ( ha and na ) and the m protein from the infected dog in australia matched those from the horse with which it had contact and did not have any of the nucleotide changes that have been identified in viruses from dogs in the united states ( 2 ) . such changes may be critical to , or a consequence of , the adaptation of eivs to dogs and may play a role in enhancing the infectivity of these viruses for dogs because there is no evidence of continuing circulation of virus in dogs in australia .

traumatic brain injury ( tbi ) is a leading cause of death and disability worldwide . yearly , about 1.5 million people die from tbi and those several millions that survive receive emergency treatment . in malaysia , the statistics for the year 2009 - 2010 reveal that the causes of death from motor vehicle accidents are head injury ( 56.5% ) followed by brain injury ( 38.1% ) , both head and brain injury ( 34% ) and skull / craniofacial fractures ( 27.9% ) . the common presentation to the ed is with an acutely altered level of consciousness that requires quick assessment , which is the crucial action of all health providers [ 3 , 4 ] . coma is the severest impairment of arousal and is defined as the inability to obey commands , speak , or open eyes to pain . the important components to assess altered level of consciousness were designed in 1974 by graham teasdale and bryan j. jennettto , called the glasgow coma scale ( gcs ) which tested 3 neurological aspects of the patient 's response : eye opening , limb movement , and vocalization . it is important to note that the scale is intended to assess level of consciousness and is not designed for following neurological deficits . this tool is used worldwide for neurological assessment of level of consciousness in nursing practice and is further enhanced with the support of best practice guidelines . it is therefore the most sensitive and reliable indicator of all neurological patient 's [ 6 , 7 ] . nurses who work in areas that care for these patients need to be competent in assessing gcs . the sample size is calculated based on 95% confidence interval and with a margin error that does not exceed 5 percent . this study was conducted in the emergency and outpatient departments of a tertiary medical centre . the ed was divided into five areas , red zone ( urgent cases ) with 5 nurses per shift , yellow zone ( acute cases ) with 7 nurses per shift , green zone ( semiacute cases ) with 5 nurses per shift , paediatric emergency with 3 nurses per shift , and trauma ward with 4 nurses per shift . outpatient department which operates from 8 p.m. to 5 p.m. has 28 nurses on duty per day . a there are 4 questions related to demographic data addressing age , level of education , gender , and years of service . part b consists of 15 multiple choice questions related to knowledge on glasgow coma scale ( gcs ) ; see the following : instrument to assess the knowledge on glasgow coma scale ( 1)the glasgow coma scale was initially devised to ( a)locate brain tumour (b)assess the depth of coma (c ) facilitate care for stroke patients (d ) monitor the extent of meningitis ( 2)what part of the brain is being assessed when you are assessing eye opening ? ( a ) cerebral cortex (b ) occipital lobe (c ) cerebellum (d ) reticular formation (e ) hypothalamus ( 3)which part of the brain is being assessed when you are assessing verbal response ? ( a ) cerebral cortex (b ) occipital lobe (c ) cerebellum (d ) reticular formation (e ) temporal lobe ( 4)which part of the brain is being assessed when you are assessing motor response ? ( a ) occipital lobe (b ) cerebellum (c ) sensorimotor pathways (d ) dermatomes (e ) reticular formation ( 5)what are the specific sections that comprise the glasgow coma scale ? ( a)eye opening , verbal response , pupil response (b)eye opening , verbal response , limb movement (c)eye opening , verbal response , motor response (d)eye opening , respiratory pattern , motor response (e)eye opening , respiratory pattern , pupil response ( 6)vital signs are a component of the glasgow coma scale . ( a ) true (b ) false ( 7)when testing the best motor response , you ( a ) record the response in the best arm . ( 8)to test motor response in a tetraplegia patients ( paralyzed in all four limbs ) , ( a ) inflict a pain stimulus in the arms until there is a response . (d ) lift the arm up and let it drop to the bed three times . ( 9)the lowest score of the glasgow coma scale is ( a ) 1 (b ) 3 (c ) 4 (d ) 10 ( 10)patients with a glasgow coma scale score of and below are considered comatose . ( a ) 1 (b ) 3 (c ) 8 (d ) 10 ( 11)in nursing practice , a reduction of the glasgow coma scale score of is seen asa deterioration in conscious level and requires informing the medical team . ( a ) 1 (b ) 3 (c ) 8 (d ) 10 ( 12)the glasgow coma scale can not assess intubated patient 's level of consciousness . ( a ) true (b ) false ( 13)on asking a patient , do you know where you are now ? the patient states he is at his daughter 's condominium . ( 14)on assessing a patient 's motor response , he is unable to comply . (d ) has abnormal extension . ( 15)you are assessing an rta ( road traffic accident ) patient , who has swollen eyes . the eye response score is ( a ) 4 (b ) c (c ) 2 (d ) 0 the glasgow coma scale was initially devised to ( a)locate brain tumour (b)assess the depth of coma (c ) facilitate care for stroke patients (d ) monitor the extent of meningitis locate brain tumour assess the depth of coma facilitate care for stroke patients monitor the extent of meningitis what part of the brain is being assessed when you are assessing eye opening ? ( a ) cerebral cortex (b ) occipital lobe (c ) cerebellum (d ) reticular formation (e ) hypothalamus reticular formation which part of the brain is being assessed when you are assessing verbal response ? ( a ) cerebral cortex (b ) occipital lobe (c ) cerebellum (d ) reticular formation (e ) temporal lobe reticular formation which part of the brain is being assessed when you are assessing motor response ? ( a ) occipital lobe (b ) cerebellum (c ) sensorimotor pathways (d ) dermatomes (e ) reticular formation sensorimotor pathways reticular formation what are the specific sections that comprise the glasgow coma scale ? ( a)eye opening , verbal response , pupil response (b)eye opening , verbal response , limb movement (c)eye opening , verbal response , motor response (d)eye opening , respiratory pattern , motor response (e)eye opening , respiratory pattern , pupil response eye opening , verbal response , pupil response eye opening , verbal response , limb movement eye opening , verbal response , motor response eye opening , respiratory pattern , motor response eye opening , respiratory pattern , pupil response vital signs are a component of the glasgow coma scale . ( a ) true (b ) false when testing the best motor response , you ( a ) record the response in the best arm . record the response in all four limbs . to test motor response in a tetraplegia patients ( paralyzed in all four limbs ) , ( a ) inflict a pain stimulus in the arms until there is a response . (d ) lift the arm up and let it drop to the bed three times . lift the arm up and let it drop to the bed three times . the lowest score of the glasgow coma scale is ( a ) 1 (b ) 3 (c ) 4 (d ) 10 patients with a glasgow coma scale score of and below are considered comatose . ( a ) 1 (b ) 3 (c ) 8 (d ) 10 in nursing practice , a reduction of the glasgow coma scale score of is seen asa deterioration in conscious level and requires informing the medical team . ( a ) 1 (b ) 3 (c ) 8 (d ) 10 the glasgow coma scale can not assess intubated patient 's level of consciousness . ( a ) true (b ) false on asking a patient , do you know where you are now ? the patient states he is at his daughter 's condominium . you are assessing an rta ( road traffic accident ) patient , who has swollen eyes . the eye response score is ( a ) 4 (b ) c (c ) 2 (d ) 0 the questionnaire used in this study is replicated from previous study in singapore with permission . one hundred and thirty - five questionnaires were distributed , to nurses that met the inclusion criteria . each nurse was given 1520 minutes to answer the questionnaire , which was then returned . data was analyzed using the statistical package of social sciences ( spss ) 20.0 version of window software . 88.9% of the nurses who participated knew what glasgow coma scale was initially devised to . to the question pertaining to the part of the brain involved in assessing eye opening , 51.9% answer correctly as opposed to 31.1% for verbal response and 40.7% for motor response . the majority ( 85.9% ) answered correctly to the question , pertaining to the components of the glasgow coma scale . only 54.8% of the participants knew that vital signs are not a component of the glasgow coma scale . only about one - fifth ( 21.5% ) of the nurses knew how to test the best motor response , but more than half ( 58.5% ) knew how to test for motor response in a tetraplegic patient . while the vast majority ( 91.1% ) knew what is the lowest score of the glasgow coma scale , however only about half ( 51.9% ) knew the score which defined comatose . 63.7% said glasgow coma scale can be assessed on an intubated patient 's and 86.7% could answer the question pertaining to patients ' verbal response . on assessing a patient 's motor response with pain stimulus only 11.9% answered correctly but when assessing rta ( road traffic accident ) patient , who has swollen eyes 84.4% , answered correctly . this showed that the nurse 's knowledge on gcs is poor in detecting deterioration of patient and in assessing the best motor response using pain stimulus . overall only 2.96% had good knowledge , scoring is 80100% ( 1215 points ) , 41.48% had satisfactory knowledge , and the knowledge of 55.56% of the nurses who participated in the questionnaire was poor ; that is , more than half had poor knowledge in assessing gcs . the result on the association between knowledge and education level shows that there was statistically significant ( significant level is p value less than 0.05 ) association between the two variables ( x = 18.412 , df = 3 , n = 135 , and p < 0.05 ) shown in table 1 . nurses with certificates have good knowledge ( 25% ) compared to post basic nursing ( 0% ) . the result on association between knowledge and age group shows that there was a statistically significant ( significance level is less than 0.05 ) association between the two variables ( x = 11.085 , df = 2 , n = 135 , and p < 0.05 ) shown in table 2 . nurses in age group of 4160 had good knowledge ( 11.1% ) compared to age group of 2030 ( 1.1% ) . glasgow coma scale ( gcs ) is a reproducible tool used by nurses in almost every healthcare facility to assess level of consciousness in a patient with a neurological problem . it is important to have the skill and knowledge when assessing and applying critical thinking to interpret the findings . our survey showed 2.96% scored had knowledge , 41.48% had satisfactory knowledge , and 55.56% had poor knowledge of gcs . this is comparable to the finding by teles et al . who found that 74.55% of the staff nurses had average knowledge and 25.45% had poor knowledge in gcs , whereas jaddoua et al . showed that all nurses ( n = 100 ) had inadequate knowledge in gcs . educational level is not the primary factor needed in assessing the gcs as shown in this study . the result on association between knowledge and education level shows that there was a statistically significant association between the two variables ( x = 18.412 , df = 3 , n = 135 , and p < 0.05 ) . similar to the study by heron et al . on interrater reliability of the gcs found there were statistically significant differences with education qualification . the result on association between knowledge and age group shows that there was statistically significant association between the two variables ( x = 11.085 , df = 2 , n = 135 , and p < 0.05 ) . similar to the study by heron et al . on interrater reliability of the gcs found there were statistically significant differences with age . further study should be conducted on all healthcare personnel practicing at the emergency and outpatient departments . this finding raises concerns on the importance of knowledge and skill in assessing gcs . continuing education and practice on use of the gcs tool are important . education and age have a correlation with satisfaction level towards nurse 's knowledge in gcs . this indicates that midage nurses with lower education level have higher skill and experience on using the gcs tool .

primary infection with varicella zoster virus ( vzv ) which is a mild to moderate self - limited disease causes varicella ( chickenpox ) in children ( 1 ) . because of the waning vzv specific cell mediate immunity , virus reactivation in relation to suppressed immune conditions or elder people , could induce herpes zoster ( 2 , 3 ) . varicella zoster is accompanied by serious complications such as post - herpetic neuralgia , viral pneumonia and encephalitis ( 4 , 5 ) . herpes zoster frequency and its most common complications are likely to increase with age ( 6 ) . the virus is usually transmitted 1 - 2 days before appearance of rashes via respiratory droplets , vesicle fluid , direct contact or aerosol exposure to the vesicular lesions of a patient with varicella . also , in hospital setting , airborne transmission has been described ( 7 , 8) . following vzv exposure ( primary infection or vaccination ) , vzv antibodies appear in serum . different studies have confirmed the increasing trend in vzv seroprevalence parallel to aging ( 9 , 10 ) . one survey that was conducted before vzv mass vaccination during 1988 - 1994 , demonstrated a high vzv seroprevalence ranging from 86% in 6 - 11 years old children to 99% in 30 years old individuals(6 ) . another study reported that 49% of 2 years old and 93% of 5 years old children were vzv seropositive before vzv vaccination ( 11 ) . vzv seroprevalence is diverse in different studies depending on race , gender , country and number of siblings in a household ( 6 , 12 , 13 ) . a highly efficient strategy to eliminate vzv - infection and its related complications is the routine varicella vaccination ( 14 ) . since 1974 a live - attenuated varicella vaccine has been proven safe and effective and universal childhood immunization has been implemented in several countries(15 ) . it is well documented universal vaccination of children would increase the prevalence of vzv protective antibodies in the population ( 16 , 17 ) . a number of studies on the prevalence of antibodies against this virus have been conducted in different countries ( 1820 ) . in france , general vaccination against varicella zoster indicated at least 80% efficacy in reduction of vzv infection ( 21 ) . besides , a survey in brazil in 2006 reported that varicella immunization developed 70 - 90% protection against any form of varicella zoster infection ( 22 ) . although varicella immunization schedule has been well established in health care systems of many countries ( 23 , 24 ) , immunization against this virus , has not been included in official vaccination program in iran . the present study aimed to compare the prevalence of anti - vzv antibody in three groups of children under 7 years old in tehran , iran , in order to obtain an informative picture of general presence of these antibodies for designing a comprehensive immunization plan . this cross - sectional study was conducted between october 2011 and july 2012 at avicenna research institute , tehran , iran . the study population included 7 months , 18 months and 6-year - old healthy children who attended to health care centers for routine vaccinations . these health care centers provide general vaccination services to children and adults with different socio - demographic status representative of an ordinary population . written informed consents were obtained from the parents of children after declaring the process of the study . the parents completed the questionnaires containing general information such as age , birth weight , and previous history of vzv infections in children . the subjects were categorized in three age groups consisting of 7 month , 18 month and 6-year - old children . it is important to note that the 7 months old children were bled again at the age of 18 months ; thus , the corresponding 7 and 18 months old children blood samples were taken from the same individuals . serological tests were conducted by elisa kit ( euroimmune anti - varicella - zoster virus igg elisa medizinische labor diagnostka ag , lubeck , germany ) . the test was defined to have a specificity of 100% and a sensitivity of 100% , according to the manufactures instructions . statistical analyses were performed using statistical package for the social sciences spss version 13.0 ( spss inc , chicago , il , usa ) . differences in antibody titers for the different age groups were assessed using chi - square , independent t - test and paired t - test . this cross - sectional study was conducted between october 2011 and july 2012 at avicenna research institute , tehran , iran . the study population included 7 months , 18 months and 6-year - old healthy children who attended to health care centers for routine vaccinations . these health care centers provide general vaccination services to children and adults with different socio - demographic status representative of an ordinary population . written informed consents were obtained from the parents of children after declaring the process of the study . the parents completed the questionnaires containing general information such as age , birth weight , and previous history of vzv infections in children . the subjects were categorized in three age groups consisting of 7 month , 18 month and 6-year - old children . it is important to note that the 7 months old children were bled again at the age of 18 months ; thus , the corresponding 7 and 18 months old children blood samples were taken from the same individuals . serological tests were conducted by elisa kit ( euroimmune anti - varicella - zoster virus igg elisa medizinische labor diagnostka ag , lubeck , germany ) . the test was defined to have a specificity of 100% and a sensitivity of 100% , according to the manufactures instructions . statistical analyses were performed using statistical package for the social sciences spss version 13.0 ( spss inc , chicago , il , usa ) . differences in antibody titers for the different age groups were assessed using chi - square , independent t - test and paired t - test . 267 serum samples were collected from september 2011 to september 2012 . from these specimens , 87 were taken from 7 month old infants , 86 of whom come back again for sampling at the age of 18 months and 94 serum samples belonged to 6 year old children . according to the filled questionnaires , none of the parents reported past history of vzv infection in their children . mean birth weights and visited - day weights of subjects in different age groups were 3.2 0.3 kg , and 5.0 0.9 kg ( 7 months ) , 3.2 0.3 kg and 11.0 1.1 kg ( 18 months ) and 3.2 0.4 kg and 20.3 2.7 kg ( 6 years ) , respectively . no significant difference was found between birth weights and visited - day weights with gender ( p > 0.05 ) ( table 1 ) . birth weights and visited- day weights in different age groups of iranian children the mean antibody concentrations against vzv were 183 781 iu / l , 327 878 iu / l in 7 months , 18 months and 6 years old subjects , respectively . the seropositive rates were 4.6% , 12.8% and 21.3% for 7 month , 18 month and 6-year - old individuals , respectively . table 2 shows antibody titers against vzv and seropositive rates of subjects in each age group and their distribution according to the gender . our findings showed no significant difference in anti - vzv antibody concentrations and seropositive rates related to gender in each of age groups ( p > 0.05 ) ( table 2 ) . positive seroprevalence rates were increased along with age increasing ( difference was not statistically significant ) mean antibody titers against vzv and seropositive rates in different age groups of iranian children comparison of antibody titers against vzv in 6 year old children with that in 7 months age ( p = 0.086 ) and 18 months age ( p = 0.506 ) groups showed no significant differences , but the rates of seropositive subjects were significantly higher in 6 year old children compared to 7 months old infants ( p = 0.001 , chi - square ) . anti - vzv antibody titers increased significantly by age when they were measured in the same individuals at 7 months and 18 months of age ( p < 0.001 , paired t - test ) . multiple linear regression did not show any significant correlation between demographic characters ( gender , birth weight , visited - day weight ) and anti - vzv antibody titers in different age groups ( table 3 ) . multiple linear regressions between demographic characters ( gender , birth weight , visited- day weight ) and anti - vzv antibody titers in different age groups of iranian children . 267 serum samples were collected from september 2011 to september 2012 . from these specimens , 87 were taken from 7 month old infants , 86 of whom come back again for sampling at the age of 18 months and 94 serum samples belonged to 6 year old children . according to the filled questionnaires , none of the parents reported past history of vzv infection in their children . mean birth weights and visited - day weights of subjects in different age groups were 3.2 0.3 kg , and 5.0 0.9 kg ( 7 months ) , 3.2 0.3 kg and 11.0 1.1 kg ( 18 months ) and 3.2 0.4 kg and 20.3 2.7 kg ( 6 years ) , respectively . no significant difference was found between birth weights and visited - day weights with gender ( p > 0.05 ) ( table 1 ) . the mean antibody concentrations against vzv were 183 781 iu / l , 327 878 iu / l and 425 107 iu / l in 7 months , 18 months and 6 years old subjects , respectively . the seropositive rates were 4.6% , 12.8% and 21.3% for 7 month , 18 month and 6-year - old individuals , respectively . table 2 shows antibody titers against vzv and seropositive rates of subjects in each age group and their distribution according to the gender . our findings showed no significant difference in anti - vzv antibody concentrations and seropositive rates related to gender in each of age groups ( p > 0.05 ) ( table 2 ) . positive seroprevalence rates were increased along with age increasing ( difference was not statistically significant ) mean antibody titers against vzv and seropositive rates in different age groups of iranian children comparison of antibody titers against vzv in 6 year old children with that in 7 months age ( p = 0.086 ) and 18 months age ( p = 0.506 ) groups showed no significant differences , but the rates of seropositive subjects were significantly higher in 6 year old children compared to 7 months old infants ( p = 0.001 , chi - square ) . anti - vzv antibody titers increased significantly by age when they were measured in the same individuals at 7 months and 18 months of age ( p < 0.001 , paired t - test ) . multiple linear regression did not show any significant correlation between demographic characters ( gender , birth weight , visited - day weight ) and anti - vzv antibody titers in different age groups ( table 3 ) . multiple linear regressions between demographic characters ( gender , birth weight , visited- day weight ) and anti - vzv antibody titers in different age groups of iranian children . this study was conducted to generate data on the epidemiology of vzv infection in a limited population of iranian children with the aim to establish a preliminary report as a basis for larger studies for health system improvement in varicella vaccination strategies . protection against vzv in neonates is dependent on maternal immunity until they are 6 months old when the production of antibody by their own immune systems begins ( 19 , 25 ) . since we did not have access to confirmed history of immunity in mothers , we selected 7 months old infants to determine their seroprevalence of anti - vzv antibodies . our results regarding similar anti - vzv prevalence rates in different genders are confirmed by previous reports ( 10 , 2628 ) . the age group specific prevalence in our survey suggests that the seropositivity of antibodies against vzv increases with age . the prevalence of vzv antibodies were 7.1% , 5.6% and 18.2% in 9 , 15 and 24 months old subjects , respectively(10 ) . also , 16% of 1 year old children and 25.1% of children between 2 - 3 years old were seropositive in a cross - sectional study in cyprus(9 ) . in thailand , sero - positivity rate increased from 15.5% in children ( between 9 months and 4 years old ) , to 75.9% in adults ( 20 - 29 years old ) ( 29 ) . reported 29 - 35% seropositivity in 1-year - old , 80.19% in 5 year old and 84.78% in 6-year - old individuals in flanders ( 30 ) . similarly , in italy , seroprevalences of varicella were reported to be 32.9% , 67% and 84% in the age classes : 2 - 4 , 5 - 9 and 10 - 14 years , respectively ( 31 ) . it could be noticeably deduced that the increasing trend in prevalence of anti - vzv antibodies with age is evident ( 27 ) , however the prevalence rates may be different from our results ( 32 , 33 ) . the elevated levels of prevalence in these countries may be related to increased exposure to virus after natural infection , since all these studies were conducted before their vaccination plans started . the elevation in vzv antibody titers following vaccination causes diminishing impact on incidence of chickenpox and zoster infection . findings of a survey demonstrated varicella vaccination could induce 34.7% decrease in the incidence of varicella among 0 - 14 year old children and 51% reduction in the number of complications associated to varicella ( 3 , 34 ) . in addition , another study reported of 55% reduction in varicella cases in the studied population that included 63% and 38% reduction in 0 - 4 and 5 - 9 years old children , respectively ( 35 ) . similar results were also reported in other studies ( 36 , 37 ) . besides , in france , sero - positivity was reported in 15% of 1 - 2 year and 89% of 7 - 8 year old children ( 20 ) . in a similar trend , in malaysia 25.6% of 1 - 5 , 47.2% of 6 - 10 and 50.8% of 11 - 15 old children were reported as seropositive ( 38 ) . this increasing pattern was also observed in india , canada and other countries ( 18 , 31 , 3942 ) . the unique characteristic of our study has been the provision of an opportunity to investigate the association between antibody titers within a person at different ages that revealed a significant increase by age . protective role of maternal antibody against vzv is durable up to 6 months ( 19 ) . with the advent of vzv vaccine , the incidence of vzv infection in children and adults has decreased and the breakthroughs are minimizing . this vaccine is not routinely used in iran , however this important subject was considered from 1988 to 1995 in japan , turky and the united states . in the usa , the combination of mmr and varicella zoster vaccine ( mmrv ) was allowed for children between 12 months and 12 years of age in 2005(42 , 43 ) . according to our results , we suggest usage of mmrv vaccine to be administered in children . our findings that , significant elevation of vzv antibodies occur from 7 to 18 months of age and rate of seronegative children support the necessity of varicella immunization at this age range in order to prevent virus infection . since serious complications and long - lasting sequels have been reported after vzv infection , it may be recommended that vzv vaccination be integrated in the iran national vaccination program . in this regard , larger and more comprehensive studies need to be conducted to warrant information required for decision - making . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

kidney cancer is currently the sixth and eighth most common cause of new cancer diagnoses in american men and women , respectively , and the incidence has increased by approximately 3% annually.1 - 5 in particular , the rate of renal cell carcinoma has steadily increased over the past several decades , and renal cell carcinomas are currently one of the most lethal tumors of the genitourinary system.6 the tumor , nodes , and metastasis ( tnm ) staging system for renal cell carcinoma has evolved over the years in an effort to be more prognostically relevant as new research has emerged.7 - 10 in 2002 , the pt1 group was subdivided into a pt1a category for tumors measuring 4 cm or less and pt1b for larger tumors measuring up to 7 cm . recently , in 2010 , the pt2 division was similarly refined with tumors measuring greater than 7 and up to 10 cm placed in a pt2a category , and those larger than 10 cm being staged as pt2b.11 in the upcoming 4 edition of the world health organization ( who ) classification of tumors of the urinary system and male genital organs , tumor size using 1.5 cm as a cutoff will be proposed to distinguish renal papillary adenoma from papillary renal cell carcinoma , though it is unclear if the tumor size should be based on fresh , postfixation , or microscopic measurement . given the fact that tumor size plays a key role in staging renal cell carcinomas , specific guidelines should be in place for tumor measurement.6 , 9 - 14 the tnm classification method has evolved from the nonspecific size distinctions of small and large to further subclassifications of specific tumor size cutoffs that may affect prognosis and treatment , but clear definitions of how to obtain these size measurements are lacking.6 , 9 - 14 we sought to quantify the effects of formalin fixation , tissue processing and mounting of histologic sections on tumor size in a prospective study . from the period of october 3 , 2013 to november 20 , 2014 , all nephrectomy cases performed by a single urologist ( cps ) at our institution were prospectively measured at different time points . at the end of surgery , the fresh specimen was sectioned and measurements of the tumor 's three dimensions were taken by the urologist ( cps ) with the assistance of pathology assistants and surgical pathologists . the most representative cross - section that included the tumor 's largest dimension was selected and marked with ink . in order to accurately measure the tumor histologically by whole mount , only postresection renal neoplasms grossly measuring 5.5 cm or less were included in the study . the kidney specimen was then placed in 10% formalin and allowed to fix for a time period ranging from 6 to 24 hours . the representative cross - section , along with the standard tissue sections taken for diagnosis and staging , underwent dehydration , clearing , paraffin wax embedding , section cutting , mounting , and staining with hematoxylin and eosin . a whole mount glass slide or standard glass slide section was made of the most representative cross - section , and two - dimensional tumor size was measured ( tt ) ( fig 1 ) . tumors that were too large to fit a whole mount slide ( fresh tumor size>5.5 cm ) , whose cross - sections did not include the largest dimension , or whose most representative cross - section was mistakenly incised and transferred to multiple slides were excluded . in addition , preoperative computerized tomography ( ct ) and magnetic resonance imaging ( mri ) images were retrospectively reviewed ( cdb ) to obtain radiologic renal tumor measurements in three dimensions . both axial and coronal / sagittal reformats were utilized in an effort to acquire the most accurate greatest dimension . clinicopathologic information , including each patient 's age , gender , type of surgery , tumor histologic subtype and pathologic staging classification , was recorded . prior to the initiation of this prospective study , the protocol was reviewed and approved by the indiana university institutional review board . all local , state , and federal guidelines for the protection of human subjects were strictly maintained throughout the course of the study . descriptive statistics were utilized to describe differences in diameter between the four stages of measurement . student t test was used to compare the percentage of tumor shrinkage between each of the measurement groups . lp , college station , tx ) and p values < 0.05 were considered statistically significant . utilizing the resulting mean shrinkage values in tumor diameter between the three stages of measurement , corresponding conversion factors were calculated based on the formula : 1/(1-(shrinkage decimal)).15 a total of 34 renal tumors were included in the study , which consisted of 23 clear cell renal cell carcinomas , 6 papillary renal cell carcinomas , 1 chromophobe renal cell carcinoma , 1 t(6;11 ) renal cell carcinoma , and 3 oncocytomas . the ages of the patients ranged from 37 to 84 years old with a mean age of 64 years . two tumors , a clear cell and a chromophobe renal cell carcinoma , were staged as pt1b . the tumor diameters for postresection ( fresh ) specimens ranged from 1 - 5.1 cm ( average , 2.7 cm ) with 7 tumors measuring less than 2 cm , 23 between 2 - 4 cm , and 4 larger than 4 cm . fixed tumor diameters ranged from 1 - 4.8 cm ( average , 2.5 cm ) with 8 tumors measuring less than 2 cm , 22 between 2 - 4 cm , and 4 larger than 4 cm . microscopic tumor diameters ranged from 1 - 4.6 cm ( average , 2.3 cm ) , and 13 tumors measured less than 2 cm , 19 between 2 - 4 cm , and 2 larger than 4 cm . the average decrease in tumor diameter for fixed specimens , as compared to fresh , was 0.1 cm ( range , 0 - 0.8 cm ) with a mean shrinkage of 4.6% ( p<0.01 ) ( range , 0 - 18.2% ) . furthermore , the average decrease in diameter for histological specimens , as compared to fixed tumors , was 0.2 cm ( range , 0 - 0.7 cm ) with a mean shrinkage of 7.1% ( p<0.01 ) ( range , 0 - 18.2% ) . the overall average decrease in tumor diameter between fresh and histological specimens was 0.3 cm ( range , 0 - 1.1 cm ) with a mean shrinkage of 11.4% ( p<0.01 ) ( range , 0 - 22.7% ) ( fig 2 ) . the tumor diameters based on radiologic imaging ranged from 1.5 - 5.7 cm ( average , 3.0 cm ) with 5 tumors measuring less than 2 cm , 24 between 2 and 4 cm , and 5 larger than 4 cm . table 1 illustrates the utilization of our calculated correction factors . in terms of tumor diameter measurements , a conversion factor of 1.08 will account for shrinkage due to histological processing alone , 1.05 will correct formalin fixation shrinkage , and 1.13 will account for shrinkage from both fixation and tissue processing . table 2is a conversion table that provides precalculated fixed and fresh tumor diameters based on microscopic measurements , utilizing the aforementioned correction factors . since the inception of staging systems for renal cell carcinomas and their evolution over the past 50 years , staging remains an important prognostic indicator.6 , 9 - 14 the first classification method was introduced by flocks and kadesky in 1958 , which concentrated on the extent of tumor spread.16 the tnm staging system was established in 1978 and introduced tumor size as a prognostic factor.10 , 11 the tnm system for renal cell carcinoma has been revised multiple times to be more prognostically precise , changing the t2 tumor size cutoff points from large to greater than 2 cm and then to greater than 7 cm.6 , 9 - 14 in addition , t1 and t2 tumors were each further subcategorized into a and b stages by the tnm system with the cutoffs placed at 4 cm and 10 cm , respectively.10 , 11 tumor size has repeatedly been shown to be a significant prognostic indicator , even independent of pathologic stage.8 , 17 larger renal cell carcinomas are significantly more likely to have renal sinus invasion , ipsilateral adrenal involvement , synchronous metastasis , and an overall decreased 5-year survival rate.18 - 22 according to bonsib and bhalodia , unrecognized retrograde vein invasion may have partially contributed to the reportedly worse prognosis associated with larger renal tumors , whose size may have been inflated with confluent nodules of intravenous cancer.23 correct renal tumor measurement remains essential for accurate prognostication . tumor size plays a key role in renal cell carcinoma staging , and its importance has grown throughout tnm 's evolution with the additions of further subdivisions according to size.6 , 9 - 14 despite this , a standardized means of tumor measurement has not been established . while , intuitively , staging a renal cell carcinoma should be based on fresh tumor measurements , most pathologists report size based on postfixation or , for small tumors , microscopic measurements . our results demonstrate that formalin fixation can shrink renal tumors , and the steps of histological processing , embedding and mounting cause even further tumor shrinkage . formalin acts by diffusing through the tissue and binding to amino groups , precipitating the formation of an extensive network of cross - linked proteins and nucleic acids , which can cause histological changes , such as cell shrinkage and distortion.24 conceivably , these cellular changes can have a global effect on the whole tissue specimen . while the effects of fixation and histological processing on specimen size have not been previously systematically studied in the kidney , there are a small number of similar studies performed on other organ systems . several of these studies also found a shrinkage effect of formalin for various tissue specimens . our mean 4.6% tumor diameter shrinkage after fixation is greater than the 2.7% found for cervical tissue , but smaller than that reported in most studies , which have ranged from approximately 8 - 20%.25 - 31 this formalin - induced shrinkage effect occurred in a range of specimens , including those from the lung , gastrointestinal system , oral cavity , vulva and breast.24 , 26 - 33 this has not been consistent , however , with several studies reporting no shrinkage after fixation for skin , breast and gastrointestinal specimens.34 - 41 one paper found an increase in one dimension for forelimb specimens , which flattened due to gravity after formalin fixation , causing an increase in width , but a decrease in height.42 the varying study results may be explained by the different types of tissues and tumors along with differences in formalin concentration and fixation time . a small subset of studies has evaluated the effects of histological processing . after formalin fixation , there are several steps that may affect tissue size as the tissue section progresses to the final staining phase . tissue shrinkage may occur as the section undergoes alcohol dehydration.15 in addition , during the embedding stage , the tissue may be compressed if the paraffin is too warm and undergo extra shrinkage during the cooling process . during section cutting , a loose or off - angled microtome knife may result in tissue compression or chatter , and the tissue may not evenly spread in a cool water bath during the mounting stage.39 in our study , the average percentage of shrinkage in tumor diameter from the fixed to the histological states was 7.1% , and the overall mean shrinkage was 11.4% between the fresh and microscopic measurements . in the previously published studies , the percentage of shrinkage from the fixed to the microscopic measurements ranged from approximately 10% to 27% , and the overall percentage of shrinkage from fresh to microscopic measurements ranged from 4.5% to 41%.15 , 25 , 31 , 32 , 39 the tissue types studied included cervical , gastrointestinal , prostatic , breast and oral cavity specimens . found both substantial increases and decreases in breast tumor size with histological processing , and johnson et al . reported that mucosal margins of oral cavity specimens did not change with slide preparation , but the deep muscular margin shrank by 9.8%.27 , 39 variations in tissue type and , even more so , in laboratory practices and processes , such as differences in water bath temperature and the time tissue sections spend in warm water15 or the practice of sectioning suboptimally fixed and processed tissue 39 , may explain the varying findings . the subjects of the published studies are highly variable and include animal models , non - neoplastic tissue , and human tumors ranging from skin to breast malignancies . while studying animals and normal tissues may provide useful background information , research involving human tumors would have the most impact on patient care . certain tumors , though , such as breast carcinomas , may have significant infiltration that can be difficult to gauge during gross tumor measurement . such measurements may underestimate the true size of the neoplasm . other tumors , such as lung and colorectal carcinomas , may be too large to lend feasibility to studies comparing fresh and fixed measurements with microscopic measurements . renal neoplasms , on the other hand , offer an ideal model for tumor measurement analysis due to their easily visualized , often circumscribed , and unifocal nature . in addition , the steady increase in renal cell carcinoma incidence due to the detection of small incidental kidney tumors by routine radiological studies translates to the finding that many renal tumors are small enough to offer a complete , representative cross - section on a standard glass or whole mount slide.43 the kidney is also a dense , solid , encapsulated organ that is neither characterized by the elasticity of skin and the gastrointestinal tract , nor the almost semisolid nature of adipose tissue . hence , the kidney often preserves the renal tumor 's overall configuration , whether solid or cystic , after removal and fixation . on the other hand , differences in renal tumor architecture , including the presence of fibrosis , cystic change , hemorrhage , and necrosis , may affect the extent of shrinkage . to the best of the authors ' knowledge , our study is the first to describe renal tumors as a model for postfixation and processing shrinkage analysis . irrespective of the differences in subject matter and tissue types of the published reports , many of these studies were implemented because tumor size or margin status is essential for staging and prognosis in their respective organ systems , and most staging systems do not specify the method for measurement . for kidney tumors in particular , two of our cases would receive a different pt1 classification depending on which measurement was used for staging . tumor size has repeatedly been shown to be a significant prognostic indicator.8 larger renal cell carcinomas included in the t1b category are significantly more likely to have renal sinus invasion , ipsilateral adrenal involvement , synchronous metastasis , and an overall decreased 5-year survival rate.18 - 21 given the importance of correctly staging renal cell carcinomas , specific guidelines should be in place for tumor measurement . intuitively , renal tumors should be measured before placement in formalin for the most accurate tumor staging . however , studies reporting on the prognostic significance of tumor size frequently do not specify how or when measurements were obtained.9 , 44 due to the fact that taking fresh tumor measurements may not always be convenient or feasible , a conversion factor would be a useful tool for practicing pathologists . for example , silverman et al . because minimum margins are essential for treatment , the authors calculated a formula to estimate in vivo margins from fixed - tissue measurements for melanoma excisions.45 furthermore , in the prostate , tumor volume is an essential staging and prognostic indicator.15 tissue shrinkage from processing has long been recognized and various correction factors have been proposed for accurate tumor volume calculations , ranging from 1.14 - 1.5.46 , 47 the methods that engendered these prostate volume correction factors varied and included computer planimetry , water displacement , transrectal ultrasound , and direct measurements.46 , 47 in terms of the kidney , our results demonstrate an average 4.6% shrinkage in diameter between fresh and fixed tumor measurements , 7.1% between fixed and glass slide measurements , and 11.4% between fresh and glass slide measurements , which correlate to correction factors of 1.05 , 1.08 , and 1.13 , respectively . the following example illustrates the correction of shrinkage effects on a kidney tumor that measures 2 cm on the glass slide . by using the linear conversion factors , the postfixation diameter would equate to 2.2 cm and the postresection diameter 2.3 cm ( table 2 ) . while fresh tumors measuring greater than 5.5 cm were excluded from the study due to decreased accuracy in terms of histologic measurement , the conversion factors may be applicable for such tumors and the measurements were extrapolated and also provided in table 2 . papillary adenomas of the kidney are defined as small , low grade , unencapsulated tumors with papillary or tubular architecture that measure no larger than 1.5 cm . this size cutoff will be proposed in the upcoming 4 edition of the who classification of tumors of the urinary system and male genital organs , but the method of measurement remains undefined . if one were to apply the correction factors to a renal papillary tumor measuring 1.4 cm in diameter on the glass slide , the corresponding postfixation diameter would equal 1.5 cm and postresection diameter 1.6 cm ( table 2 ) . using the microscopic measurement , the tumor in our example but by utilizing the conversion factor to account for shrinkage from the fresh state , the tumor could be diagnosed as a papillary renal cell carcinoma . currently , small papillary tumors of the kidney are likely to be diagnosed using gross , postfixation or microscopic measurements . when the definition of a benign tumor versus a malignant neoplasm is so narrow , correction factors are especially pertinent for accurate diagnosis . kidney tumors provide an ideal model to analyze the shrinkage effects of fixation and processing . our results suggest that renal cell carcinomas with diameters at tnm staging thresholds may be understaged if postfixation or microscopic measurements are utilized . we recommend using a tumor diameter conversion factor of 1.05 for postfixation gross measurements or 1.13 for microscopic measurements to approximate the fresh tumor size more accurately . furthermore , with an average difference of 12.1% in diameter , our results demonstrate that tumor measurements based on radiologic imaging may overestimate the size of renal neoplasms when compared to the gold standard ( fresh ) measurements . our outcomes are in line with those of previous studies that found radiographic measurements of renal masses to be larger than pathologic measurements , especially for smaller tumors.48 - 58 while the size differences between radiologic and pathologic measurements may be statistically significant in a majority of these studies , the clinical significance in terms of treatment outcomes is uncertain.49 , 52 , 53 , 57 nevertheless , the utilization of correction factors for pathologic specimens would increase accuracy for correlations with presurgical imaging measurements often used in clinical studies . most importantly , conversion factors would be valuable for the placement of renal cell carcinomas in the appropriate pathologic tumor stage . while a standard means of renal tumor measurement has not been established , intuitively , tumor size should be based on fresh measurements . the shrinkage effects of formalin fixation and histological processing may result in understaging of renal cell carcinomas . it would be helpful if future reports of the prognostic significance of pathologic stage for renal cell carcinomas included details on the method for tumor size measurement , especially for small renal tumors .

small nontranslated rnas perform a multitude of functions in the cell , e.g. , transfer rnas function as adapters in translation , small nuclear rnas are involved in mrna splicing , and small nucleolar rnas direct the modification of ribosomal rnas . recently , two other groups of small rnas have received much attention : small interfering rnas ( sirnas ) and strnas . both types of rnas are 2125 nt long and both mediate downregulation of gene expression . however , despite these and other similarities ( for review see banerjee and slack , 2002 ) , their respective modes of action appear to be quite different . sirnas mediate rnai , a process in which target mrnas are degraded ( elbashir et al . , 2001 ; for review see sharp , 2001 ) . strnas , in contrast , downregulate expression of target rnas after translation initiation without affecting mrna stability ( lee et al . , 1993 : moss et al . , 1997 ; olsen and ambros , 1999 ; reinhart et al . , 2000 ; slack et al . , 2000 ) . although our understanding of the mechanisms of rnai has evolved over the last few years , its physiological role remains to be explored . the opposite is true for strnas ; although their mode of action is largely obscure , their role in the control of developmental timing in the nematode c. elegans has been well established . lack of expression or overexpression of either of the two known strnas , lin-4 and let-7 , causes heterochronic phenotypes ( for reviews see banerjee and slack , 2002 ; rougvie , 2001 ) , i.e. , changes in the timing of developmental events . lin-4 and let-7 rnas both act as repressors of their respective target genes , lin-14 , lin-28 , and lin-41 ( lee et al . , 1993 ; moss et al . , 1997 ; slack et al repression in all these cases requires the presence of strna complementary sequences in the 3 untranslated regions ( utrs ) of the target mrnas , suggesting that mrna / strna hybrids can form ( fig . 1 , a and b ; reinhart et al . , 2000 ; slack et al . , 2000 ; wightman et al . , 1991 , 1993 ) . the presence of these strna - binding elements is indeed sufficient to confer strna - dependent expression on unrelated reporter genes ( ha et al . , 1996 ; lee et al . , 1993 ; moss et al . , 1997 ; slack et al how downregulation is achieved through the putative rna / rna hybrid is , however , unclear . evidence with lin-4 suggests that mrna stability and polyadenylation level as well as translation initiation are not affected ( moss et al . , 1997 ; ( a ) lin-41 is the major target of let-7 and contains two slightly varying elements in its 3 utr that are complementary to the let-7 strna as well as one element that is complementary to the second strna , lin-4 . ( b ) examples of potential rna / rna duplexes between let-7 and lin-41 , and lin-4 and lin-14 . in the case of lin-41 , a consensus sequence for the two slightly varying 3 utr elements is shown . the arrow points to a conserved bulged c that is essential for lin-4 function and might constitute a binding site for additional factors . the bulged a in lin-41 is present in only one of the two elements and thus shown in brackets . ( c ) strnas and mirnas are transcribed as precursors of 6070 nt that can be folded into a stem - loop structure . processing releases a mature rna of 22 nt ( shaded ) . for lin-4 and let-7 , which are shown here , the mature rna is released from the 5 arm of the precursor ; however , mirnas have been identified in which the 3 arm ( or even both arms ) is stable . in addition to sharing a similar size and , presumably , mode of action , lin-4 and let-7 are also both synthesized as longer precursors that , in silico , can be folded into stem - loop structures ( fig . processing into the mature forms involves the rnase iii like protein dicer ( dcr-1 in c. elegans ) as well as the ago1/rde-1 family members agl-1 and agl-2 ( grishok et al . , 2001 ; hutvagner et al . , 2001 ; ketting et al . , 2001 ) . this is another notable parallel to rnai , where sirna biogenesis likewise requires dicer and distinct members of the ago1/rde-1 family . since functional homologues of lin-4 were only found in related caenorhabiditis species ( lee et al . , 1993 ) , an open question after the initial identification of lin-4 almost a decade ago was whether strnas are unique to nematodes or more widely used . the latter idea proved correct upon the identification of a second c. elegans strna , let-7 ( reinhart et al . , 2000 ) . perfect homologues of let-7 were found in various other organisms including the fly , zebrafish , and human and in some of these organisms , stage - specific expression of let-7 was observed ( pasquinelli et al . , 2000 ) . in addition , putative orthologues of let-7 's major target , lin-41 , were identified in vertebrates and invertebrates ( slack et al . , 2000 ) . together , these data suggested that a posttranscriptional regulatory mechanism of gene expression might operate in a wide variety of other organisms besides c. elegans . this view has now received strong support through the identification of some 90 small , 22 nt long rnas from c. elegans , drosophila , and human cell cultures ( table i ) , nine of which are found in more than one phylum ( table ii , lagos - quintana et al . , 2001 ; lau et al . , 2001 ; lee and ambros , 2001 ) . to identify novel small rnas , all three groups used a biochemical approach based on purification of rnas after size selection ( lagos - quintana et al . , 2001 ; lau et al . , 2001 ; lee and ambros , 2001 ) or a bioinformatic approach ( lee and ambros , 2001 ) centering on the conservation of intergenic regions of dna between two related caenorhabditis species . the expression of 2/3 of the thus identified rnas was verified by northern blot experiments ( table i ) . 99 unique sequences from the three organisms account for 91 novel mirnas after correction for putative orthologues . numbers in brackets give the numbers of mirnas for which a transcript of 22 nt was detected in this analysis . letters in parentheses indicate variations in otherwise identical mirnas from different organisms or variant genes within one organism . several lines of evidence suggest that these rnas are indeed mirnas and not degradation products of longer rnas or sirnas . first , all mirnas are encoded in regions of the genome for which no genes had been predicted so far , although the biochemical protocols would be expected to yield a rather unbiased sample of rnas of the appropriate size . second , when genomic sequences surrounding the identified 22-nt rnas were examined , computer analysis predicted mirna precursors capable of forming stem - loop structures similar to those of the lin-4 and let-7 precursors ( fig finally , in two cases where this was examined , an accumulation of these precursors could be detected in a dcr-1 mutant , similar to the previously reported accumulation of pre lin-4 and pre , these data thus strongly suggest that lin-4 and let-7 are just two of a vast group of mirnas . ( 2001 ) used a biochemical procedure that was tailored to identify dicer products , i.e. , sirnas as well as strnas . given further that size was the critical determinant used for selective rna cloning by the other two groups ( lagos - quintana et al . , 2001 ; lee and ambros , 2001 ) , it comes as a surprise that not a single sirna was recovered . mirnas rather than sirnas might , under unstressed conditions , thus be the prevalent agent of small rna - mediated regulation of gene expression . the small rna processing machinery centered on dicer might then , likewise , principally serve mirna biogenesis . lending credence to this claim is the finding that dcr-1 mutants display heterochronic developmental defects and fertility defects ( grishok et al . , 2001 ; ketting et al . , 2001 ; knight and bass , 2001 ) . computer analysis suggested that some of the mirnas were highly conserved in other organisms . in agreement with this idea , one mirna was identified by all three groups independently , revealing its expression in drosophila , c. elegans , and humans ( lagos - quintana et al . , 2001 ; lau et al . , 2001 ; lee and ambros , 2001 ) , and > 2/3 of the mirnas identified from human hela cells were found to be expressed in at least one other vertebrate species ( lagos - quintana et al . , 2001 ) . in addition , most of the mirnas identified in c. elegans were also found in a related nematode species , c. briggsae and , occasionally , in other , more distantly related species including man ( lau et al . they might be ; after all , there is both precedence and room for function of novel mirnas as strnas . both lin-4 and let-7 are essential components of the c. elegans heterochronic pathway , with lin-4 controlling transition from the first ( designated l1 ) to the second ( l2 ) larval stage ( chalfie et al . , 1981 ; lee et al . , 1993 ) and let-7 having a similar role in the l4 to young adult transition ( reinhart et al . , 2000 ) . no such switches have yet been identified for the l2-to - l3 and l3-to - l4 transitions , respectively ; by analogy , leaving some attractive space for more strna function . moreover , although the heterochronic pathway is well understood as far as larval development is concerned , little is known about embryonic development . intriguingly , expression data suggest that many of the mirnas are highly expressed in embryos with levels dropping after hatching ( lau et al . , 2001 ) . however , the sheer number of mirnas identified make it unlikely that they function exclusively in developmental timing . also , although northern blot analysis revealed some differences in mirna expression levels when comparing embryos to adult animals , only a few mirnas yielded signs of distinct up- or downregulation upon transition from one developmental stage to the next ( lagos - quintana et al . , 2001 ; lau et al . , 2001 ) , a hallmark of lin-4 and let-7 ( feinbaum and ambros , 1999 ; reinhart et al . , 2000 ) . if not developmental timing , what then ? as noted earlier , the similarities between the control of spatial and temporal development are striking ( slack and ruvkun , 1997 ; see also banerjee and slack , 2002 ) . in a particularly revealing example , hunchback and krppel , known for their roles in spatial patterning , were also found to define temporal identities of neuroblasts in the drosophila central nervous system ( isshiki et al . , 2001 ) . it is thus conceivable that mirnas might regulate genes involved in spatial organization of development and fertility . although it may at first seem unlikely that key molecules in the well - understood pathway of spatial patterning may have been missed , one should consider the small size of the strnas that makes them poor targets in forward genetic screens . given the large number of mirnas and we predict that the screens have not yet been saturated even more specialized functions are conceivable . clearly , an important next step will be the identification of targets of all these small rnas through genetical and bioinformatics approaches . the abundance of stem - loop structures in pre - mirnas , as well as the significant conservation of these structures in multiple organisms where homologous mirnas were found ( lagos - quintana et al . , 2001 ; lau et al . , 2001 ; lee and ambros , 2001 ) , suggests that these structures might be a prerequisite for mirna maturation and possibly for function . one attractive hypothesis is that transcription as a precursor might allow another level of regulation of mirna activity . indeed , in one case northern blot analysis suggested that the pre - mirna might be continuously transcribed throughout development , but that maturation into the 22-nt form occurs only at distinct stages of development ( lau et al . , 2001 ) . this seems also to be true of let-7 in sea urchin development ( pasquinelli et al . , 2000 ) . the stem - loop structure of the pre - mirna in combination with its size ( 70 nt ) also brings to mind another group of small rnas , trnas . it is tempting to speculate that the structured pre - mirna may actually be the substrate for nuclear export in which the dedicated trna nuclear export factor , xpo - t ( for review see grosshans et al . , 2000 ) , might thus be involved through specific recognition of the pre - mirna . processing of the pre - mirna through dicer might then occur in the cytoplasm with subsequent targeting of the mature mirna to its site of function . given what we know about the intricate coupling of maturation steps in other rna biogenesis pathways , e.g. , those that involve mrna or trna ( wolin and matera , 1999 ; grosshans et al . , 2000 ; reed and magni , 2001 ) , we speculate that biogenesis of mirnas might indeed occur in a highly coordinated channeling pathway ( fig . 2 ) . nascent or freshly transcribed pre - mirna might be recognized by the export machinery and , following nuclear export , be handed over to the processing machinery in the cytoplasm . proteins that participate in the processing might also be involved in the delivery of the mature mirnas to their sites of action so that mirnas are handed over from one biogenesis factor to the next without intermittent release . although no direct evidence is currently available to support this model , it would well explain why mirnas appear to be surprisingly stable . it would also allow for several levels of regulation a feature one would anticipate for a regulatory pathway . the future identification of additional components of this pathway , as well as the subcellular localization of the known ones ( such as dicer ) , will help to establish the validity of such a model . the model ilustrates important stages in the life cycle of a mirna , all of which might be regulated . ( 1 ) mirnas are transcribed as precursors by an , as yet , unidentified rna polymerase ( pol ) . ( 2 ) following its export to the cytoplasm by a likewise unidentified factor the precursor is processed ( 3 ) into the mature mirna . ( however , it should be noted that the site of pre - mirna processing is currently unknown so that it is also possible that processing occurs in the nucleus and the mature mirna is the substrate for nuclear export . ) pre - mirna processing involves dicer and , presumably , one or more cofactors such as agl-1/agl-2 . these factors might then also be involved in delivery of the mirna to its site of action , i.e. , its target mrna . ( 4 ) binding of the mirna to the mrna 3 utr inhibits translation presumably at a point after translation initiation . ( however , the nascent polypeptide chain ( purple ) exiting the ribosome in this model is solely for illustrative purposes , the presence of proteins stalled in elongation has not been demonstrated . ) downregulation of translation is thought to involve additional cofactors that presumably recognize the bulged - out nucleotides of the mrna / strna duplexes . one mystery of mirna biogenesis has been further complicated by the new data : how does dicer recognize which side of the precursor molecule it has to excise ? although production of sirnas by dicer results in symmetric duplex structures , only one stem of the strna precursors remains stable ( fig . the finding that with both lin-4 and let-7 the 5 arm of the duplex survives suggested that the loop 3 of the mature strna might be part of the solution . however , the majority of the newly identified mature mirnas are derived from the 3 arm of the stem - loop ( table i ) , and in one case mature mirnas have even be identified from both arms . it is likely that accessory factors tell the dicer machinery which arm of the stem - loop is to be preserved . mutations in two of these genes , agl-1 and agl-2 , have been shown to cause an accumulation of pre lin-4 similar to the phenotypes observed for mutants lacking dicer , whereas prelet-7 levels remained unaffected ( grishok et al . , 2001 ) . it would therefore be highly interesting to see whether other pre - mirna , in particular those where the mature mirna is encoded on the 3 arm , would accumulate in these mutants or in mutants of other members of the ago1/rde-1 family . the large number of mirnas suggests that these rnas might constitute an abundant component of the gene regulatory machinery . although the first two such rnas were discovered through their role in developmental timing , the sheer abundance of novel mirnas as well as , in many cases , their apparent lack of developmental regulation suggest that mirnas might be involved in other pathways as well . clearly , we are only at the beginning of our exploration of mirna versatility and function and many question marks remain regarding both mirna biogenesis and function ( fig . 2 ) . what are the functions and targets of all these mirnas ? do all mirnas function through a similar mechanism , and if so , what exactly is this mechanism the abundance and conservation of mirnas in animal genomes suggests that we have just begun to glimpse an important and so far underappreciated mechanism of gene regulation .

forty adolescent girls ( aged 1218 years ) with diabetes ( 20 with type 1 diabetes and 20 with type 2 diabetes ) were identified from our hospital adolescent diabetes database and invited to participate : 11 patients with type 1 and 8 patients with type 2 diabetes agreed to participate . twenty healthy , nondiabetic girls were recruited from the schools of the diabetic participants and formed two control subject groups : lean and overweight / obese based on age- and sex - specific bmi equivalents ( 13 ) . no participants had diabetes complications , coexisting cardiovascular disease , or a history of diabetic ketoacidosis in the past 12 months . participants with type 2 diabetes were not receiving insulin therapy : in all patients , diabetes was treated either by diet alone and/or by metformin . all nondiabetic participants had a1c levels < 6% and fasting glucose < 5.5 mmol and were pubertal stage 4 or 5 and postmenarchal . all participants had reported low physical fitness ( <3 h / week of moderate / intense physical activity ) . this study was approved by the auckland northern regional ethics committee , and all participants provided written consent before participating ; those aged < 16 years required parental consent to participate in the study . a fasting venous blood sample was collected for determination of a1c and fasting blood glucose ( fbg ) . during the same visit , body composition and echocardiography body surface area ( bsa ) was calculated using the dubois formula ; bmi was calculated as weight in kilograms divided by the square of height in meters . dual - photon x - ray absorptiometry ( ge lunar prodigy ; ge healthcare , madison , wi ) was performed to determine body composition including fat - free mass ( ffm ) , fat mass , bone mineral content , and total weight using standard analytical software . all images were digitally acquired ( philips ie33 or philips hdi-5000 ; phillips medical systems , bothell , wa ) according to a standardized protocol by trained research sonographers and measured off - line ( digiview ; digisonics , houston , tx ) without knowledge of the patients ' clinical details or group allocation . a comprehensive research echocardiogram was performed , including standard transthoracic views , as well as protocol - specific measurements ( below ) . images were obtained in the parasternal short - axis view at the midpapillary muscle level and left ventricular mass was calculated using the area - length method recommended by the american society of echocardiography ( 14 ) . left ventricular mass mitral valve inflow pulsed - wave doppler recordings were made between the leaflet tips , isovolumic relaxation time ( ivrt ) recordings were made from the left ventricular outflow tract , and tissue doppler imaging recordings were obtained from the medial and lateral aspects of the mitral annulus in the apical four - chamber view . all doppler signals were acquired using a 5-mm sample volume , optimized , and recorded at 100 mm / s sweep speed . measurements were the following : 1 ) m - mode measurements : left ventricular end - diastolic dimension and left ventricular end - systolic dimension ; 2 ) two - dimensional images : left atrial area ; 3 ) doppler measurements : mitral valve early filling velocity ( e ) , late - filling velocity ( a ) , mitral e wave deceleration time , and ivrt , and 4 ) tissue doppler measurements ( average of lateral and medial ) : mitral annular e velocity ( ea ) , mitral annular a velocity ( aa ) , and mitral annular s velocity ( sa ) . the following variables were calculated : e - to - a ratio , left ventricular ejection fraction , and e - to - ea ratio ( average ) . comparisons between groups for continuous normally distributed variables were performed using student 's t test or anova for more than two groups . nonparametric continuous data were analyzed using wilcoxon and kruskall - wallis tests , where appropriate . body surface area ( bsa ) was calculated using the dubois formula ; bmi was calculated as weight in kilograms divided by the square of height in meters . dual - photon x - ray absorptiometry ( ge lunar prodigy ; ge healthcare , madison , wi ) was performed to determine body composition including fat - free mass ( ffm ) , fat mass , bone mineral content , and total weight using standard analytical software . all images were digitally acquired ( philips ie33 or philips hdi-5000 ; phillips medical systems , bothell , wa ) according to a standardized protocol by trained research sonographers and measured off - line ( digiview ; digisonics , houston , tx ) without knowledge of the patients ' clinical details or group allocation . a comprehensive research echocardiogram was performed , including standard transthoracic views , as well as protocol - specific measurements ( below ) . images were obtained in the parasternal short - axis view at the midpapillary muscle level and left ventricular mass was calculated using the area - length method recommended by the american society of echocardiography ( 14 ) . left ventricular mass mitral valve inflow pulsed - wave doppler recordings were made between the leaflet tips , isovolumic relaxation time ( ivrt ) recordings were made from the left ventricular outflow tract , and tissue doppler imaging recordings were obtained from the medial and lateral aspects of the mitral annulus in the apical four - chamber view . all doppler signals were acquired using a 5-mm sample volume , optimized , and recorded at 100 mm / s sweep speed . measurements were the following : 1 ) m - mode measurements : left ventricular end - diastolic dimension and left ventricular end - systolic dimension ; 2 ) two - dimensional images : left atrial area ; 3 ) doppler measurements : mitral valve early filling velocity ( e ) , late - filling velocity ( a ) , mitral e wave deceleration time , and ivrt , and 4 ) tissue doppler measurements ( average of lateral and medial ) : mitral annular e velocity ( ea ) , mitral annular a velocity ( aa ) , and mitral annular s velocity ( sa ) . the following variables were calculated : e - to - a ratio , left ventricular ejection fraction , and e - to - ea ratio ( average ) . comparisons between groups for continuous normally distributed variables were performed using student 's t test or anova for more than two groups . nonparametric continuous data were analyzed using wilcoxon and kruskall - wallis tests , where appropriate . thirty - nine girls completed all assessments : 11 with type 1 diabetes , 8 with type 2 diabetes , 9 lean control subjects , and 11 overweight / obese control subjects . age and height were similar , but significant differences were seen across the groups in weight , bsa , bmi , and percent body fat ( table 1 ) . weight , bsa , and bmi were highest in the type 2 diabetic group . no significant differences in body size were observed between the type 1 diabetic group and lean control subjects . percent body fat was highest in the type 2 diabetic group and the overweight control subjects but not different between them . although the type 1 diabetic group exhibited significantly lower percent body fat , it remained higher than that for the lean control subjects ( p < 0.05 ) . duration of diabetes was longer in the type 1 diabetic group ( p = 0.004 ) . both fbg and a1c were significantly elevated in the two diabetic groups , with significantly higher fbg in the type 1 diabetic group ( p < 0.05 ) , but no difference in a1c was seen between the two diabetic groups nor were any echocardiographic measures correlated with a1c . baseline demographics and body composition of the four groups values are means sd or median ( interquartile range ) . the type 2 diabetic group had the largest hearts ( left ventricular diameter , left ventricular mass , and la area ) ( table 2 ) . the largest left ventricular diameters were observed in the type 2 diabetic group : significant differences in end - diastolic left ventricular dimension were observed between the type 2 diabetic groups and both the type 1 diabetic group and the lean control subjects ( both p < 0.05 ) . left atrial size was also higher in the type 2 diabetic group and overweight control subjects . the highest left ventricular mass occurred in the type 2 diabetic group ( p < 0.001 ) ; this was significantly different from that in both the lean control subjects and type 1 diabetic group ( both p < 0.05 ) but similar to that in the overweight control subjects . these relationships persisted when left ventricular mass was indexed to height ( p = 0.02 ) but not when left ventricular mass was indexed to ffm ( p = 0.54 ) ( fig . echocardiographic measurements of heart size values are means sd or n ( % ) . lv , left ventricular ; lvedd , left ventricular end - diastolic dimension ; lvesd , left ventricular end - systolic dimension . left ventricular ( lv ) mass by group : lv mass ( a ) , lv mass / height ( b ) , and lv mass / ffm ( c).t1 dm , type 1 diabetes ; t2 dm , type 2 diabetes . specifically , there was a decline in the e - to - a ratio across the groups , with dominant late filling in both diabetic groups , a pattern of diastolic filling usually seen in older individuals . this decline was driven primarily by significant differences in late filling ( mitral a velocity ) ( table 2 ) . these abnormalities in diastolic function were confirmed by prolonged isovolumic relaxation in all groups , except the lean control subjects ( p = 0.04 ) , and a trend toward reduced left ventricular longitudinal relaxation ( ea velocity ) , ( p = 0.057 ) . left ventricular filling pressure ( estimated from the e - to - ea ratio ) was different across the groups : it was significantly higher in the type 2 diabetic group ( all p < 0.05 ) . longitudinal left ventricular systolic function ( measured by longitudinal annular motion [ sa ] ) was lowest in the type 2 diabetic group , different across the groups , and different between the two diabetic groups ( p < 0.05 ) . in summary , tissue doppler velocities indicated lower diastolic and systolic longitudinal myocardial motion and higher left ventricular filling pressure in the type 2 diabetic group ( fig . average ea velocity ( a ) , average sa ( b ) , and average e : ea ( c ) . t1 dm , type 1 diabetes ; t2 dm , type 2 diabetes ; tdi , tissue doppler imaging . compared with published normal values for adult women ( 14 ) , 63% of the type 2 diabetic group met the criteria for left ventricular dilatation , compared with 11% of the lean control subjects and 27% of the overweight control subjects ( table 2 ) ; 75% met the published criteria for lvh , compared with 11% of the lean control subjects and 36% of the overweight control subjects ; 38% had larger left atrial areas , compared with 9% of the type 1 diabetic group and 27% of the overweight control subjects ; and 50% fell outside the range of normal left ventricular filling pressure ( e - to - ea ratio 9 cm / s ) ( 15 ) , of whom three of four subjects had dilated left ventricles and dilated left atria and met the criteria for lvh . importantly , just one type 2 diabetic subject had no abnormalities detected and one subject had a single abnormality present . half of the overweight control subjects had a single abnormality detected , and in all subjects this was lvh . nearly all of the type 1 diabetic and lean control subjects had no abnormalities detected . the type 2 diabetic group had the largest hearts ( left ventricular diameter , left ventricular mass , and la area ) ( table 2 ) . the largest left ventricular diameters were observed in the type 2 diabetic group : significant differences in end - diastolic left ventricular dimension were observed between the type 2 diabetic groups and both the type 1 diabetic group and the lean control subjects ( both p < 0.05 ) . left atrial size was also higher in the type 2 diabetic group and overweight control subjects . the highest left ventricular mass occurred in the type 2 diabetic group ( p < 0.001 ) ; this was significantly different from that in both the lean control subjects and type 1 diabetic group ( both p < 0.05 ) but similar to that in the overweight control subjects . these relationships persisted when left ventricular mass was indexed to height ( p = 0.02 ) but not when left ventricular mass was indexed to ffm ( p = 0.54 ) ( fig . 1 ) . echocardiographic measurements of heart size values are means sd or n ( % ) . lv , left ventricular ; lvedd , left ventricular end - diastolic dimension ; lvesd , left ventricular end - systolic dimension . left ventricular ( lv ) mass by group : lv mass ( a ) , lv mass / height ( b ) , and lv mass / ffm ( c).t1 dm , type 1 diabetes ; t2 dm , type 2 diabetes . the type 2 diabetic group demonstrated differences in diastolic filling and systolic function . specifically , there was a decline in the e - to - a ratio across the groups , with dominant late filling in both diabetic groups , a pattern of diastolic filling usually seen in older individuals . this decline was driven primarily by significant differences in late filling ( mitral a velocity ) ( table 2 ) . these abnormalities in diastolic function were confirmed by prolonged isovolumic relaxation in all groups , except the lean control subjects ( p = 0.04 ) , and a trend toward reduced left ventricular longitudinal relaxation ( ea velocity ) , ( p = 0.057 ) . left ventricular filling pressure ( estimated from the e - to - ea ratio ) was different across the groups : it was significantly higher in the type 2 diabetic group ( all p < 0.05 ) . longitudinal left ventricular systolic function ( measured by longitudinal annular motion [ sa ] ) was lowest in the type 2 diabetic group , different across the groups , and different between the two diabetic groups ( p < 0.05 ) . in summary , tissue doppler velocities indicated lower diastolic and systolic longitudinal myocardial motion and higher left ventricular filling pressure in the type 2 diabetic group ( fig . average ea velocity ( a ) , average sa ( b ) , and average e : ea ( c ) . t1 dm , type 1 diabetes ; t2 dm , type 2 diabetes ; tdi , tissue doppler imaging . compared with published normal values for adult women ( 14 ) , 63% of the type 2 diabetic group met the criteria for left ventricular dilatation , compared with 11% of the lean control subjects and 27% of the overweight control subjects ( table 2 ) ; 75% met the published criteria for lvh , compared with 11% of the lean control subjects and 36% of the overweight control subjects ; 38% had larger left atrial areas , compared with 9% of the type 1 diabetic group and 27% of the overweight control subjects ; and 50% fell outside the range of normal left ventricular filling pressure ( e - to - ea ratio 9 cm / s ) ( 15 ) , of whom three of four subjects had dilated left ventricles and dilated left atria and met the criteria for lvh . importantly , just one type 2 diabetic subject had no abnormalities detected and one subject had a single abnormality present . half of the overweight control subjects had a single abnormality detected , and in all subjects this was lvh . nearly all of the type 1 diabetic and lean control subjects had no abnormalities detected . this study is the first to demonstrate that important cardiac abnormalities occur early in adolescent - onset type 2 diabetes and highlights the potential for higher cardiovascular risk . by comparing type 2 diabetic girls with age - matched girls with type 1 diabetes and lean and overweight control subjects , we found that obesity was associated with increased cardiac dimensions and higher left ventricular mass , both of which were further augmented by the addition of type 2 diabetes . furthermore , in the type 2 diabetic group there was evidence of impaired diastolic and systolic function , increased left ventricular filling pressure , and larger left atrial volume , a marker of long - standing diastolic disease . usually a disease of older patients , type 2 diabetes often coexists with multiple other cardiovascular risk factors , including hyperlipidemia , hypertension , and obesity and is associated with poor cardiovascular outcome ( 3 ) . lvh , one of the first signs of diabetic cardiomyopathy , may eventually progress to left ventricular dysfunction and clinical heart failure ( 16 ) and is an independent risk factor for cardiovascular morbidity and mortality ( 17 ) . hypertrophy of cardiac myocytes occurs in response to increased load , leading to increased left ventricular wall thickness and mass . adolescent obesity has been previously associated with increased left ventricular volumes and mass ( 11 ) , but obesity is also associated with diastolic dysfunction ( 18 ) and may independently lead to heart failure ( 19 ) . although left ventricular mass was similar between the type 2 diabetic group and the overweight control subjects and different from that of the lean and type 1 diabetic control groups , left ventricular dilatation and functional differences were only seen in the type 2 diabetic group , suggesting that obesity may be an important factor contributing to structural changes in the heart and that the presence of diabetes augments these changes . the overweight control subjects in our study displayed important left ventricular structural changes , comparable with those of the type 2 diabetic subjects : both had higher left ventricular mass and this remained true when left ventricular mass was indexed to height . because left ventricular mass is related to body composition , in particular ffm ( 20 ) , the higher left ventricular mass in these groups might simply reflect normal physiological growth in response to higher ffm . although ffm may be the ideal indexing variable for left ventricular mass ( 20 ) , few data exist regarding the relationship between ffm and lvh in overweight and obese subjects . indeed , when left ventricular mass was indexed to ffm , no differences were detected among the groups in our study . however , the functional changes observed in the type 2 diabetic group suggest that the higher lv mass observed may be abnormal . elevated left ventricular mass leads to alterations in both myocardial relaxation and passive filling of the left ventricle ( 16 ) , and adults with type 2 diabetes exhibit a high prevalence of diastolic dysfunction ( 8) as do patients with type 1 diabetes ( 21 ) . in the current study , a dependence upon active rather than passive diastolic filling was observed in both diabetic groups , despite the shorter duration of diabetes in the type 2 diabetic group . we hypothesized that this finding in type 2 diabetic participants reflects a longer exposure to obesity and other cardiovascular risk factors rather than diabetes per se . obesity is related to hypertension as well as to diabetes , and it is difficult to measure exposure to either in retrospect , perhaps explaining the lack of association between duration of diabetes and cardiac adaptation . this is the first study to document the direct structural and functional effects of type 2 diabetes on the heart . obesity in adolescents and young adults has been linked with hypertension ( 22 ) and cardiovascular disease ( 23 ) in later life and there are some data about the direct cardiovascular effects of obesity : in a study of young women ( mean age 30 years ) , obesity ( average duration 12 years ) was associated with higher left ventricular mass and diastolic and systolic abnormalities similar to those seen in the type 2 diabetic group in the current study ( 12 ) . our subjects were younger with shorter exposure to obesity and yet displayed comparable structural and functional changes . adolescent - onset type 2 diabetes is a relatively new disease and increasingly prevalent ; thus , long - term data are limited and the prognostic implications of the abnormalities detected in this study are unknown . however , many of these parameters predict cardiovascular outcome in other population cohorts , and these adverse changes may be the precursor of future cardiovascular disease and are unlikely to reverse without significant intervention . in fact , these adolescents may already be progressing toward development of the clinical syndrome of heart failure . recent american college of cardiology / american heart association heart failure guidelines ( 24 ) describe heart failure as a continuum that begins with risk factors , including diabetes and hypertension ( stage a ) , followed by the development of structural heart disease ( stage b ) and eventually leading to the clinical syndrome of heart failure ( stages c and d ) . because the diagnosis of type 2 diabetes is a recognized contributor to stage a heart failure , the additional findings of structural and functional abnormalities ( lvh and diastolic and systolic dysfunction ) suggest that some of these girls could and should be considered to have stage b heart failure already . substantial data exist to support the use of antihypertensive therapy ( especially ace inhibitors and angiotensin receptor blockers ) for lowering blood pressure and regressing lvh , but many therapies are not registered for use nor proven in children . significant weight reduction , such as might be achieved through bariatric surgery or severe caloric restriction , may result in regression of left ventricular mass and reversal of functional ( 10 ) and cardiovascular changes ( 23 ) . furthermore , physical exercise is recommended by the american diabetes association to delay the onset of cardiovascular disease in type 2 diabetes . thus , a strategy to promote exercise and sustain weight loss may reverse this premature myocardial aging . we found large and clinically significant differences among the groups and a high proportion of subjects with echocardiographic measurements that fell outside of the normal range using adult female normal values . we used adult normal values to ensure that we did not overdiagnose abnormalities by using adolescent normal ranges . if anything , the use of adolescent normal values would have increased the number of subjects with abnormal echocardiographic measurements of left ventricular size and hypertrophy . the diabetic subjects in this study were not matched for duration of diabetes , nor was it possible to match them by a1c , although both groups had elevated a1c . it is possible that the abnormalities detected in this study may be related to both disease duration and glycemic control , as well as to other cardiovascular risk factors ; however , it was not possible to determine predictors of these abnormalities . the results of this small study require confirmation in larger cohorts with longitudinal data to determine whether these abnormalities progress . in summary , to our knowledge this is the first study to report potentially important left ventricular structural and functional changes associated with adolescent - onset type 2 diabetes . compared with age - matched control subjects , girls with type 2 diabetes exhibited significant lvh and a high prevalence of abnormal echocardiographic measurements . if left unchecked , it is likely that these changes will lead to the development of clinically overt cardiovascular disease . we found large and clinically significant differences among the groups and a high proportion of subjects with echocardiographic measurements that fell outside of the normal range using adult female normal values . we used adult normal values to ensure that we did not overdiagnose abnormalities by using adolescent normal ranges . if anything , the use of adolescent normal values would have increased the number of subjects with abnormal echocardiographic measurements of left ventricular size and hypertrophy . the diabetic subjects in this study were not matched for duration of diabetes , nor was it possible to match them by a1c , although both groups had elevated a1c . it is possible that the abnormalities detected in this study may be related to both disease duration and glycemic control , as well as to other cardiovascular risk factors ; however , it was not possible to determine predictors of these abnormalities . the results of this small study require confirmation in larger cohorts with longitudinal data to determine whether these abnormalities progress . in summary , to our knowledge this is the first study to report potentially important left ventricular structural and functional changes associated with adolescent - onset type 2 diabetes . compared with age - matched control subjects , girls with type 2 diabetes exhibited significant lvh and a high prevalence of abnormal echocardiographic measurements . if left unchecked , it is likely that these changes will lead to the development of clinically overt cardiovascular disease .

veterinary drugs are widely used in medical and veterinary practices to treat and prevent disease as well as improve feed efficiency and increase animal growth rates . pesticides are also widely used to enhance food production by protecting food crops from potentially harmful and destructive pests . however , the resulting occurrence of contaminants and/or residues in the human diet represents an issue of high concern . according to the european union , the maximum residue limit ( mrl ) in dairy milk is 100 g / kg for tetracycline and sulfenamide , 50 g / kg for macrolides and quinolones , and 10 g / kg for pesticides . sensitive analytic methods have been developed to monitor and detect the mrl values in the dairy milk . there are ultra - high pressure liquid chromatography mass spectrometry ( uhplc - ms / ms ) methods reported to detect multiple residues of -lactams [ 4 , 5 ] , as well as pesticides and mycotoxins , and some antihelminthic drugs and phenylbutazone . milk is a complex food that is high in fat and protein , and such ingredients may cause interactions in the analytical processes . formerly , sample preparation methods were based on a few compounds or a single class of such drugs . applying common extraction procedures and developing chromatographic conditions are difficult in multiclass and multiresidue analyses . solid phase extraction methods have been applied , after the phases of protein precipitation and centrifugation , in order to observe the fluoroquinolones , veterinary drugs , mycotoxins , and pesticides in milk [ 6 , 10 ] . however , these methods are generally found to be time - consuming and require large volumes of organic solvents . multiresidue veterinary drugs that were developed for milk tests depend on various extraction and cleanup principles . one of the most accepted approaches is to dilute a sample of milk with a solvent like acetonitrile and then to centrifuge and evaporate the obtained supernatant organic extract [ 11 , 12 ] . some multiclass analytical method applications by lc - ms / ms or lc - tof / ms , related to homogenized or raw milk , that have the ability to specify undesirable chemicals , such as tetracycline , quinolone , sulfonamide , peptide , hormone , nonsteroidal anti - inflammatory anthelmintic drugs , mycotoxin , and pesticides , can be found in the literature . yet most of these methods are unable to offer satisfactory recovery of a large range of compounds of different polarities [ 13 , 14 ] . most methods for the analysis of veterinary residues have some disadvantages , including high solvent consumption , tedious spe cleanup steps that require extended time for analysis , and high costs . the quick easy cheap effective rugged safe ( quechers ) methodology , which was originally developed for pesticide analysis , has recently been proposed for the analysis of veterinary drugs using different matrices [ 1518 ] . however , quechers was found to be inconvenient for the recovery of polar veterinary drugs , including penicillin , tetracycline , and quinolone [ 13 , 18 , 19 ] . therefore , there is still a great need for simple and rapid multiresidue analytical methods for simultaneously determining veterinary drug and pesticide residues in milk . in this study , we prepared milk samples by using a procedure based on a simple liquid - to - liquid extraction . this method utilized a simple and quick sample preparation procedure using a single extraction step . through this method , milk samples were analyzed for the determination of both veterinary drugs and pesticide residues by utilizing liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . as a result , the reduced use of chemicals and steps in the sample preparation phase , together with the avoidance of a sample cleanup step , simplified the sample pretreatment and reduced the overall total cost . finally , in addition to reducing analyses costs , the method provided a higher recovery of compounds of various polarities and improved the simplicity of detection efforts . hplc grade acetonitrile ( acn ) , methanol , ethyl acetate ( etoac ) ( lichrosolv , purity 99.9 ) , and glacial acetic acid ( emprove , 100% ) were purchased from merck ( darmstadt , germany ) . the water used to prepare the solutions was purified in a milli - q plus system ( emd millipore , billerica , ma ) . magnesium sulfate , sodium chloride , supelclean primary secondary amine ( psa ) , pure tetracyclines , sulfonamides , quinolones , macrolides , and antibiotics were provided from sigma aldrich ( st . all pasteurized whole milk samples were purchased from local markets . also , raw milk was used for interference and specificity / selectivity as a blank . individual stock solutions of the veterinary drugs and pesticides were prepared in acetonitrile at a concentration of 1000 mg / kg . a mixed intermediate standard solution was prepared by diluting the stock standard solutions of the veterinary drugs and pesticides in acetonitrile at a concentration of 10 mg / kg . stock and intermediate standard solutions were stored at 4c in amber flasks and were found stable for at least 6 months . milk samples , upon arrival at our laboratory , were kept at refrigerator temperature ( 10 4c ) until analysis . for the preparation an aliquot of approximately 5 ml milk sample was pipetted in a 50 ml polypropylene centrifuge tube . then , 200 mcl acetic acid was added to 10 ml of ethyl acetate . after vortex for 3 minutes , the mixture was centrifuged at 5000 rpm for 10 minutes . the upper phase was taken in 15 ml centrifuge tube and was dried under a gentle stream of nitrogen , and the residue was reconstituted with 1000 mcl of mobile phase a / mobile phase b ( 80/20 ) . the extract was filtered through a 0.45 m filter prior to lc - ms / ms analysis . approximately 5 ml milk sample was pipetted in a 50 ml polypropylene centrifuge tube . then , 10 ml of acetonitrile and 200 mcl acetic acid were added to milk . after mixing by a vortex stirrer for 3 minutes , the upper phase was taken in 15 ml centrifuge tube and was dried under a gentle stream of nitrogen , and the residue was reconstituted with 1000 mcl of mobile phase a / mobile phase b ( 80/20 ) . the extract was filtered through a 0.45 m filter prior to lc - ms / ms analysis . approximately 5 ml milk sample was pipetted in a 50 ml polypropylene centrifuge tube . then , 2 g of magnesium sulfate and 1 g of sodium acetate were added to milk samples . then , 10 ml of acetonitrile and 100 mcl acetic acid were added to milk samples . after vortex for 3 minutes , the mixture was centrifuged at 5000 rpm for 10 minutes . the upper phase was taken in 15 ml centrifuge tube and was dried under a gentle stream of nitrogen , and the residue was reconstituted with 1000 mcl of mobile phase a / mobile phase b ( 80/20 ) . the extract was transferred to a 2 ml eppendorf microtube containing 50 mg psa and 200 mg magnesium sulfate . the extract was filtered through a 0.45 m filter prior to lc - ms / ms analysis . the chromatographic analyses were performed using an hplc system consisting of a binary pump ( shimadzu uflc lc-20ad model ) , shimadzu automatic injector ( autosampler sil-20a ht model ) , and a column oven ( cto-20ac ) . analytical columns , symmetry c18 2.1 150 mm i d , 5 m particle size ( waters , milford , ma ) , and waters xterra c18 150 mm 2.1 mm i d , 5 m particle size ( waters , milford , ma ) , were tested . chromatographic separation of veterinary drugs and pesticides was carried out on a waters symmetry c18 column . the method used a gradient mobile phase containing 0.1% acetic acid water and mobile phase b containing methanol . the column temperature was maintained at 40c with a flow rate of 0.3 ml / min . the gradient profile was scheduled as follows : initial proportion ( 98% a and 2% b ) for 0.3 minutes , linear increase to 80% ( b ) until 7 minutes , and hold of 80% ( b ) for 3 minutes . the chromatographic system was coupled to electrospray ionization ( esi ) source followed by an applied biosystems mds sciex 4500 q trap mass spectrometer . the ms / ms detector conditions were as follows : curtain gas 20 ml / min , exit potential 10 v , ion source gas 1 and ion source gas 2 set at 50 ml / min , ion spray voltage 5500 v , and turbo spray temperature set at 550c . ms data were acquired in the positive ion esi mode using two alternating ms / ms scan events . the selected molecular ion and optimized collision voltages of product ions used for quantification , confirmation , and ion ratio were summarized in table 1 . applied biosystems sciex quantification was by comparison with a six - point calibration ( 0.0 , 0.01 , 0.025 , 0.05 , 0.1 , and 0.2 mg / kg ) in matrix - matched calibration . the analytical method developed for determination of veterinary drug and pesticide residues in milk was validated according to eu decision 2002/657/ec and sante/11945/2015 . the following parameters were evaluated in the validation procedure : selectivity , sensitivity , linearity , precision ( intraday and interday reproducibility ) , accuracy and cc and cc , lod , and loq . ethyl acetate extraction without salt procedure was chosen to be performed in this study because of its advantages . there was no need to use salt and it could give lower detection limit in terms of volatile characteristic of ethyl acetate . recovery values showed no difference among three different extraction procedures ( acetonitrile extraction , quechers extraction , and ethyl acetate extraction without salting procedure ) ( figure 1 ) . the recovery values expressed as recovery % are all within the reference range of 70120% . comparing three procedures , etoac without salt provided recoveries between 100% and 120% for a higher number of veterinary drugs and pesticides ( 26 veterinary drugs and 134 pesticides ; total of 160 compounds ) than quechers ( 82 compounds ) and acn ( 100 compounds ) , as it can be observed in figure 2 . in terms of extraction recoveries , etoac was found to be a suitable extraction procedure for all 26 veterinary drugs and most of the pesticides analyzed in this study . accuracy was evaluated in terms of relative standard deviation ( rsd ) by spiking blank samples with the corresponding volume of the multicompound working standard solution . rsd was evaluated at 50 g / kg by spiking six blank samples at each level for three procedures that provided similar rsd values . these values were within 1 < rsd < 10 for 75% of each analyte in the three procedures . these results indicated that the etoac without salt method was precise , accurate , and reliable for the analysis of the veterinary drug and pesticide compounds in the milk samples as an alternative method . formic acid in acetonitrile and water was used as a mobile phase in . according to analyte intensities , the dried residue was redissolved in a mixture of meoh / water with 0.1% acetic acid to test different reconstitution solvents . this composition produced better peak shapes for all analytes compared with water - methanol ( 80 : 20 ) that gave lower response . increasing acetic acid to 1% in the mixture did not improve chromatography but caused extra peaks in the background noise . the selectivity of the method was assessed by duplicate analysis of 10 blank milk samples . no peaks of interfering compounds were observed within the intervals of the retention time of the analytes in any of these samples . linearity was evaluated from the calibration curves by triplicate analyses of blank milk samples fortified with the analytes at six ( 0.0 , 0.01 , 0.025 , 0.05 , 0.1 , and 0.2 mg / kg ) concentration levels . linearity was expressed as the coefficient of linear correlation ( r ) and from the slope of the calibration curve . the linearity of the analytical response across the studied range was excellent , with correlation coefficients higher than 0.997 for all analytes , which was similar to the findings in . the authors found correlation coefficients higher than 0.992 for all analytes , which was a lower score than ours . cc is defined as the limit at and above which it can be concluded with an error probability of that a sample is noncompliant . cc is defined as the smallest content of the substance that may be detected , identified , and/or quantified in a sample with an error probability of . the cc and cc were determined by analysis of 10 blank milk samples and the signal - to - noise ( s / n ) ratio is calculated at the time window in which the analyte is expected . the cc was calculated by analyzing 10 blank samples spiked with concentration at cc. then the cc value was added up to 1.64 times the corresponding standard deviation . then , a preliminary experiment was conducted to check if all compounds were detected when spiked at their cc level ( table 2 ) . in figure 3 , the lowest loq value was 50 g / kg for tetracyclines and for sulfonamides 20 g / kg in veterinary drugs in while it was 10 g / kg for both of them in our study except ciprofloxacin and quinolone . figure 3 shows mrm chromatograms of milk samples at the lowest validation concentration at loq level . the accuracy was evaluated by recovery tests , analyzing fortified blank samples at the same concentration levels used in the precision tests ( 0.01 , 0.025 , and 0.05 mg / kg ) . the accuracy and precision of the method results ( table 2 ) confirmed the values given in decision 2002/657/ec . thus , the mean accuracy values obtained in the recovery tests were between 61 and 130% . the precision of the method was determined in two stages : repeatability ( intraday ) and intermediate precision ( interday ) . repeatability was expressed by the rsd of the results from six replicates analyzed on the same day by the same analyst using the same instrument . the intermediate precision was expressed by the rsd of the results of eighteen analyses performed on three different days ( n = 3 ) , six analyses / day , by the same analyst using the same instrument . the relative standard deviation ( rsd ) of interday values of veterinary drugs and pesticides analyzed by the present method was 2 to 13% and for the intraday test 519% ( table 2 ) , while relative standard deviation ( rsdr ) of intraday values was 426% in . evaluation of matrix effect is important during validation of analytical methods using the lc - ms / ms technique . the ionization efficiency of the analytes in esi source may be affected by matrix interference . in order to evaluate the degree of ion suppression or signal enhancement , matrix - induced effects were assessed by comparing the slopes of these calibration curves using the following formula : matrix effect ( me ) = 1 ( amatrix / astandard ) 100 , where amatrix and astandard are the slopes of calibration straight lines for standard and matrix - matched calibration graphs . the matrix - matched calibration curves were constructed using milk samples ( 5 g / ml matrix equivalent ) prepared in meoh - water solution with 0.1% acetic acid and spiked with veterinary drug and pesticides at concentration levels of 0.01 , 0.025 , and 0.05 mg / kg . matrix effect was further evaluated for ion suppression between the standards prepared in pure solvent and standards prepared in matrix and the matrix effect was found to be in a range of 1525% . these results showed that standard calibration which was simpler and less time - consuming compared with matrix - matched calibration can effectively be used for quantitation of veterinary drug and pesticides in milk ( table 2 ) . the method used analyzed more than 220 milk samples submitted to the laboratory for veterinary drug and pesticide residues by the local markets . two transition ion pairs were monitored for each of the analytes and the ion ratios of detected samples were compared well with those of standards . retention times of analytes were also confirmed by addition of known standards in detected samples . eight samples out of 220 milk samples were found to contain residues of veterinary drug and pesticide residues ( 4% incidence was positive ) . sulfadiazine ( veterinary drug ) residue amount was found between 0.075 and 0.125 mg / l in 2 samples and tetracycline ( veterinary drug ) amount was found to be 0.0150.100 mg / l in 4 samples . a multiclass / multiresidue procedure with lc - ms / ms detection has been developed and validated to determine and quantify veterinary and pesticide residues in milk . a simple sample preparation method involved liquid extraction salting out procedures in ethyl acetate system , without cleanup steps , and shortening the sample preparation time . the method was characterized by good results in terms of recovery , reproducibility , and repeatability allowing the detection of veterinary drug and pesticide residues below the recommended analytical level . based on these results , lc - ms / ms method with ethyl acetate extraction showed the suitability for sensitive quantification of veterinary and pesticide residues in milk samples for food safety applications . this short protocol can be applicable to a large number of samples for routine analysis and rapid detection .

micrornas ( mirs ) are endogenous 22 nucleotide noncoding rnas which suppress gene expression at posttranscriptional levels by binding to the 3-untranslated region of specific mrna targets through base pairing . a number of recent studies reveal that mirs have critical functions in key biological processes such as cell proliferation and cell death during development , fat metabolism [ 3 , 4 ] , or insulin secretion . regulated expression of mirs is scarcely understood but is of special interest in the context of innate immunity and inflammation . expression profiling of more than 200 mirs in human monocytic thp-1 cells revealed that mir-146a is substantially inducible by lps or other toll - like receptor ligands . moreover , promoter analysis of the mir-146a gene suggests a role of nf-b in lps - dependent upregulation of mir-146a . modulation of the lps reactive oxygen species nf-b signalling pathway and dependent mir-146a expression could be an approach in order to fine - tune the inflammatory response . the importance of such a modulation is of special interest because the balance between pro- and anti - inflammatory signals is critical in certain inflammatory diseases such as atherosclerosis . here we hypothesized that the reduced form of coenzyme q10 ( qh2 , ubiquinol-10 ) affects lps - inducible expression of the anti - inflammatory mir-146a . the hypothesis was based on functions of coq10 as a potent antioxidant and putative modulator of inflammation via gene expression [ 8 , 9 ] . in the present work initially , we utilized the human monocytic cell line thp-1 as a well - established model of lps - induced mir-146a expression and applied ubiquinol-10 . additionally , liver samples of lps - injected mice supplemented with ubiquinol-10 were used to strengthen the hypothesis that ubiquinol-10 has a relevance in fine - tuning lps - induced mir-146a expression levels . ubiquinol-10 aqueous solution ( peg-60 hydrogenated castor oil , ubiquinol-10 , glycerol , water ) and corresponding vehicle ( no ubiquinol-10 supplement ) were purchased from kaneka corporation , japan . lipopolysaccharide ( lps , e. coli o55:b5 ) was obtained from sigma - aldrich ( germany ) . thp-1 cells were routinely cultivated in rpmi medium 1640 supplemented with 10% fcs and 1% antibiotics ( penicillin / streptomycin ) in a humidified incubator containing 5% co2 at 37c . twenty four hours before preincubation , cells were plated at a density of 1 10 cells per well in a 6-well plate . subsequently , cells were preincubated with 10 m ubiquinol-10 or the respective vehicle control . after 24 hours , cell culture medium was removed and fresh lps - containing medium ( 1 g / ml ) was added for 4 hours . finally , cells were collected in qiazol - lysis buffer ( qiagen , germany ) for isolation of total rna or alternatively scraped in phosphate buffered saline ( pbs ) for hplc - analysis . male c57/bl6j mice ( 1012 weeks old , 25 g weight ) were purchased from charles river lab . animals were separated into two groups : ( 1 ) intervention group ( n = 6 ) , which was given a diet enriched with ubiquinol-10 ( qh2 , 250 mg / kg / d ) for one week and ( 2 ) the control group ( n = 6 ) , which received a diet prepared by using corn oil in equal proportions to 1% ( v / w ) of the diet as a vehicle . in other respects all animals were maintained on a standard laboratory diet ( powdered ce-2 , crea japan ) and housed under conditions at 22 2c with a 12-hour light / dark cycle . food intake and body weight were monitored daily but indicated no relevant differences between animals . after the 7-day supplementation or control diet period , an intraperitoneal injection ( 1 mg / kg bw ) of lipopolysaccharide ( lps , e. coli , o55:b5 , sigma - aldrich , japan ) was administered for further 4 hours . however , for hplc - experiments , only saline - injected mice were used . in all other aspects , animal treatment was identical . subsequently , all mice were sacrificed , and liver samples were collected and stored at 80c in rnalater storage solution ( qiagen , japan ) until use . determination of cell viability was performed using the cell - titer glo luminescent assay . thus , the total atp levels were measured as an index of the viable cell number . data ( figure 1 ) are means sd of three biological experiments performed in triplicate . the different treated cells of each well were washed with phosphate buffered saline ( pbs ) and counted with trypan blue at 80c before measurement of cellular coq10 . liver homogenates of 10 qh2-supplemented or nonsupplemented mice ( 5 from each group , resp . ) were stored at 80c ( in 0.9% sodium chloride , 10 mg / ml ) until further analysis . the method is based on high - pressure liquid chromatography ( hplc ) with electrochemical detection and internal standardisation using ubihydroquinone-9 and ubiquinone-9 as standards and is described elsewhere . in brief , as internal standard 15 pmol of ubihydroquinone-9 in 50 l ethanol were added to a 100 l monocyte or liver homogenate suspension . the cells and homogenates were disintegrated by adding of 300 l of cold methanol . subsequently , the sample was mixed for 1 minute , and the suspension was immediately extracted with 500 l hexane after mixing for further 2 minutes . after centrifugation ( 1000 xg , 5 minutes , 4c ) , 300 l of the supernatant were transferred to a separate tube and dried under a stream of argon . finally , the dried residue was redissolved in 40 l ethanol and injected into the hplc system . for each liver homogenate sample , the analyzed coq10 concentration was related to its respective protein level . for calculation of differences in sample preparation , protein concentration was determined in liver homogenate samples ( in g / ml ) . thus , homogenate samples were collected into net - buffer ( 50 mm tris [ ph 7.5 ] , 150 mm nacl , 1 mm edta [ ph 8.0 ] , 0.5% np-40 ) . in each case , homogenate solutions were treated with ultrasonics ( n vs. m ) and then centrifuged by 14.000 rpm at 4c for 20 minutes . the protein concentration was determined by the bradford method according to the manufacturer 's instructions . total rna was isolated by using the mirneasy isolation kit ( qiagen , germany ) , and cdna was converted by the taqmanmicrorna reverse transcription kit ( applied biosystems ) . mirna-146a expression was measured and quantified by using the taqmanmicrorna assays ( applied biosystems ) according to the manufacturer 's protocol and normalised by snorna202 ( applied biosystems ) . quantitative rt - pcr reaction was performed on an applied biosystems 7300 real - time pcr system . total rna was isolated with qiazol lysis reagent obtained with the mirneasy isolation kit ( qiagen , germany ) . cdna was converted by the taqman microrna reverse transcription ( rt ) kit ( applied biosystems ) . the rt reaction product was diluted 10 times in water and subsequently used for rt - pcr amplification of mirna-146a by using the taqman microrna assays ( applied biosystems ) according to the manufacturer 's protocol and normalized by snorna202 ( applied biosystems ) . a 6-fold total rna - dilution series from a control - treated ( + lps ) mouse liver served as standard to ensure a linear range of the amplification . quantitative rt - pcr reaction was performed on an applied biosystems 7300 real - time pcr system . results were analyzed by an unpaired , two- or one - sided student 's t - test using spss 11.5 for windows and graphpad prism 4.0 software . to exclude cytotoxic side effects in our experimental set - up , vitality of thp-1 cells was determined after incubation with ubiquinol-10 . as shown in figure 1 , incubation of thp-1 cells with increasing ubiquinol-10 concentrations ( 0.1100 m ) for 24 hours led to no significant effects on cell vitality . thus , in thp-1 cells no cytotoxicity was found for ubiquinol-10 at physiological ( 1.0 m ) , supraphysiological ( 10 m ) , and pharmacological ( > 10 m ) concentrations . the putative effects of extracellular ubiquinol-10 on mir expression depend on its capability to reach cellular concentrations above background level . therefore , we determined the cellular concentration of coq10 as a function of medium ubiquinol-10 . as shown in table 1 , as mainly the reduced form of coq10 can function as an antioxidant , we determined the proportion between the oxidized and reduced form of coq10 . depending on the extracellular ubiquinol-10 concentration , about 7590% of cellular coq10 was present in its reduced form ( table 1 ) . moreover , our previous results have shown that monocytic cells are able to convert oxidized coq10 effectively into its reduced form . thus , at physiological and supraphysiological coq10 levels in the medium ( 0.110 m ) , the intracellular coq10 distribution is clearly in favor of the reduced form ( 7590% ) . other in vitro and in vivo studies also revealed an intracellular incorporation of coq10 after supplementation in blood cells , thereby leading , for example , to a reduction of dna strand breakdowns [ 11 , 13 ] . taken together , we were able to increase the cellular ubiquinol-10 concentration without any cytotoxic side - effects in human thp-1 cells . in order to study the effect of ubiquinol-10 on lps - induced mir-146a expression , appropriate conditions were established . for this purpose , the lps - induced response of thp-1 cells was examined . unstimulated thp-1 cells did not secrete relevant amounts of tnf- into the medium ( 4.22 pg / mg protein sd ) . however , stimulation with 1 g / ml lps for 4 hours resulted in an increase of medium tnf- levels ( 351.94 pg / mg protein sd ) . thus , we obtained a substantial and anticipated lps - response of thp-1 cells . as already described in the literature , the expression level of mir-146a is upregulated by lps . as shown in figure 2 , in comparison to unstimulated thp-1 cells , lps challenge induced a 19-fold induction of mir-146a expression ( p = .0007 ) . with respect to our results regarding cytotoxicity and cellular accumulation of ubiquinol-10 , thp-1 cells were preincubated with 10 m ubiquinol-10 for 24 hours as an effective dose . this is nearly in accordance to other in vitro studies performed with coq10 [ 15 , 16 ] and is only 2-fold higher than coq10 serum levels after supplementation in humans . thereafter , cells were stimulated with 1 g / ml lps for 4 hours and the resulting steady - state expression level of mir-146a was determined . as shown in figure 2 , preincubation of thp-1 cells with ubiquinol-10 reduced the lps - induced expression level of mir-146a to 78.9 13.22% . although these effects were not statistically significant ( p .05 ) , the reduced expression levels of mir-146a in the ubiquinol-10-pretreated cells ( qh2 + lps ) might be a first hint for a fine - tuning mechanism of qh2 on mir-146a expression when compared to the high significant induction levels in control cells after lps - stimulation ( veco lps ) . to test putative effects of coq10 on mirna 146a regulation in a more physiological manner however , to mediate these effects , a tissue - specific accumulation of coq10 is essential . therefore , total coq10 levels were determined in liver homogenate samples of qh2-supplemented and control mice . as shown in figure 3(a ) , total coq10 levels increased about 12-fold ( p = .0193 ) in liver tissues of qh2-supplemented mice when related to control samples . because coq9 is the predominant coq form in rodents , coq9 was used as an internal standard for hplc - analysis thus , the coq9 level was not significantly changed between treatment and control group ( p = .51 , data not shown ) . accordingly , the coq9/coq10 ratio was significantly different between groups , corresponding to a 6.5-fold increase in control tissue samples ( p = .0019 , figure 3(b ) ) . in general , all hplc measurements were related to protein levels in the respective tissue homogenate samples . in summary , we could significantly increase the coq10 concentration in liver tissues of ubiquinol-10-supplemented mice . an effective uptake of exogenously applied coq10 in liver tissues of rodents has been already described earlier . to test the relevance of a putative ubiquinol-10-dependent downregulation of lps - inducible mir-146a expression in vivo , we used mice receiving either a diet enriched with ubiquinol-10 ( qh2 , 250 mg / kg / d ) or a respective control diet for seven days . this dosage is in accordance with previous coq10 studies in mice [ 2022 ] . thereafter , an intraperitoneal injection of lipopolysaccharide ( lps , 1 mg / kg bw ) was administered for further 4 hours . in contrast to the control non - lps - injected mice ( no detectable tnf- levels ) , tnf- levels increased significantly in the serum of lps - treated animals ( 612.46 sd ) . finally , mice were sacrificed and livers were collected for mir isolation and determination of mirna-146a expression levels . as shown in figure 4(a ) , the lps - induced expression level of mir-146a is generally lower in animals supplemented with ubiquinol-10 . in average , ubiquinol-10 reduces the lps - induced mir-146a expression to 78.12 21.25% ( + qh2/+lps ) when compared to control animals ( qh2/+lps ) ( figure 4(b ) ) . this effect was statistically not significant but was consistent to those obtained in cell culture experiments . regulation of mirs by certain compounds or nutrients is of general interest , because this class of noncoding rnas is involved in central biological processes such as development , inflammation and innate immunity , and signalling networks [ 1 , 23 ] . so far , only a small number of environmental modulators of mir expression have been identified . expression levels of several mirs ( i.e. , mir-15a / b , mir-16 , mir-107 ) are regulated by retinoic acid - induced differentiation in human acute promyelocytic leukemia patients and cell lines . compounds such as sulphate , phosphate , and amino acids regulate the expression of special mirs in plants and human liver cells . toll - like receptor ( tlr ) ligands such as lps induce the expression of mir-146a significantly in human monocytes / macrophages . preincubation of thp-1 cells with ubiquinol-10 reduced the lps - induced expression level of mir-146a . these results are consistent to our in vivo data , where the expression of mir-146a was reduced in liver samples of mice supplemented with ubiquinol-10 before lps - injection ( + qh2/+lps ) when compared to control animals ( + lps ) . although the observed effects are statistically not significant , we postulated a fine - tuning mechanism of ubiquinol-10 on the inflammatory response via a moderate reduction of mir-146a expression . according to the superior function of mir-146a in the inflammatory response , the observed moderate reduction of its expression by ubiquinol-10 seems to be desirable based on the following mechanisms . first , the lps induced upregulation of mir-146a in human monocytes / macrophages functions as a negative regulator of the innate immune response because mir-146a targets traf6 , a regulator protein within the tlr - signalling pathways involved in the formation and accumulation of reactive oxygen species . second , we have recently shown that ubiquinol-10 lowers the lps - stimulated release of some proinflammatory cytokines and chemokines relevant in inflammatory processes . the observed effects were comparable to those of the potent and characterized antioxidants n - acetyl - cysteine ( nac ) or pyrrolidine - dithiocarbamate ( pdtc ) [ 12 , 14 , 27 ] . moreover , mir-146a has been shown to be induced by proinflammatory cytokines such as tnf- , interleukin 1-beta ( il-1 ) , and tlrs [ 7 , 28 , 29 ] . mir-146a was also detected in tissues related to inflammatory diseases including , for example , synovial fibroblasts and rheumatoid synovial tissue . thus , we conclude that ubiquinol-10 reduces both the secretion of proinflammatory agents and the expression of the anti - inflammatory mir-146a . as a consequence , ubiquinol-10 acts as an anti - inflammatory compound but perpetuates the essential inflammatory response via moderate reduction of mir-146a expression . this dual effect could be due to the radical scavenging activity of ubiquinol-10 since reactive oxygen species are involved in the tlr - signalling pathways . in conclusion , the consistent in vitro and in vivo data suggest that ubiquinol-10 may fine - tune the inflammatory response via moderate reduction of mir-146a expression .

finding effective methods to diagnose bacterial vaginosis ( bv ) has taken on increased urgency since bv was associated with a greater than 3-fold increased risk of female - to - male hiv-1 transmission and a doubling of risk for acquiring sexually transmitted infections [ 14 ] . bv has also been implicated in neonatal morbidity , preterm delivery [ 68 ] , and low birth weight infants . bv is usually diagnosed using amsel 's criteria or nugent scoring of gram - stained vaginal smears both of which require microscopy . in countries like india where access to laboratory services is often limited , bv is typically managed using a syndromic approach for vaginal discharge , a method with low sensitivity and specificity . point - of - care ( poc ) testing may improve diagnosis of bv by providing accurate results without laboratory or on - site microscopy . rapid assays detecting the presence of proline amino peptidase [ 14 , 15 ] have been found to have high sensitivity and specificity . others which rely on detection of trimethylamine and high vaginal ph have not been compared favorably with amsel 's criteria or nugent scoring [ 1618 ] . bvblue , a chromogenic poc , diagnoses bv based on elevated levels of sialidase , an enzyme produced by anaerobic flora including bacteroides , prevotella , and gardnerella species [ 19 , 20 ] . in other studies , this approach has been shown to have excellent sensitivity , specificity , and predictive values in several populations [ 2124 ] . this paper describes a study comparing the performance of bvblue point - of - care test ( osom bvblue test , gryphus diagnostics , birmingham , al , usa ) with amsel 's criteria and nugent scoring of gram - stained vaginal smears among women attending a reproductive health clinic in mysore , india . between august 2009 and january 2010 , a consecutive sample of 347 nonpregnant , sexually active women were recruited prospectively from a reproductive health clinic in mysore , india , into a cross - sectional study comparing the performance of the bvblue poc test to amsel 's criteria and nugent scoring of gram - stained vaginal smears . to be included in the study , participants were required to be 18 years of age or older , have had vaginal intercourse at least once in the previous three months , be willing to undergo a pelvic examination , and have a vaginal ph over 4.5 . vaginal ph was measured by placing a self - collected vaginal swab on a bdh ph test strip and comparing the color to a ph chart provided by the manufacturer ( vwr international , west chester , pa , usa ) . the committee for protection of human subjects at university of california , berkeley , ca , usa and the institutional review board at public health research institute of india ( phrii ) , mysore , india , approved the study protocol . after undergoing an informed consent process , trained interviewers collected information on demographics and reproductive / sexual health using a standardized questionnaire . biological samples , questionnaires , and clinician checklists were labeled with a unique identifier to ensure confidentiality of participants . a trained study clinician performed a pelvic examination and collected three swabs of vaginal fluid from the posterior fornix of the vagina in a random order to test for bv , trichomonas vaginalis , and vaginal candidiasis . in addition , clinical signs from external and internal examination were recorded on a medical chart . signs of vaginal discharge including amount , odor , color , and consistency were noted . diagnosis was based on amsel 's criteria : presence of any three of four clinical features : a characteristic homogeneous white adherent vaginal discharge , a vaginal ph greater than 4.5 , a positive amine test , and presence of 35 clue cells per high power field on wet - mount microscopy . symptomatic women diagnosed with bv by amsel 's criteria were treated according to standard indian treatment guidelines . all tests were performed by three trained laboratory research assistants in a blinded manner to prevent bias . saline wet - mount preparation of vaginal fluid was examined microscopically within five minutes of collection for clue cells , motile trichomonads , and yeast buds / hyphae . the vaginal swab was placed in a test tube containing 3 drops of sterile normal saline at the time of pelvic examination by the clinician . after agitation , 1 drop of solution was placed on a glass slide , covered with a cover slip , and observed at 10x and 40x magnifications . another drop of the saline solution was placed on a sterile glass slide with an added drop of koh solution . the slide was used for detection of amine odor ( whiff test ) . following the whiff test , a cover slip was placed on the slide and read for detection of budding yeast or hyphae . wet - mount examinations were part of the routine clinical care at this clinic and hence the time interval between specimen collection and microscopy was less than five minutes . a second vaginal swab was placed in the bvblue test vessel containing the chromogenic substrate of bacterial sialidase and the mixture was gently swirled . one drop of developer solution was added to the bv test vessel and the mixture was swirled gently again . results were read immediately ; a blue or green color in the bv test vessel or on the head of the swab was considered positive and a yellow color in the bv test vessel was considered negative . if the results were not blue / green or yellow , then the test was repeated . a positive result indicated an elevated level of sialidase activity and a negative result indicated a normal level . the third vaginal swab was smeared on a glass slide and air - dried before fixing and gram - staining at the phrii laboratory . the swab was then used to inoculate inpouchtv culture kit ( biomed diagnostic , white city , or , usa ) , for detection of t. vaginalis infection . the swab was inserted in the upper chamber , agitated in the medium , and discarded , and the pouch was sealed . the contents of the upper chamber were immediately expressed into the lower chamber by rolling down . the pouch was then transported to the laboratory within four hours and placed in a 37c incubator . it was read without opening the culture or sampling the contents for five days or until trichomonads were detected using a microscope at 10x and 40x magnification . each gram - stained slide was scored by two different trained laboratory assistants masked to the other test results to minimize bias . in cases of discrepancy , the slide was then scored by a third reader blinded to the scores of the first two readers . the nugent score is a standardized 010-point scoring system based on the presence of three bacterial morphotypes : large gram - positive rods ( lactobacillus spp . ) , small gram - negative or gram - variable coccobacilli ( gardnerella and anaerobic spp . ) , and curved gram - variable rods ( mobiluncus spp . ) . a nugent score ( ns ) of 03 is classified as the presence of normal flora , 46 as the presence of intermediate flora , and 710 as bv . this method is still considered the gold standard for diagnosis of bv . kits were refrigerated between 2 and 8 degrees celsius and kept out of direct sunlight . all bv vessels were stored inside the box as suggested by the manufacturer , and kits were brought to room temperature before use . for quality control purposes , test vessels were checked before use to ensure that they contained only a colorless liquid without sediments . the bvblue test result was only reported if there was appearance of blue / green or yellow color in the testing vessel . we also conducted 10% random quality control checks by an experienced microscopist who has expertise in bv for nugent 's scoring and wet - mount preparation readings for clue cells and found high concordance . data were entered and stored in microsoft access and analyzed using stata 10.1 ( stata corporation , college station , tx ) . proportions were compared using chi - square and fisher 's exact tests where appropriate , and 95% confidence intervals ( cis ) were calculated . participants were excluded from the analysis if complete clinical information or specimens were not available . sensitivity , specificity , and predictive values were calculated using a traditional standard defined as nugent score of 710 for bv positivity . analyses were carried out in two ways : first , women with an ns of 46 ( intermediate flora ) were classified as negatives ; second , women with intermediate flora were excluded , and the performance of the rapid test was calculated for each analysis . we also stratified the women based on their complaints into symptomatic ( excess vaginal discharge , odor , burning , and itching ) and asymptomatic cases to examine the performance of the rapid test . sensitivity and specificity were calculated for each testing method using the following formulas : sensitivity : ( number of true positives/(number of true positives + number of false negatives))100 ; specificity : ( number of true negatives/(number of true negatives + number of false positives))100 . figure 1 describes the number of women eligible who did not participate in the study and the reasons for not participating . ( seven were missing bvblue results , and five nugent score . ) among the total participants , 221 ( 71% ) women reported symptoms of abnormal vaginal discharge , pruritus , burning , or odor at the time of enrollment . the median age of the patients was 33 years and the vast majority ( 87% ) reported their religion as hindu . eighty - seven ( 58.3% ) women diagnosed with bv complained of vaginal symptoms , and 62 ( 41.6% ) were asymptomatic . about 206 ( 63.7% ) women had abnormal vaginal flora with an ns of 410 , and 149 ( 46% ) were diagnosed with bv ( ns 710 ) ( figure 1 ) . using amsel 's criteria , 111 ( 35% ) women were diagnosed with bv and 68 ( 21.9% ) by the bvblue poc test . t. vaginalis infection was common among women with bv ( ns : 710 ) as compared to women without bv ( 18% versus 8.6% ; p = 0.02 ) . a positive bvblue test result was strongly associated with two of amsel 's criteria : the presence of clue cells ( p < 0.0001 ) and amine odor ( p < 0.0001 ) . table 2 shows the bvblue poc test performance compared to wet - mount microscopy , amsel 's method , and nugent scoring among a subset of women where women with intermediate flora were excluded from the analysis . table 3 shows the performance of bvblue test as compared to amsel 's criteria and nugent score . with bv positive being defined as ns of 710 and all other results considered negative including the intermediates ( ns : 06 ) , bvblue performance was not very different among symptomatic women as compared to all women . among symptomatic women with complaint of any vaginal symptom , amsel 's criteria had the best sensitivity followed by wet mount microscopy and bvblue with nugent score being considered the gold standard . the bvblue test performance did not improve ( in terms of sensitivity and specificity ) among symptomatic women as compared to its performance among all women ( tables 2 and 3 ) . used alone , a positive amine odor ( whiff test ) was highly specific ( 87.9% ) but had a low sensitivity ( 59.7% ) . combined use of a whiff test and the bvblue poc test , however , improved the sensitivity of bv diagnosis to 64.4% . diagnosing bv with a bvblue poc test and/or amine test result was found to be the best performing approach with high sensitivity and minimal loss of specificity as compared to amsel 's criteria . our study examined the performance of the bvblue poc test as compared to amsel 's criteria and nugent scoring of gram stains among women with and without symptoms of bv . our results showed that bvblue test had poor sensitivity in detecting bv ( 38.1% ) but was highly specific ( 92.7% ) in a population of women attending a reproductive health clinic in mysore , india . in previous research , bvblue has been shown to perform well compared with conventional diagnostic methods for the diagnosis of bv in populations in canada , australia , thailand , malaysia , and china [ 21 , 2426 ] . sensitivity ranged from 88% to 100% and specificity from 95% to 97.8% using nugent gram stain as a gold standard . the only study from north india showed that the bvblue test had 97.6% sensitivity and 97.5% specificity as compared to nugent score of gram stain . however , our findings were similar to another study that examined the performance of bvblue for diagnosis of bacterial vaginosis in symptomatic and asymptomatic women in the us . that study found bvblue test was less sensitive than gram stain for diagnosis of bv and not statistically different from amsel 's criteria . because our results were substantially different from previous evaluations of the bvblue , we considered and eliminated several alternative explanations for the difference . our samples were collected by a trained study clinician who was an experienced obstetrician / gynecologist and processed within five minutes of the time that the physical examination was finished . our team had been extensively trained and recently completed several studies using nugent score reading of several thousand gram stains , so we ruled out systematic human error in the diagnosis of bv [ 2833 ] . we had conducted quality control checks by an experienced microscopist for nugent scoring and wet mount readings for clue cells and found high concordance . finally , we ruled out manufacturing or handling problems with bvblue kits by checking refrigerator logs and contacting the manufacturer to enquire about known quality issues with the manufacture or handling of the kits . having examined and dismissed those alternative explanations , we speculate that the bvblue poc kit was not sensitive in this population because of differences in the composition and diversity of the vaginal bacterial flora among women with bv in our sample . recent studies using broad - range 16s rrna gene pcr and pyrosequencing have shown that bv is a highly heterogeneous condition marked by greater species richness and diversity than previously thought , with no single species universally present . since the bvblue kit operates on the principle that bv is associated with elevated levels of sialidases , it is possible that the vaginal biota of women with bv in our study population may have sialidase - negative g. vaginalis strains or contain a low number of anaerobic gram - negative rods such as prevotella spp . and bacteroides spp . that are common sources of sialidases in bv previous studies have shown that sialidase activity was detected in only 75% to 84% of women [ 35 , 36 ] with bv , suggesting that the presence of sialidase is not uniform . studies have also demonstrated that the composition of bv flora varies by race / ethnicity , raising the possibility that bv - related bacteria in this population may be different from those found in other racial and ethnic groups [ 34 , 37 ] . first , we did not evaluate the performance of the rapid test using molecular methods such as polymerase chain reaction ( pcr ) for diagnosis of bv . currently , nugent scoring of gram stains continues to be the gold standard for diagnosis of bv in research studies , but it is possible that results might have been different if molecular diagnostic methods had been used . second , corroborating low sialidase levels in the vaginal fluid of participants would have been useful in helping to explain the poor sensitivity of the bvblue poc test , but it was beyond the scope of this study . finally , we were not able to definitively establish the fitness of bvblue kits used in the study since we were not provided with an external control by the manufacturer . there is an important public health need for development of poc tests that facilitate diagnosis of bv in settings without adequate laboratory infrastructure . however , bvblue test does not appear to be a good screening test in our population . unfortunately , the effectiveness of different methods may depend on the bacterial composition of bv flora in different populations giving added importance to the need for wide evaluation of poc tests . furthermore , given the heterogeneity of bv , culture - based methods for characterizing the vaginal biota may have to give way to more exacting molecular methods to detect and characterize a greater range of organisms . additional research should also be conducted to analyze differences in vaginal biota of different populations in india and other parts of the world .

cardiometabolic risk factors ( cmrfs ) such as elevated fasting glucose , elevated blood pressure , low high - density lipoprotein cholesterol , elevated fasting triglycerides , and abdominal obesity often coexist in the same individual . the presence of multiple cmrfs has been broadly termed as the metabolic syndrome.1 although definitions of metabolic syndrome from different organizations vary regarding the exact number of criteria and cut points for each parameter , the presence of these risk factors often results in increased risk of cardiovascular ( cv ) disease - related morbidity and mortality.27 cv disease still poses a significant burden in the us and accounted for one in three deaths and annual costs for treating cv disease were ~us$320.1 billion in 2011.8 health interventions in the us have reduced the prevalence of elevated fasting triglycerides and elevated blood pressure from 1999 to 2010.8 however , prevalence of elevated glucose and elevated waist circumference has continued to rise over this same time period , particularly among children and adolescent populations . age - adjusted national us prevalence of metabolic syndrome has decreased for women but prevalence remains high for men . given the increasing prevalence of elevated glucose and waist circumference among children and adolescents , and an aging population , the prevalence and economic burden of cmrfs will continue to rise . prior to the onset of cv disease , concomitant cmrfs are associated with a significant increase in medical expenditures and lost productivity.912 several studies suggest that a higher number of cmrfs and specific combinations of cmrfs ( eg , diabetes and obesity ) are associated with significant cv - related events and costs.1316 however , there is a lack of evidence reporting both direct medical costs and lost productivity associated with incremental increases in cmrfs regardless of concomitant conditions . the primary objective of this study was to estimate the economic burden as a function of direct medical costs and lost productivity associated with incremental increases in the number of cmrfs using recently available nationally representative data from the us noninstitutionalized population . we utilized the nationally representative , publicly available panel database , the medical expenditure panel survey ( meps ) . the meps tracks individual and household health - related , demographic , and socioeconomic characteristics.17 to provide estimates that are representative of a national us population , the household component of the meps ( meps - hc ) panels have oversampled subgroups of individuals such as african - americans , hispanics , asians , low - income households , and those likely to incur high medical expenditures . at the time of the analysis , we combined the 20102012 meps full - year consolidated , medical , and pharmacy utilization data files to generate an analytical cohort with robust sample size . the colorado multiple institutional review board considers this research to be exempt from requiring board approval because meps is a publicly available data source . the study cohort consisted of noninstitutionalized adult us individuals ( 18 years of age and above ) with at least one and up to four cmrfs . the study cohort was defined using a combination of international classification of diseases , ninth revision ( icd-9 ) codes , national drug codes , and survey responses . we calculated the number of cmrfs for each patient and stratified the population into four mutually exclusive and exhaustive groups : those with zero ( comparison group ) , one , two , and a group with three or four cmrfs . the following cmrfs were used to calculate the number of cmrfs : abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year.elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year.elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18 elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year . elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year . elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . demographic variables available in meps include sex ( male , female ) , age in years ( 1834 , 3549 , 5064 , 6579 , 80 + ) , race ( white , black , american indian , and other race ) , ethnicity ( hispanic / non - hispanic ) , health insurance type ( private , public , dual covered , and uninsured ) , highest degree completed ( no degree , less than bachelors , bachelors , masters , and above ) , family income ( classified using percentage of federal poverty level ) , and region of the country ( northeast , midwest , south , west ) . clinical variables included other chronic medical conditions coded as numerical counts of conditions other than the four cmrfs described earlier . the numerical count of other conditions was used as an adjustment variable in the regression analyses . outcome measures in this study were health care utilization , expenditures , and lost productivity . health care utilization included the number of outpatient visits , number of emergency room visits , number of inpatient visits , inpatient length of stay , and number of prescriptions including refills . all - cause health care expenditures in meps consist of direct payments for all health care utilization during the calendar year , including out - of - pocket payments and payments made by private and public insurance . health care expenditures were estimated as total health care expenditures ( pharmacy + medical ) . all expenditures were estimated in 2014 us dollars using the medical care component of consumer price index . two measures of workplace productivity were estimated : number of days missed at work and employed or not employed during ( including part - time employment status ) the entire year . descriptive statistics were estimated for utilization , expenditures , lost productivity , and other clinical and demographic variables . regression models for health care utilization , expenditures , and lost productivity reported incremental differences for those with one , two , three , or four of the cmrfs defined in this study compared with the general population with zero cmrfs . regression models adjusted for all relevant clinical and demographic variables , including age , sex , race , ethnicity , region , education , number of other chronic conditions , family income , and insurance status . negative binomial models were used for the adjusted health care utilization analysis and the lost - productivity analysis for the number of missed days at work . health care expenditures often contain a large percentage of zero costs and are heavily right - skewed . to account for these statistical issues , we used generalized linear modeling with log link and gamma family distribution for health care expenditures . adjusted differences in health care expenditure outcomes were reported as marginal effects ( dy / dx ) as compared with those with zero cmrfs . a logistic regression model was developed to estimate the adjusted difference in employment between groups and reported as odds ratios . all analyses were conducted using stata version 12 ( statacorp lp , college station , tx , usa ) incorporating meps survey design variables ( eg , probability weights ) . we performed a sensitivity analysis by splitting the three or four cmrfs group into three and four cmrfs separately . we utilized the nationally representative , publicly available panel database , the medical expenditure panel survey ( meps ) . the meps tracks individual and household health - related , demographic , and socioeconomic characteristics.17 to provide estimates that are representative of a national us population , the household component of the meps ( meps - hc ) panels have oversampled subgroups of individuals such as african - americans , hispanics , asians , low - income households , and those likely to incur high medical expenditures . at the time of the analysis , we combined the 20102012 meps full - year consolidated , medical , and pharmacy utilization data files to generate an analytical cohort with robust sample size . the colorado multiple institutional review board considers this research to be exempt from requiring board approval because meps is a publicly available data source . the study cohort consisted of noninstitutionalized adult us individuals ( 18 years of age and above ) with at least one and up to four cmrfs . the study cohort was defined using a combination of international classification of diseases , ninth revision ( icd-9 ) codes , national drug codes , and survey responses . we calculated the number of cmrfs for each patient and stratified the population into four mutually exclusive and exhaustive groups : those with zero ( comparison group ) , one , two , and a group with three or four cmrfs . the following cmrfs were used to calculate the number of cmrfs : abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year.elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year.elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18 elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year . elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year . elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . demographic variables available in meps include sex ( male , female ) , age in years ( 1834 , 3549 , 5064 , 6579 , 80 + ) , race ( white , black , american indian , and other race ) , ethnicity ( hispanic / non - hispanic ) , health insurance type ( private , public , dual covered , and uninsured ) , highest degree completed ( no degree , less than bachelors , bachelors , masters , and above ) , family income ( classified using percentage of federal poverty level ) , and region of the country ( northeast , midwest , south , west ) . clinical variables included other chronic medical conditions coded as numerical counts of conditions other than the four cmrfs described earlier . the numerical count of other conditions was used as an adjustment variable in the regression analyses . outcome measures in this study were health care utilization , expenditures , and lost productivity . health care utilization included the number of outpatient visits , number of emergency room visits , number of inpatient visits , inpatient length of stay , and number of prescriptions including refills . all - cause health care expenditures in meps consist of direct payments for all health care utilization during the calendar year , including out - of - pocket payments and payments made by private and public insurance . health care expenditures were estimated as total health care expenditures ( pharmacy + medical ) . all expenditures were estimated in 2014 us dollars using the medical care component of consumer price index . two measures of workplace productivity were estimated : number of days missed at work and employed or not employed during ( including part - time employment status ) the entire year . descriptive statistics were estimated for utilization , expenditures , lost productivity , and other clinical and demographic variables . regression models for health care utilization , expenditures , and lost productivity reported incremental differences for those with one , two , three , or four of the cmrfs defined in this study compared with the general population with zero cmrfs . regression models adjusted for all relevant clinical and demographic variables , including age , sex , race , ethnicity , region , education , number of other chronic conditions , family income , and insurance status . negative binomial models were used for the adjusted health care utilization analysis and the lost - productivity analysis for the number of missed days at work . health care expenditures often contain a large percentage of zero costs and are heavily right - skewed . to account for these statistical issues , we used generalized linear modeling with log link and gamma family distribution for health care expenditures . adjusted differences in health care expenditure outcomes were reported as marginal effects ( dy / dx ) as compared with those with zero cmrfs . a logistic regression model was developed to estimate the adjusted difference in employment between groups and reported as odds ratios . all analyses were conducted using stata version 12 ( statacorp lp , college station , tx , usa ) incorporating meps survey design variables ( eg , probability weights ) . we performed a sensitivity analysis by splitting the three or four cmrfs group into three and four cmrfs separately . the following cmrfs were used to calculate the number of cmrfs : abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year.elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year.elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . abdominal obesity : defined as body mass index ( bmi ) 27 kg / m . previous research has suggested treatment of overweight individuals when other cmrfs are present should begin at a bmi 27 kg / m.18 elevated blood pressure : defined as having at least one icd-9 code for hypertension ( 401.x ) and at least one prescription for an antihypertensive agent ( angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , beta blocker , calcium channel blocker , diuretic , direct renin inhibitor , alpha blocker , alpha agonist , aldosterone antagonist , or direct arterial vasodilator ) during a survey year . elevated triglycerides : defined as having at least one prescription for a triglyceride - lowering medication ( fibrate , niacin , or omega-3 fatty acids ) during a survey year . elevated glucose : defined as having at least one icd-9 code for diabetes ( 250.x ) and at least one prescription for an antihyperglycemic medication ( biguanide , sulfonylurea , thiazolidinedione , insulin , alpha - glucosidase inhibitor , incretin mimetic , or dipeptidyl peptidase-4 inhibitor ) during a survey year . demographic variables available in meps include sex ( male , female ) , age in years ( 1834 , 3549 , 5064 , 6579 , 80 + ) , race ( white , black , american indian , and other race ) , ethnicity ( hispanic / non - hispanic ) , health insurance type ( private , public , dual covered , and uninsured ) , highest degree completed ( no degree , less than bachelors , bachelors , masters , and above ) , family income ( classified using percentage of federal poverty level ) , and region of the country ( northeast , midwest , south , west ) . clinical variables included other chronic medical conditions coded as numerical counts of conditions other than the four cmrfs described earlier . the numerical count of other conditions was used as an adjustment variable in the regression analyses . outcome measures in this study were health care utilization , expenditures , and lost productivity . health care utilization included the number of outpatient visits , number of emergency room visits , number of inpatient visits , inpatient length of stay , and number of prescriptions including refills . all - cause health care expenditures in meps consist of direct payments for all health care utilization during the calendar year , including out - of - pocket payments and payments made by private and public insurance . health care expenditures were estimated as total health care expenditures ( pharmacy + medical ) . all expenditures were estimated in 2014 us dollars using the medical care component of consumer price index . two measures of workplace productivity were estimated : number of days missed at work and employed or not employed during ( including part - time employment status ) the entire year . descriptive statistics were estimated for utilization , expenditures , lost productivity , and other clinical and demographic variables . regression models for health care utilization , expenditures , and lost productivity reported incremental differences for those with one , two , three , or four of the cmrfs defined in this study compared with the general population with zero cmrfs . regression models adjusted for all relevant clinical and demographic variables , including age , sex , race , ethnicity , region , education , number of other chronic conditions , family income , and insurance status . negative binomial models were used for the adjusted health care utilization analysis and the lost - productivity analysis for the number of missed days at work . health care expenditures often contain a large percentage of zero costs and are heavily right - skewed . to account for these statistical issues , we used generalized linear modeling with log link and gamma family distribution for health care expenditures . adjusted differences in health care expenditure outcomes were reported as marginal effects ( dy / dx ) as compared with those with zero cmrfs . a logistic regression model was developed to estimate the adjusted difference in employment between groups and reported as odds ratios . all analyses were conducted using stata version 12 ( statacorp lp , college station , tx , usa ) incorporating meps survey design variables ( eg , probability weights ) . we performed a sensitivity analysis by splitting the three or four cmrfs group into three and four cmrfs separately . there were 43,037 people with at least one cmrf ; 30,849 had zero cmrfs ; 29,647 had one cmrf ; 9,392 had two cmrfs ; and 3,998 had three or four cmrfs ( table 1 ) . among the same sample , the most prevalent cmrf was abdominal obesity ( n=36,638 ) followed by elevated blood pressure ( n=16,830 ) ( table 2 ) . those with a higher number of cmrfs had a higher number of other chronic conditions ( ie , excluding cmrfs ) as compared with the average meps population . the number of other chronic conditions was highest for people with elevated triglycerides ( 3.05 ) , elevated glucose ( 2.61 ) , and elevated blood pressure ( 2.54 ) as compared with the average meps population ( 1.35 ) . significant increases in unadjusted and adjusted utilization and expenditures were observed as the number of cmrfs increased ( table 3 and figure 1 ) . as compared with people with zero cmrfs , people with one , two , and three or four cmrfs had 1.15 ( 95% ci : 1.06 , 1.24 ) , 1.37 ( 95% ci : 1.25 , 1.51 ) , and 1.39 ( 95% ci : 1.22 , 1.57 ) times higher expected rate of emergency room visits , respectively , after adjusting for clinical and demographic characteristics . overall adjusted annual health care expenditures were approximately double for those with three or four cmrfs at us$7,640 compared with us$3,523 and us$3,940 for those with zero cmrfs and one cmrf , respectively ( figure 1 ) . people with three or four cmrfs reported employment of 60% compared with 80% in people with zero cmrfs ( table 4 ) . after adjusting for clinical and demographic characteristics , on average people with three or four cmrfs had 1.38 ( 95% ci : 1.17 , 1.63 ) times higher odds of reporting not being employed as compared with those with zero cmrfs . after adjusting for clinical and demographic characteristics , people with one , two , and three or four cmrfs had 1.21 ( 95% ci : 1.09 , 1.34 ) , 1.55 ( 95% ci : 1.31 , 1.82 ) , and 1.75 ( 95% ci : 1.42 , 2.17 ) times higher expected rate of number of missed days at work due to illness or injury , respectively , compared with people with zero cmrfs . when three or four risk factors were split into separate categories , outcome measures were not significantly different from each other ; therefore , we kept the three or four cmrf groups merged together for the main findings . the results of this study suggest the economic burden associated with cmrfs increases as the number of cmrfs increase . our findings are similar to previous research that found a significant economic burden of cmrfs independent of the cost of cv disease . sullivan et al estimated the medical cost of cmrf clusters in the us using meps data from 2000 and 2002.10 the authors grouped four risk factors ( ie , bmi 25 kg / m , diabetes , hypertension , and hyperlipidemia ) to estimate overall utilization and expenditure differences for those with and without cmrfs , and to compare attributable cmrf expenditures by insurance type . the authors found adjusted annual total health care expenditures of $ 5,477 in 2005 us dollars attributable to cmrfs when including bmi 25 kg / m , and at least two cmrfs out of diabetes , hypertension , and hyperlipidemia . using recently updated meps data , our study , when analyzed differently , found a similarly large difference of us$4,117 in adjusted annual health care expenditures for those with at least three or four cmrfs vs those with zero cmrfs . sullivan et al published a follow - up study using the same data ( meps 2000 and 2002 ) and assumptions ( bmi 25 kg / m ) but focused solely on lost productivity - related outcomes.11 the annual number of missed work - days due to illness or injury attributable to cmrfs when including bmi 25 kg / m , and at least two cmrfs out of diabetes , hypertension , and hyperlipidemia , increased by a factor of 1.79 when compared with those without cmrfs . these results are similar to our findings as individuals with three or four risk factors had an increase in missed work - days due to illness and injury by a factor of 1.75 . but our study differs from these two studies by sullivan et al in that we used the most recently updated meps - hc files . we categorized and estimated cmrfs by the number of cmrfs as opposed to a population with and without any cmrfs , and we used a different bmi stratification ( bmi 27 kg / m ) . our results are also similar to previous studies that have analyzed medical costs by the number of cmrfs in combination with specific conditions such as coronary artery disease ( cad ) , diabetes , and obesity . using a claims data set , tamariz et al analyzed health care paid claims by cad and by the number of cmrfs from one to four.15 their popula tion included individuals with at least one of the following conditions : diabetes , hypertension , abnormal lipid panel , and obesity . health care expenditures were estimated with and without cad and by the number of cmrfs . although not directly comparable to our results , the observed trend of increasing expenditures associated with cmrfs is consistent with our findings . for example , adjusted health care expenditures in 20032004 us dollars were $ 4,117 , $ 4,968 , $ 6,173 , and $ 9,276 for one , two , three , and four cmrfs , respectively . sullivan et al conducted a cmrf - related study using meps data but estimated health care expenditures and lost productivity attributable to cmrfs and comorbid obesity ( ie , bmi 30 kg / m).16 their findings suggested significantly higher medical expenditures and lost productivity for normal weight individuals with diabetes , dyslipidemia , or hypertension as compared with those without those conditions . for example , obese individuals with three risk factors had an increase of nearly $ 10,000 2007 us dollars in annual total adjusted health care expenditures as compared to normal weight individuals with zero risk factors . boudreau et al conducted a cmrf study in a managed care population , estimating health care costs with and without cmrfs , and by the number of cmrfs stratified by diabetes diagnosis.12 cmrfs included hypertension , weight risk ( bmi > 27 kg / m ) , high triglycerides , low high - density lipoprotein , and diabetes . the most expensive group of cmrfs included diabetes , dyslipidemia , and hypertension that had total annual costs of $ 9,650 in 2005 us dollars . a 24% increase in total annual health care costs per additional risk factor was demonstrated with similar trends observed when stratified by diabetes status . although the percentage increase and stratification of diabetes status were not similar to our study , these results are consistent with our findings that health care expenditures significantly increase for people with at least two to four cmrfs as compared with people without cmrfs . boudreau et al suggested focusing on specific metabolic syndrome risk factor combinations for health care resource planning . our study differs from these studies as our primary focus is on the number of cmrfs , regardless of a specific comorbid condition such as obesity , diabetes , or cad . moreover , we used the most recently available nationally representative survey data from meps and present lost - productivity outcomes as an additional source of the economic burden attributable to cmrfs . this study was cross - sectional where cmrf stratification and economic burden outcomes were measured contemporaneously . this may result in higher than actual health care expenditure estimates considering that cmrf definitions were based on utilization of health care resources such as prescription medications . we did not estimate health care resource utilization and expenditures when cmrfs were linked with cv disease events , or other conditions such as diabetes . there has been previous research suggesting that composite measures of the metabolic syndrome ( ie , all cmrfs included ) are not significant predictors of health care resource utilization.12,19 rather , some have suggested individual cmrfs such as abdominal obesity , comorbid conditions such as diabetes , or specific combinations of cmrfs and comorbid conditions ( eg , hypertension and diabetes ) are the drivers of high utilization . our study findings suggest that regardless of specific cmrfs , significant health care resources are consumed when patients have a combination of cmrfs as compared with patients with no cmrfs . this may represent an underreporting of included conditions20 and thus an underestimate of the economic burden . however , prevalence estimates reported here are similar , in some cases lower or higher , to other studies relying on recent self - reported data.8,21 for example , as compared with data using national health and nutrition examination survey ( nhanes ) , we found a higher prevalence estimate of abdominal obesity , yet slightly lower prevalence of elevated blood pressure and elevated glucose . overweight individuals specifically may underestimate their weight and overestimate their height.22,23 furthermore , meps does not contain information on undiagnosed conditions such as nhanes does , which may also lead to underestimated prevalence of these cmrfs . this study contributes to the body of literature suggesting as the number of cmrfs increase , regardless of specific underlying conditions , the economic burden increases for both direct health care expenditures and lost productivity . these results underscore the value of providing incentives to reduce the burden of these cmrfs from an economic and productivity standpoint . more attention should focus on treating the underlying metabolic syndrome instead of focusing on one condition alone .

bone - replacement graft materials have played an important role in regenerative dentistry for many years . several types of filling biomaterials have been evaluated for bone regeneration , and the choice of the biomaterial mostly depends on its features and application site . the grafts could be classified according to their origin as autologous , homologous , heterologous and alloplastic materials , and according to their mechanism as osteogenic , osteoconductive , and osteoinductive materials . osteogenic materials directly stimulate bone cells to synthesize bone tissue ; osteoconductive materials facilitate cell proliferation , migration and new bone apposition ; osteoinductive materials induce differentiation of mesenchymal cells into osteoblasts . since 1978 , autologous material has been used for bone regeneration and presently it is considered the gold standard in bone grafts since it has osteogenetic , osteoconductive and osteoinductive features . the advantage of autogenous bone is that it maintains bone structures such as minerals and collagen , as well as viable osteoblasts and bone morphogenetic proteins ( bmp ) ; furthermore , there is no immunological response to autologous grafts . homologous grafts , are composed by non - vital osseous tissue taken from one individual , stocked in bone banks , and transferred to another individual of the same species . there are three forms of homologous bone or allograft : fresh frozen , freeze - dried bone allograft ( fdba ) and demineralized freeze - dried bone allograft ( dfdba ) . homologous graft is thought to be osteoinductive and osteoconductive , but the amount of bmps in any single allograft has shown dramatic variability , and contradictory opinions about its biological properties are still present in literature [ 13 - 17 ] . heterologous grafts consist of deproteinized cancellous skeletal bone tissue that is harvested from one species and transferred to the recipient site of another species ; bovine bone being the most common source . heterologous materials have been used in several types of bone defects with satisfactory results ; the advantage is the maintenance of the physical dimension , and the disadvantage is that they are only osteoconductive . alloplastic materials are synthetic materials that have been developed to replace human bone and that are available in different sizes , forms and textures . they are biocompatible and are the most common type of graft materials utilized . the varying nature of commercially available synthetic graft materials , such as porosity , geometries , different solubility , and densities , determines their biological features and their resorption times . there are several types of alloplastic substances in clinical use nowdays : calcium phosphate - based ( caps ) , other ceramics ( e.g. hydroxyapatite - ha ) , biphasic calcium phosphate ( bcp ) , tricalcium phosphate ( tcp ) , calcium sulfate , and biocompatible composite polymers . calcium phosphate cement composites ( cpcs ) are osteoconductive materials rapidly integrate into the bone structure and are transformed into new bone by the action of bone cells responsible for the local bone remodelling . however , in spite of these good properties , synthetic materials have limitations due to their poor mechanical properties and slow biodegradation in vivo . in view of the biological limitations associated individually with graft materials , surgeons have attempted to augment the activity and physical properties with composite grafts combining molecular , cellular , and genetic tissue engineering technologies [ 21 - 23 ] . the molecular approach using bmps has received the most attention over the past decade . bmps are differentiation factors that are part of the transforming growth factor superfamily , but many other factors also contribute , such as transformating growth factor ( tgf-s ) , insuline - like growth factor ( igfs ) , fibroblast growth factor ( fgfs ) , plateled - derived growth factor ( pdgfs ) . the commercial availability of these growth factors ( gf ) has given oral and maxillofacial surgeons an additional option for the reconstruction of bony defects , but despite their potential usefulness , gf are still not available for routine use in practice . another gf approach is to use the patient 's own blood , separating out the platelet - rich plasma ( prp ) and adding this concentrated group of autologous gfs to the grafting material . prp is considered to be a rich source of autologous gfs , and the contribution of prp formulations to the bone healing process is thought to be based on the gfs contained . the addition of prp to autogenous grafts showed a more rapid and dense bone formation compared to autogenous grafts used alone for bone augmentation . prp has been also used in conjunction with allografts as a source of autologous gfs , but an improvement in bone formation when prp is added to these graft materials has not been demonstrated clearly . recently has been proposed to surgeons an highly purified bovine allograft characterized by preservation of the type i collagen matrix associated with spindle - shaped hydroxypatite crystals ( laddec , biohorizons , birmingham , usa ) . the results of preliminary studies suggest that presence of type i collagen fibbers in the matrix of a bone biomaterial could be of major interest to determine cell attachment , spreading and orientation of osteoblasts and that type i collagen can bind osteoblasts via specific cell surface receptors , the integrins . hence , the present study was undertaken to assess the role of platelet rich plasma with this highly purified bovine allograft in regeneration of osseous defects of jaws caused by cysts enucleation . the present study included 20 consecutively treated patients , between 34 and 68 years of age , who needed cystectomy for some kind of pathology in the oral cavity ( figures 1 - 4 ) . in all the patients , after cystectomy , prp mixed with laddec was used for bone regeneration . subjects with systemic diseases , renal disorders , regional malignancies , and respiratory problems were excluded from the study . all patients were examined with panoramic radiographs ( promax , planmeca.helsinky , finland ) converted into digital images using the computed system regius ( konica , minolta , tokyo , japan ) . periapical radiographs ( ultra - speeda , eastman kodak co , rochester ny , usa ) , by means of 65 kv dental x - ray unit equipped with a longcone ( oralix 65 s , gendex dental system s.r.l . , milano , italy ) were used before surgery for preoperative evaluation of size lesion , and at 1 month , 2 months , 4 months , and 6 months postsurgery , respectively , to assess the rate of bone regeneration . a silicone index material was fixated to the adjacent teeth , and a radiograph holder was constructed for each patient . this technique ensures that the same position of the radiograph could be reproduced at each visit . all these radiographs were compared with preoperative radiographs to check the height of bone regenerated ( defect bone fill ) from the base of the defect to cemento - enamel junction ( cej ) of the adjacent teeth . example of radiographic cystic cavity shrinkage : a = preoperative , b = 6 months postoperative . the nature of the study was explained to the patients and informed consent was obtained . the study was conducted in full accordance with ethical principles , including the world medical association declaration of helsinki , the protocol and consent form were approved by the institutional ethics committee of university of catania ( italy ) . the prp was performed at the department of hematology hospital " canizzaro " catania . prior to the start of the surgery , 300 ml of blood was drawn intravenously from patients and collected in sterile plastic vacuum tube coated with anti - coagulant citric acid and dextrose . automated centrifugation machine was used for obtaining prp with a speed of 1300 rpm for 10 min . after centrifugation , 3 layers were obtained : 1 ) an upper straw coloured fluid - ppp ( platelet poor plasma ) ; 2 ) a middle buffy coat rich in platelets ; 3 ) a lower layer rich in red blood cell ( rbc ) . the straw coloured plasma was collected along with buffy coat and 1 ml of the rbc layer . the prp was obtained in the form of a red button at the bottom of the test tube . this was collected with the help of a pasture pipette and transferred into a sterile tube . prp obtained after second centrifugation was placed in a sterile tube . for activation , 6 ml of calcium chloride and thrombin were added to prp . a first layer of prp gel was introduced into the residual bone cavity after cystectomy to stimulate the capillary regeneration in wound healing , and a second layer was mixed with laddec in a volume preparation of 1:1 . histological and histomorphometric evaluation 3 months after surgery a bone biopsy was performed in all patients . specimens were taken through the use of a milling cutter of 2.5 mm diameter trephine ( figure 5 ) . the biopsies were immediately fixed in 4% formaldehyde in a buffered solution of 0.1 m phosphate ( ph 7.3 ) at 4 c for 24 hours to their dispatch to the laboratory . samples were hydrated gradually with ethanol and soaked in epon 812 ( shell chemical co. , new york , ny , usa ) . decalcified sections of a thickness of 30 10 m were obtained by cutting , by means of buehler isomet ( buehler , an itw co , lake bluff , il , usa ) along the vertical axis of the cylinder marrow . the bone sections were stained with toluidine blue and were used for qualitative histological analysis and for quantitative histomorphometric analysis , carried out with fomi iii ( carl zeiss , ovberkochen , germany ) equipped with a microscope with image resolution ( dc 280 leica , wetzlar , germany ) . the height of regenerated bone ( defect bone fill ) in mm at four follow - up periods was compared with that from the preoperative periapical radiographs . the data set was analysed with the aid of the spss 13.0 package ( spss , chigaco , il , usa ) and a student t - test was used for comparision between values of different time periods . in the periapical radiographs , it was observed that the mean preoperative defect size was 22.5 mm with standard deviation of 4.5 when calculated from the base of the defect to the cej of the adjacent tooth . in the first month , the defect size reduced to 9.4 1.1 mm , the difference from the preoperative radiograph was 13.1 4.2 mm , and the size of the defect was filled by 56% . in the second month the defect size reduced to 8.4 0.6 mm , the difference from the preoperative radiograph was 14.1 4 mm , and the size of the defect was filled by 62% . in the fourth month , the defect size reduced to 4.7 1.5 mm , the difference from the preoperative radiograph was 17.8 4.4 mm , and the size of the defect was filled by 74% . in the sixth month , the defect size reduced to 1.1 2 mm , the difference from the preoperative radiograph was 21 4.5 mm , and the size of the defect was filled by 92% . the difference was significant between the postoperative 4 months and 6 months results ( table 1 ) . observation for the height of regenerated bone ( defect bone fill ) with platelet rich plasma + laddec application seen on periapical radiograph sd = standard deviation . the difference was significant between the post - operative 4 months and 6 months results . the postoperative successive periapical radiographs showed adequate consolidation ( regeneration ) of the bone , as manifested by homogeneous radiopacity . on observation for comparison of height of regenerated bone , it was noticed that , by first postoperative month about 56% of the defect was filled ; this gradually increased in each month and showed about 92% of defect fill at 6 months . in all the specimens , histological analysis showed a significant presence of bone tissue and vessels . both , histological and histomorphometric analysis , showed newly formed bone in contact with anorganic bone particles . the mean volume of vital bone was 68 1.6% and the mean percentage of vital bone was 48 2.4% . the mean percentage of inorganic particles in tissues was 20 1.2% of the total volume . all the samples analyzed did not evidence the presence of inflammatory cells ( figures 6 - 9 ) . roberto crespi ) . increased cellular activity with lines of newly formed bone tissue . cystectomy includes the removal of all inflamed soft tissues and sometimes application of different biomaterials to enhance new bone formation in the defect site . various bone grafts and barrier membranes can be used to achieve optimal healing and regeneration of the cystic cavity . recently it has been developed a procedure for bony defects regeneration utilizes prp in addition to bone grafts , and several studies have showed that a combination of prp with bone grafts promote bone regeneration [ 36 - 42 ] . however , other authors still argue the lack of scientific evidence for defending the use of prp associated with bone grafts in bone regeneration and recommend the surgeon to maintain a critical mind regarding its efficacy . in our opinion , the controversy found in the literature regarding the use of this technique could be probably related to a lack of standardization in the different prp formulations , and in the protocols , experimental models and surgical techniques employed . the cellular events responsible for healing are controlled and regulated by specific signalling molecules , growth factors , and cytokines . tgf - b1 , bone morphogenetic protein-2 ( bmp-2 ) , and pdgf - a are secreted by cells recruited to the healing wound which are released in response to wounding stimuli detected at the cell surface . the local availability of these growth factors is enhanced by about threefold or greater in concentration by addition of autologous prp . it has been also reported that during the early stages of wound healing prp has a strong stimulant effect on capillary regeneration . particularly , the publications , which point out positive features of prp over the last years , have stressed : importance of controlled release systems of growth and differentiation factors using biomaterials in combination with prp ; enhancement of osteogenesis and angiogenesis ; inhibition of osteoclast activation ; the enhancement of bone density adding prp to a suboptimal dose of recombinant human bmp-2 ( rhbmp-2 ) ; a significant increase of early bone marrow stromal cells ( bmscs ) proliferation and differentiation using the combination of rhbmp-2 and bfgb ( one of the signalling molecules of prp ) ; relevance of pdgf and transforming growth factors ( tgf - al and tgf32 ) for bone regeneration . our study used these principles for enhancing the osteoconductive property of a new highly purified bovine allograft ( laddec ) by addition of autologous prp in regeneration of osseous defects of jaws caused by cystectomy . radiographic assessments of present study indicated that this association induced a fast new bone growth in the cystic cavities . it was observed that the defect was filled by 56% at the first month , and after a time interval of 6 months postoperatively the defect was filled by 92% , showing a significant increase in vertical height on radiographs . the clinical efficacy of the association prp and laddec reported from our study is also supported by histological data that we have documented , since the mean volume of vital bone at 6 months was found to be 68 1.6% and the mean percentage of vital bone 48 2.4% . these data are in accordance with and in support of what has been previously suggested by others studies that have shown that adding prp to graft material significantly accelerates the rate of bone formation and improves trabecular bone density as compared to sites treated with only graft material . contrary to this , there are also reports that suggest that the use of prp in combination with anorganic bovine bone mineral does not benefit bone regeneration [ 53 - 60 ] , highlighted that osteoblasts have difficulties in adhering to allograft smooth surfaces . since some allograft has a smooth surface , most attempts of creating new bone using bovine allografts may not be able to provide close contact between bone and the bovine material under reproducible conditions . basl and co - workers suggested that treatments applied to bovine allografts to prevent immunological , inflammatory , bacteriological or virological adverse responses , may also interact with type i collagen , to which can bind osteoblasts via specific cell surface receptors , the integrins . the authors have compared in vitro two different bovine allografts that displayed similar architectural organization with connected plates and rods and similar surface topography and roughness . they differed by the presence or not of collagen type i. the first one was characterized by preservation of the type i collagen matrix associated with spindle - shaped hydroxypatite crystals and the second was solely composed by heat - modified apatite crystals . osteoblast - like cells ( saos-2 ) were cultured on both biomaterials and examined in scanning and transmission electron microscopy after 7 and 14 days . both biomaterials were cytocompatible as demonstrated by good ultrastructural cell preservation . at the surface of the collagen containing biomaterial , cells were elongated in shape and oriented according to the trabecular architecture and to the superficial collagen network . after 14 days of culture , cells were confluent and the biomaterial surface was hidden by the cell sheet . the beta 1 integrin subunit was detected by immunogold in transmission electron microscopy in close relationship with the superficial collagen fibbers of the biomaterial and with the outer cell surface . when cultures were carried out in presence of anti beta 1 integrin subunit , cells were packed and piled up with lack of specific orientation . at the surface of the deproteinized biomaterial , cells were globular without specific disposition and often partially attached to the surface . after 14 days of culture , large areas of the biomaterial surface remained uncovered . anti beta 1 subunits conjugated with gold particles were detected around the cells but with no specific association with the deproteinized biomaterial . these results strongly suggest that the chemical nature of the surface of bovine allografts directly influences adhesion process , shape , and spatial organization of cultured osteoblastic cells . furthermore , the presence of type i collagen fibbers in the matrix seems to be of major interest to determine cell attachment , spreading and orientation via interaction between type i collagen and beta 1 integrin subunit of osteoblasts . in contrast , at the surface of the single mineral matrix , cells were round shaped with random disposition . this data has been also confirmed by another research in which has been documented that the beta 1-integrin subunit was localized at the outer surface of cells , in close association with collagen and at the contact points between cells and laddec allograft . these in vitro results are still limited and must be confirmed by other studies , but they could support favourable outcomes concerning newly grown bone achieved in our present clinical study performed using a combination of prp and laddec . we have to add that it is not possible to compare the published clinical studies and animal trials concerning the association between prp and heterologous grafts , due to the varying methodologies applied and due to the varying nature and biological features of commercially available bovine graft materials . the results of our clinical and histological study seems however to confirm that preservation of the type i collagen matrix associated with spindle - shaped hydroxaypatite crystals in bovine graft bone substitutes may promote the biomaterial - prp interaction . within the limits of this study , the treatment with a combination of prp and laddec bovine graft seems to lead to significantly favourable and fast bone regeneration after grafting enucleated mandibular cyst cavities , however further studies are necessary to assess the long - term effectiveness of prp - laddec association , and a larger sample size is recommended . the results of this study showed that laddec in association with platelet rich plasma has a potential for routine clinical use for regeneration of cystic bony defects . roberto crespi ( ateneo vita salute san raffaele , milano , italy ) for histological and histomorphometric analysis .

self - expanding metal stents ( semss ) are a useful therapy to palliate malignant and benign luminal gastrointestinal ( gi ) obstruction.1 although sems placement has been widely reported for colonic , esophageal , and gastric obstructions,2 - 5 little is known about its applicability for small bowel strictures . , we present a balloon overtube technique that enabled successful sems placement in the distal duodenum . an 80-year - old woman who underwent a pylorus - preserving whipple procedure 6 years previous for pancreatic head cancer and recurrent disease with duodenal obstruction of the previous placement of two palliative semss was referred to our institution for bowel obstruction distal to the ligament of treitz . the previously placed sems was obstructed owing to ingrowth with tissue and the small bowel kinking distally to the stent ( fig . a large amount of impacted food was removed by flushing and suctioning . using endoscopic and fluoroscopic visualization , deep enteroscopy was then performed , and no other obstructions were found in the afferent or efferent limbs . the roux - en - y anastomosis was located approximately 50 cm distal to the pylorus . a new sems ( 90-mm length , 10-mm diameter ; boston scientific , natick , ma , usa ) was inserted across the previous two sems using the herein described balloon overtube technique using the following six steps . first , the enteroscope was kept across the stenosis in the upper jejunum ( fig . second , a jagwire guidewire ( boston scientific ) was inserted in the jejunum ( figs . 2 , 3 ) . third , the scope was removed under fluoroscopic guidance and the wire was left in place ( fig . fourth , the overtube , which was looped and kinked in the j - shaped stomach , was straightened ( fig . a trick was used to straighten the overtube without losing its position ; that is , the balloon of the overtube was inflated distally to the stent . this maneuver allowed the overtube tip to get hooked to the distal part of the overtube while being pulled ( i.e. , straightened ) . the overtube straightening was essential to advance the stent through the wire inside the overtube and across the previous stents ( fig . sixth , the distal part of the stent was deployed under fluoroscopic guidance while the now deflated overtube was carefully brought back into the stomach through the old stent , thus permitting the new stent to deploy distal to the old stent and the overtube tip across the obstruction ( figs . 6 , 7 ) . the patient is asymptomatic 3 months after the procedure . malignant small bowel obstruction usually results from primary small bowel malignant neoplasm , invasive locoregional tumors or metastasis.1,6 - 9 semss represent a potential palliative measure.6 - 9 in an interesting study , lee et al.6 describe the usefulness of the dbe technique to advance a wire through malignant small bowel strictures to enable sems placement . in that study , the authors passed another scope along the wire to visualize and aid the stent insertion . in our case , we used an overtube as an extralarge working channel . although a similar technique using spiral single - balloon enteroscopy and spiral enteroscopy was previously described,7 - 9 our case is of additional interest because we describe in detail all the steps necessary to perform a balloon overtube placement of a sems into the proximal small bowel . in addition , limited data are available on this technique.10 sems placement within the small bowel is challenging for both anatomic and mechanical reasons . the technical challenge includes the tortuous endoscope position and limitations of the stent delivery devices , which are often stiff and inflexible , that disallows easy travel through angled or long curves of the gi tract , such as a j - shaped stomach and the duodenal sweep . in this case , we show how a balloon overtube helped to overcome most of this challenges , allowing for accurate sems placement . use of the dbe permitted the placement of the overtube distal to the previously placed metal stent . inflation of the overtube balloon allowed for the tube to be straightened while remaining hooked distal to the stent . the straightened overtube allowed us to advance the stent over a previously placed floppy guidewire . although stent placement through the endoscopic channel is feasible , the previously placed sems and the angulated position of the distal duodenum prevented us from choosing this option . although several overtubes are available for use during endoscopy , our technique using the balloon overtube is unique , adds to the interventional endoscopist 's armamentarium , and represents another useful option for sems placement in the small bowel . in summary , we recommend that clinicians consider using an overtube or balloon overtube when placing stents into difficult locations within the gi tract .

gatti and colleagues reported the first successful allogenic marrow graft in a patient with severe combined immunological deficiency using a sibling donor in 1968 . with the refinement of hla testing , anti - cancer drugs , intensive care units , the spectrum of hematopoietic stem cell transplant ( hsct ) has widened to include malignant and non - malignant hematological conditions , and connective tissue disorders . with increasing availability and affordability , hsct is being performed with refinement of the procedure and post - transplant care , the quantum of long - term survivors in ever increasing . with increased survival after hsct , more long - term complications are being observed . these can arise as a consequence of the underlying disease , chemotherapy and radiotherapy used in preparative regimens , the hsct itself , immunosuppressive drugs , graft versus host disease ( gvhd ) , induction of autoimmunity . the recipient might develop endocrine organ dysfunction in the acute period after transplant or at a later stage . the endocrine concerns after hsct are quite different in the pediatric and adult recipients . in the pediatric population , the growth and puberty are the main concerns , while in adults fertility is the main concern . although hsct has been performed in india at many centers , there is no data on endocrine dysfunction among patients receiving hsct . hence , this study was carried out with the aim to evaluate endocrine dysfunction among patients undergone hsct in the past . patients who had received steroids in the preceding 6 months prior to endocrine testing were excluded . all patients had received busulphan ( 16 mg / kg in 4 days ) and cyclophosphamide ( 120 mg / kg in 2 days ) . the hsct carried out at our center used a steroid free protocol and none of the patient received total body irradiation ( tbi ) . the study group was divided into two groups : group - i comprised of children and adolescents ( < 18 years , n = 22 ) and group - ii comprised of adults ( > 18 years , n = 28 ) . this group was divided into two subgroups in order to see the effect of puberty age 10 years ( 45% ) and > 10 years ( 55% ) . all subjects were analyzed according to duration post hsct : 1 year ( 46% ) and > 1 year ( 54% ) , type of hsct allogenic ( 78% ) and autologous ( 22% ) , presence ( 30% ) or absence ( 70% ) of gvhd . samples for hormonal estimation were collected in fasting state at 0800 hours and kept at 80c . stimulation tests for cortisol and growth hormone ( gh ) were interpreted based on peak values achieved during insulin tolerance test ( itt ) , which is the gold standard . this study was conducted according to the guidelines laid down in the declaration of helsinki and all procedures involving human subjects / patients were approved by the institutional human ethics committee . written informed consent was obtained from all subjects / patients . hormone levels were measured using commercial kit provided by immunotech , beckman coulter company , france . primary and subclinical hypothyroidism was defined as low free thyroxin ( ft4 ) ( normal 0.8 - 2.1 ng / ml ) with raised thyroid - stimulating hormone ( tsh ) ( normal 0.5 - 6.5 iu / ml ) and normal ft4 with raised tsh , respectively . a peak cortisol < 18 g / dl after itt constituted adrenal insufficiency and peak gh level < 10 ng / ml in children and adolescent ( 18 years ) and < 5 ng / ml in adults defined gh deficiency ( ghd ) . gonadal failure was defined as elevated luteinizing hormone ( lh ) and follicular - stimulating hormone ( fsh ) ( > 15 gonadal failure was further classified as germ cell ( gc ) failure ( elevated fsh , normal lh ) or leydig cell ( lc ) failure ( elevated lh ) which was called compensated if testosterone levels were normal ( 3 - 10 ng / ml ) or decompensated if testosterone levels were <3 ng / ml . insulin resistance and secretion were calculated with homeostatic model assessment ( homa - ir = fasting glucose in mmol / l*fasting insulin in iu / l divided by 22.5 ; homa- = 20*fasting insulin in iu / l divided by fasting glucose in mmol / l 3.5 ) . statistical analysis was carried out using epi info 3.5.3 ( cdc , atlanta , ga , usa ) . all parametric data were analyzed by student 's t - test . if barlett 's chi - square test for equality of population variances was < 0.05 , then the kruska if value in any cell was < 5 then the fisher exact test was used . this study consisted of 50 patients who have undergone hsct . basic characteristic of study population is given in table 1 . twenty - seven ( 54% ) patients had malignant hematopoietic diseases and 23 ( 46% ) had non - malignant hematopoietic diseases [ table 1 ] . adrenal insufficiency ( ai ) was present in 60% , hypergonadotropic hypogonadism ( hh ) in 60% , ghd in 54% , hypothyroidism in 4% , hyperprolactinemia in 4% , new onset diabetes after transplant ( nodat ) in 4% , and impaired fasting glucose ( ifg ) in 6% in the total study group . twenty - eight percent had single hormone deficiency , 66% had two , and 16% had three hormonal deficiencies [ figure 1 ] . basic characteristics of study population endocrinal abnormalities according to age groups spectrum of endocrine deficiency in the study group growth velocity was evaluated in group - i patients who had successfully completed 1 year of transplant ( 11 patients ) . only two of these patients had growth velocity of less than 4 cm and were started on growth hormone therapy [ table 3 ] . the mean height standard deviation score ( sds ) was not statistically significant in those who had ghd ( 0.46 0.85 ) when compared with subjects without ghd ( 0.87 0.98 , p value 0.42 ) . seventy six percent allo - hsct recipients were diagnosed to have ghd and the only patient who had an auto - hsct also had ghd . those who had ghd , mean peak gh value was 3.76 3.09 ng / ml [ figure 2 ] . twelve of the 17 children had severe ghd ( peak gh < 5 ng / ml ) , out of these 8 were tm and the other 2 were all who had received prophylactic cranial irradiation . patients with tm had increased risk for ghd ( risk ratio 2.86 , 95% ci : 1.0 - 8.18 ; p = 0.016 ) . ninety - one percent , who had received hsct more than a year ago , had ghd compared to 64% than those whose hsct duration was less than 1 year ( risk ratio 1.94 , 95% ci : 0.95 - 3.98 ; p = 0.15 ) . eighty - three percent of patients who had gvhd had ghd when compared to those without gvhd ( 75% ) , this was not statistically significant ( p = 0.58 ) [ table 4 ] . growth parameters in group - i who had successfully completed 1 year of transplant hormonal values in the study group . a : basal and peak cortisol after insulin tolerance test ( itt ) ; b : peak growth hormone after itt in children and adults ; c : gonadal hormone pattern in children ; d : gonadal hormone pattern in females ; e : gonadal hormone pattern in males ; e : thyroid function test characteristics of the hormonal defi ciency in the study group in group - ii ( n = 28 ) , 36% had ghd . those who had ghd , mean peak gh response was 2.14 1.78 ng / ml . the type of transplant ( p = 0.48 ) and gvhd ( p = 0.60 ) had no bearing on the occurrence of ghd . those who had been transplanted more than a year back had higher risk of developing ghd ( risk ratio 1.67 , 95% ci : 0.58 - 4.78 ; p = 0.26 ) . in group - i , 36% had hypogonadism . the mean fsh and lh values were 54.75 36.25 iu / l and 33.05 18.83 iu / l , respectively , in those with hh [ figure 2 ] . in those who were 10 years when they were transplanted only one had definitive evidence of hypogonadism and in the rest no comment can be made due to immaturity of gonadal axis . in those who were > 10 years , 58% had hh . in contrast to group - i , 79% in group - ii had gonadal dysfunction . hh was seen in all female recipients , both fsh and lh were elevated in all and none of them had recovered ovarian function . seventy - nine percent of males had gc dysfunction , and all of them had azoospermia . lc dysfunction was present in 89% of males , and 79% of them had compensated lc dysfunction . the mean fsh , lh , and testosterone values were 33.78 15.60 iu / l , 27.44 17.63 iu / l , and 5.66 3.66 pg / ml , respectively . the duration after hsct ( p = 0.57 ) , type of transplant ( p = 0.20 ) , and gvhd ( p = 0.43 ) had no impact on development gonadal dysfunction [ table 4 ] . those who underwent allo - hsct had a statistically significant risk of developing ai ( p = 0.015 ) , so were patients whose transplant duration was less than 1 year ( p = 0.032 ) . history of steroid intake in past was not associated with increased risk of ai ( risk ratio 1.07 , 95% ci : 0.76 - 1.51 , p = 0.94 ) . though patients with gvhd had higher risk for development of ai ( risk ratio 1.26 , 95% ci : 0.89 - 1.79 ) , this was not statistically significant the mean peak cortisol value was 12.75 4.16 g / dl [ figure 2 ] . mean peak cortisol was not significantly different according to duration of hsct ( 12.18 4.01 vs. 13.62 4.40 g / dl ( p = 0.36 ) . patients with ai had significantly lower high - density lipoprotein cholesterol ( hdl - c ) than those had normal adrenal functions ( 30.9 7.48 mg / dl v / s 40.30 9.45 ; p value 0.0003 ) , but low density lipoprotein cholesterol ( ldl - c ) levels were similar ( 93.11 31.15 vs. 84.20 22.62 mg / dl ; p = 0.27 ) . one patient who had received auto - hsct for progressive systemic sclerosis developed acute reversible autoimmune hypothyroidism five months after transplant . a four and half year old tm patient developed subclinical hypothyroidism with mild elevation of anti - tpo ab . two patients had nodat and three had ifg . the mean body mass index ( bmi ) , homa - ir , and homa- in our cohort was 18.2 3.6 kg / m , 1.07 1.67 , and 77.81 116.15 , respectively . the mean bmi ( 19.7 1.3 vs. 18.6 3.8 kg / m ; p = 0.52 ) and homa- ( 148.87 143.56 vs. 69.92 111.85 ; p = 0.15 ) were comparable in patients with or without dysglycemia . homa - ir was significantly higher ( 3.04 2.37 vs. 0.85 1.44 ; p = 0.004 ) in patients with dysglycemia than those with normoglycemia . the various parameters concerning glucose metabolism in patients with dysglycemia are shown in table 5 . growth velocity was evaluated in group - i patients who had successfully completed 1 year of transplant ( 11 patients ) . only two of these patients had growth velocity of less than 4 cm and were started on growth hormone therapy [ table 3 ] . the mean height standard deviation score ( sds ) was not statistically significant in those who had ghd ( 0.46 0.85 ) when compared with subjects without ghd ( 0.87 0.98 , p value 0.42 ) . seventy six percent allo - hsct recipients were diagnosed to have ghd and the only patient who had an auto - hsct also had ghd . those who had ghd , mean peak gh value was 3.76 3.09 ng / ml [ figure 2 ] . twelve of the 17 children had severe ghd ( peak gh < 5 ng / ml ) , out of these 8 were tm and the other 2 were all who had received prophylactic cranial irradiation . patients with tm had increased risk for ghd ( risk ratio 2.86 , 95% ci : 1.0 - 8.18 ; p = 0.016 ) . ninety - one percent , who had received hsct more than a year ago , had ghd compared to 64% than those whose hsct duration was less than 1 year ( risk ratio 1.94 , 95% ci : 0.95 - 3.98 ; p = 0.15 ) . eighty - three percent of patients who had gvhd had ghd when compared to those without gvhd ( 75% ) , this was not statistically significant ( p = 0.58 ) [ table 4 ] . growth parameters in group - i who had successfully completed 1 year of transplant hormonal values in the study group . a : basal and peak cortisol after insulin tolerance test ( itt ) ; b : peak growth hormone after itt in children and adults ; c : gonadal hormone pattern in children ; d : gonadal hormone pattern in females ; e : gonadal hormone pattern in males ; e : thyroid function test characteristics of the hormonal defi ciency in the study group in group - ii ( n = 28 ) , 36% had ghd . those who had ghd , mean peak gh response was 2.14 1.78 ng / ml . the type of transplant ( p = 0.48 ) and gvhd ( p = 0.60 ) had no bearing on the occurrence of ghd . those who had been transplanted more than a year back had higher risk of developing ghd ( risk ratio 1.67 , 95% ci : 0.58 - 4.78 ; p = 0.26 ) . the mean fsh and lh values were 54.75 36.25 iu / l and 33.05 18.83 iu / l , respectively , in those with hh [ figure 2 ] . in those who were 10 years when they were transplanted only one had definitive evidence of hypogonadism and in the rest no comment can be made due to immaturity of gonadal axis . in those who were in contrast to group - i , 79% in group - ii had gonadal dysfunction . hh was seen in all female recipients , both fsh and lh were elevated in all and none of them had recovered ovarian function . seventy - nine percent of males had gc dysfunction , and all of them had azoospermia . lc dysfunction was present in 89% of males , and 79% of them had compensated lc dysfunction . the mean fsh , lh , and testosterone values were 33.78 15.60 iu / l , 27.44 17.63 iu / l , and 5.66 3.66 pg / ml , respectively . the duration after hsct ( p = 0.57 ) , type of transplant ( p = 0.20 ) , and gvhd ( p = 0.43 ) had no impact on development gonadal dysfunction [ table 4 ] . those who underwent allo - hsct had a statistically significant risk of developing ai ( p = 0.015 ) , so were patients whose transplant duration was less than 1 year ( p = 0.032 ) . history of steroid intake in past was not associated with increased risk of ai ( risk ratio 1.07 , 95% ci : 0.76 - 1.51 , p = 0.94 ) . though patients with gvhd had higher risk for development of ai ( risk ratio 1.26 , 95% ci : 0.89 - 1.79 ) , this was not statistically significant ( 73% vs. 80% ; p = 0.47 ) . the mean peak cortisol value was 12.75 4.16 g / dl [ figure 2 ] . mean peak cortisol was not significantly different according to duration of hsct ( 12.18 4.01 vs. 13.62 4.40 g / dl ( p = 0.36 ) . patients with ai had significantly lower high - density lipoprotein cholesterol ( hdl - c ) than those had normal adrenal functions ( 30.9 7.48 mg / dl v / s 40.30 9.45 ; p value 0.0003 ) , but low density lipoprotein cholesterol ( ldl - c ) levels were similar ( 93.11 31.15 vs. 84.20 22.62 mg / dl ; p = 0.27 ) . one patient who had received auto - hsct for progressive systemic sclerosis developed acute reversible autoimmune hypothyroidism five months after transplant . a four and half year old tm patient developed subclinical hypothyroidism with mild elevation of anti - tpo ab . two patients had nodat and three had ifg . the mean body mass index ( bmi ) , homa - ir , and homa- in our cohort was 18.2 3.6 kg / m , 1.07 1.67 , and 77.81 116.15 , respectively . the mean bmi ( 19.7 1.3 vs. 18.6 3.8 kg / m ; p = 0.52 ) and homa- ( 148.87 143.56 vs. 69.92 111.85 ; p = 0.15 ) were comparable in patients with or without dysglycemia . homa - ir was significantly higher ( 3.04 2.37 vs. 0.85 1.44 ; p = 0.004 ) in patients with dysglycemia than those with normoglycemia . the various parameters concerning glucose metabolism in patients with dysglycemia are shown in table 5 . hsct has come a long way in curing many hematological and non - hematological diseases . with increasing survival many studies have evaluated endocrine complications , but these studies reported endocrine dysfunctions as a part of all post hsct complications or had not used the prevailing gold standards to document endocrine deficiencies . in this study , bucy regimen was used uniformly in hsct protocol and this study is among the largest cohort to evaluate endocrine functions in this subset of hsct patients [ table 6 ] . comparison of the endocrine dysfunction of few prominent studies with the present study in the present study , though most of the children had ghd , only two required gh therapy , based on growth velocity . important factors for adequate growth in our cohort were steroid - free transplant regimen and the absence of tbi in the protocol . this in coherence with an earlier study which showed that chemotherapy - only based regimen does nt disturb growth in children , in fact they noted spurt in growth after hsct . however , another study had shown comparable growth deceleration after hsct when either bucy or tbi / cy protocols were used . there was no significant difference in mean height standard deviation score ( sds ) for patients with and without ghd . the interval between the onset of a hematological disorder and the transplant is less as compared to other end stage disease for which transplant is performed . the high prevalence of ghd in this group can be attributed to the fact that 68% of the patients in this group had tm . those who had tm had higher risk of developing ghd ( odds ratio 5.33 , 95% ci : 1.27 - 22.32 ; p = 0.016 ) than other patients . whether ghd was present before transplant , as is expected in tm due to hemosiderin deposition in pituitary , ca nt be commented due to unavailability of pre - transplant endocrine work - up . brauner et al . reported that those who had received tbi had a decrease in growth velocity in spite of normal peak gh , and those who had received chemotherapy alone had catch up growth . however , in our cohort two patients who were successfully treated for gvhd had normal growth velocity , which has also been reported by cohen et al . bakker et al . found unexplained growth disturbance in 35% of children without growth limiting disorder , who had received busulfan - based preparative regimen . there have not been many studies which have evaluated the growth hormone secretion pattern in adults post hsct . we also speculate that ghd was a late complication after hsct and busulfan preconditioning can be a major factor . however , they used gh increment after ( < 5 g / dl ) ghrh stimulation test as the basis for diagnosis of ghd . in our study , we have used itt , the gold standard for diagnosing ghd in adults . the high prevalence of ghd requires attention due to increased morbidity and poor quality of life associated with it in adults . these patients probably require further follow - up and re - evaluation to recommend growth hormone ( gh ) therapy . none of them have received gh therapy . in group-1 , 36% and in group - ii 79% had hh . neither the sex , duration after transplant , nor gvhd had any significant effect on the occurrence of hh . noted gonadal functions in long - term survivors of childhood hsct65.5% of females and 64.3% of males had gonadal dysfunction , which is similar to observed in the present study . they observed that risk of gonadal dysfunction was significantly higher with bucy preconditioning in females . fsh and lh elevated in 100% and 89% of boys aged more than 14 years and 75% and 75% in females aged more than 13 years . however , all but three of these had normal pubertal development . in the present study , among three hypogonadal girls , one had secondary amenorrhea , one had delayed puberty , and another had entered puberty . of the four hypogonadal boys , de sanctis et al . noted that in prepubertal tm patients receiving hsct , 80% of girls went on to develop gonadal failure , but all boys had fsh and lh in the normal range . in the present study , of the 15 tm , 6 children were in the pubertal group and out of these , 2 girls and 1 boy had definite evidence of hh . studied ovarian function in 31 girls who had received hsct , 81% had ovarian failure , and fsh was elevated in 100% , and lh in 79% of the recipients . most of them had received either busulfan - based regimen or tbi as the preparative regimen . among post - pubertal patients , all males with hh had azoospermia and all females had hh with secondary amenorrhea . the pattern of development of hh is characteristic in males , the gc are predominantly involved , and lc damage is minimal as evidenced by low testosterone in only two patients . anserini et al . demonstrated a high incidence of azoospermia ( 70% ) , but they also interestingly showed reversal of azoospermia in a significant proportion . studied 40 allo - hsct patients of whom 95% of women had ovarian insufficiency and 47% male had abnormal spermatogenesis . interestingly we found that history of steroid intake did nt increase the risk of ai and so was history of gvhd . ai was present in 78% patients with hsct duration < 1 year than 44% in those with hsct duration > 1 year . mean peak cortisol also increased with increasing interval after hsct ( 12.17 v / s 13.62 g / dl ) and more number of subjects had cortisol < 10 g / dl with duration < 1 year ( 5 vs. 1 ) . the acth stimulation test might not be an appropriate test to detect secondary ai in acute setting . none of the studies have commented the type of ai post - hsct and it has been assumed to be due to steroids . assuming that chances of secondary ai is high , an itt would be the most ideal test in this situation . unfortunately , we were unable to measure plasma acth . in one of the earliest studies , sanders et al . noted ai in 24% of patients , but the methodology used remains questionable . used itt in tbi - based hsct to demonstrate ai in 2 of the 31 evaluated patients . found ai in 4 of the 40 patients evaluated had secondary adrenal insufficiency and all of them were on steroids . however , they diagnosed ai on the basis of basal cortisol values which can be erroneous . tauchmanov and colleagues found ai in 30% of auto - hsct recipients when evaluated within 1 year of transplant . they had nt performed any stimulation test and all patients had received steroids in past . all these patients had no symptoms related to adrenal insufficiency , hence early assessment post - hsct is important to start supplement and prevent acute adrenal crisis at the time of stress like infections , which they are predisposed to being immune compromised status . the mean hdl - c value in ai group was significantly lower compared to non - ai group . ai patients also had a hdl - c values lower than mean for indians i.e. , ~44 mg / dl . the low incidence of thyroid dysfunction in our study may be due to the absence of tbi in our preparative regimen . in one of the largest studies till date subclinical hypothyroidism was seen in 15% , overt hypothyroidism in 1.3% , central hypothyroidism in 9.3% , and hyperthyroidism in 2.9% . found overt hypothyroidism in 0.03% , subclinical hypothyroidism in 26.5% , and hyperthyroidism in one subject . siekierska - hellmann et al . found that at 1 year none of patients developed overt hypothyroidism in hsct recipients prepared with chemotherapy . somali et al . in non - tbi - based hsct protocol found overt hypothyroidism in 6% of male patients and 5% of female patients and subclinical hypothyroidism in 13% of males and 5% of females . in the present study , two patients developed nodat and three had ifg [ table 5 ] . both the patients of nodat had history of steroid intake and their glycemic control was initially maintained by basal bolus insulin regimen and subsequently on metformin . reported a very high incidence of nodat-30% with 2 years of transplant and also showed reversal in 70% of them . however , low prevalence of nodat in the present study could be due to steroid - free immunosuppression and inclusion of children in the total cohort . there have been very few studies which have addressed insulin resistance ( ir ) and secretion in post - hsct patients . one of the early studies had utilized the intravenous glucose tolerance test to show that after hsct , ir is common . another study when evaluating bone health in post - hsct patients found high homa - ir and homa-. an italian group had also shown elevated homa - ir in their post - hsct cohort . these results indicate that post - hsct recipients are insulin sensitive ; however , those who develop dysglycemia tend to have ir as a pathophysiologic event . however , we were unable to compare these values with age- and sex - matched controls . comparison of metabolic parameters in our group with few of those reported in literature following were the limitations of our study . firstly , the present study was a cross - sectional study without the control group . secondly , pre - transplant endocrine status of the cohort was not available , and hence the findings can not be completely attributed to hsct . the large number of tm patients in group - i can bias the effect of hsct , as tm by itself is known to be associated with many endocrine dysfunction . finally , ai could nt be classified as primary or secondary as plasma acth could nt be measured due to local constraints . in the present study , though most of the children had ghd , only two required gh therapy , based on growth velocity . important factors for adequate growth in our cohort were steroid - free transplant regimen and the absence of tbi in the protocol . this in coherence with an earlier study which showed that chemotherapy - only based regimen does nt disturb growth in children , in fact they noted spurt in growth after hsct . however , another study had shown comparable growth deceleration after hsct when either bucy or tbi / cy protocols were used . there was no significant difference in mean height standard deviation score ( sds ) for patients with and without ghd . the interval between the onset of a hematological disorder and the transplant is less as compared to other end stage disease for which transplant is performed . the high prevalence of ghd in this group can be attributed to the fact that 68% of the patients in this group had tm . eighty percent of tm had ghd . those who had tm had higher risk of developing ghd ( odds ratio 5.33 , 95% ci : 1.27 - 22.32 ; p = 0.016 ) than other patients . whether ghd was present before transplant , as is expected in tm due to hemosiderin deposition in pituitary , ca nt be commented due to unavailability of pre - transplant endocrine work - up . brauner et al . reported that those who had received tbi had a decrease in growth velocity in spite of normal peak gh , and those who had received chemotherapy alone had catch up growth . however , in our cohort two patients who were successfully treated for gvhd had normal growth velocity , which has also been reported by cohen et al . found unexplained growth disturbance in 35% of children without growth limiting disorder , who had received busulfan - based preparative regimen . there have not been many studies which have evaluated the growth hormone secretion pattern in adults post hsct . we also speculate that ghd was a late complication after hsct and busulfan preconditioning can be a major factor . however , they used gh increment after ( < 5 g / dl ) ghrh stimulation test as the basis for diagnosis of ghd . in our study , we have used itt , the gold standard for diagnosing ghd in adults . the high prevalence of ghd requires attention due to increased morbidity and poor quality of life associated with it in adults . these patients probably require further follow - up and re - evaluation to recommend growth hormone ( gh ) therapy . neither the sex , duration after transplant , nor gvhd had any significant effect on the occurrence of hh . noted gonadal functions in long - term survivors of childhood hsct65.5% of females and 64.3% of males had gonadal dysfunction , which is similar to observed in the present study . they observed that risk of gonadal dysfunction was significantly higher with bucy preconditioning in females . fsh and lh elevated in 100% and 89% of boys aged more than 14 years and 75% and 75% in females aged more than 13 years . however , all but three of these had normal pubertal development . in the present study , among three hypogonadal girls , one had secondary amenorrhea , one had delayed puberty , and another had entered puberty . of the four hypogonadal boys , de sanctis et al . noted that in prepubertal tm patients receiving hsct , 80% of girls went on to develop gonadal failure , but all boys had fsh and lh in the normal range . in the present study , of the 15 tm , 6 children were in the pubertal group and out of these , 2 girls and 1 boy had definite evidence of hh . studied ovarian function in 31 girls who had received hsct , 81% had ovarian failure , and fsh was elevated in 100% , and lh in 79% of the recipients . most of them had received either busulfan - based regimen or tbi as the preparative regimen . among post - pubertal patients , all males with hh had azoospermia and all females had hh with secondary amenorrhea . the pattern of development of hh is characteristic in males , the gc are predominantly involved , and lc damage is minimal as evidenced by low testosterone in only two patients . anserini et al . demonstrated a high incidence of azoospermia ( 70% ) , but they also interestingly showed reversal of azoospermia in a significant proportion . studied 40 allo - hsct patients of whom 95% of women had ovarian insufficiency and 47% male had abnormal spermatogenesis . interestingly we found that history of steroid intake did nt increase the risk of ai and so was history of gvhd . ai was present in 78% patients with hsct duration < 1 year than 44% in those with hsct duration > 1 year . mean peak cortisol also increased with increasing interval after hsct ( 12.17 v / s 13.62 g / dl ) and more number of subjects had cortisol < 10 g / dl with duration < 1 year ( 5 vs. 1 ) . the acth stimulation test might not be an appropriate test to detect secondary ai in acute setting . none of the studies have commented the type of ai post - hsct and it has been assumed to be due to steroids . assuming that chances of secondary ai is high , an itt would be the most ideal test in this situation . unfortunately , we were unable to measure plasma acth . in one of the earliest studies , sanders ogilvy - stuart et al . used itt in tbi - based hsct to demonstrate ai in 2 of the 31 evaluated patients . found ai in 4 of the 40 patients evaluated had secondary adrenal insufficiency and all of them were on steroids . however , they diagnosed ai on the basis of basal cortisol values which can be erroneous . tauchmanov and colleagues found ai in 30% of auto - hsct recipients when evaluated within 1 year of transplant . they had nt performed any stimulation test and all patients had received steroids in past . all these patients had no symptoms related to adrenal insufficiency , hence early assessment post - hsct is important to start supplement and prevent acute adrenal crisis at the time of stress like infections , which they are predisposed to being immune compromised status . the mean hdl - c value in ai group was significantly lower compared to non - ai group . ai patients also had a hdl - c values lower than mean for indians i.e. , ~44 mg / dl . the low incidence of thyroid dysfunction in our study may be due to the absence of tbi in our preparative regimen . in one of the largest studies till date , subclinical hypothyroidism was seen in 15% , overt hypothyroidism in 1.3% , central hypothyroidism in 9.3% , and hyperthyroidism in 2.9% . of the 147 allo - hsct recipients , ishiguro et al . found overt hypothyroidism in 0.03% , subclinical hypothyroidism in 26.5% , and hyperthyroidism in one subject . siekierska - hellmann et al . found that at 1 year none of patients developed overt hypothyroidism in hsct recipients prepared with chemotherapy . somali et al . in non - tbi - based hsct protocol found overt hypothyroidism in 6% of male patients and 5% of female patients and subclinical hypothyroidism in 13% of males and 5% of females . in the present study , two patients developed nodat and three had ifg [ table 5 ] . both the patients of nodat had history of steroid intake and their glycemic control was initially maintained by basal bolus insulin regimen and subsequently on metformin . majhail et al . reported a very high incidence of nodat-30% with 2 years of transplant and also showed reversal in 70% of them . however , low prevalence of nodat in the present study could be due to steroid - free immunosuppression and inclusion of children in the total cohort . there have been very few studies which have addressed insulin resistance ( ir ) and secretion in post - hsct patients . one of the early studies had utilized the intravenous glucose tolerance test to show that after hsct , ir is common . another study when evaluating bone health in post - hsct patients found high homa - ir and homa-. an italian group had also shown elevated homa - ir in their post - hsct cohort . these results indicate that post - hsct recipients are insulin sensitive ; however , those who develop dysglycemia tend to have ir as a pathophysiologic event . however , we were unable to compare these values with age- and sex - matched controls . comparison of metabolic parameters in our group with few of those reported in literature following were the limitations of our study . firstly , the present study was a cross - sectional study without the control group . secondly , pre - transplant endocrine status of the cohort was not available , and hence the findings can not be completely attributed to hsct . the large number of tm patients in group - i can bias the effect of hsct , as tm by itself is known to be associated with many endocrine dysfunction . finally , ai could nt be classified as primary or secondary as plasma acth could nt be measured due to local constraints . endocrine manifestations are common after hsct ; they can occur as early or late complications . however , this is hindered by non - availability of trained endocrinologist . by this study we were able to emphasize the need for pre- and post - transplant endocrine evaluation of all patients requiring hsct at our center . ai , ghd , and hypogonadism are equally prevalent ( ~60% ) after bucy - based hsct . all children with ghd might not require therapy , as growth velocity can be normal after hsct even in the presence of ghd . the most common pattern of gonadal dysfunction in adult males is germ cell failure with compensated leydig cell failure . all recipients in reproductive age should be considered for preservation of gonadal functions prior to hsct .

biocompatibility of root canal filling materials is of particular importance particularly when an inadvertent intimate contact occurs between the extruded root canal filling material and the adjacent periapical tissue cells [ 1 , 2 ] . on the other hand , a substantial number of teeth requiring root canal treatment have necrotic pulps in which contamination with bacterial toxins is a common finding . it has been well established that gram - negative anaerobic species are the predominant etiologic bacteria in endodontic infections . lps , also called endotoxin , stimulates the inflammatory mediators , activates the complement system and macrophages and causes host cell toxicity and bone resorption after being released during bacterial cell proliferation or death [ 5 , 6 ] . it has also been demonstrated that filled root canals allow leakage of lps in vitro . root canal fillings are currently performed by a semisolid material as a core in conjunction with various types of sealers . gutta percha is considered the most widely used core material ; but recently , resilon ( llc , madison , connecticut ) was introduced in endodontics to bring bonding technology into the root fillings . although the literature is rich on the physical properties of resilon - filled root canals , studies concerning its biological properties are infrequent . the aim of this study was to compare the cytotoxic effects of these two materials in presence or absence of escherichia coli endotoxin on human monocytes in vitro . thp-1 human monocyte cell line was obtained from the cell bank of pasteur institute , tehran , iran and cultured in a laboratory setting in 25 mm culture flasks which contained rpmi 1640 ( gibco brl , grand island , ny ) , supplemented with penicillin ( 100 u / ml ) ( gibco brl , grand island , ny ) , streptomycin ( 100 g / ml ) ( gibco brl , grand island , ny ) , l - glutamine ( 2 mm ) ( sigma chemical co. , st . louis , mo ) , and 10% fetal bovine serum ( fbs ) ( gibco brl , grand island , ny ) at 37 c with 5% co2 . the cells were stored in their best growth state in nitrogen tanks so that 35 million cells in 90% fbs and 10% dimethyl sulfoxide were stored at 20c for 1 hour and then at 70c for another 24 hours and eventually preserved in liquid nitrogen at 137c . more than 90% viable cells were obtained , 105 cells per well were seeded into flat - bottom 96-well culture plates ( nunc - immuno - plate maxisorp , nunc , denmark ) for evaluation . the root canal filling materials used in this study were sterile size # 20 , 0.02 taper resilon ( llc , madison , connecticut ) and the same size gutta percha ( meta biomed , korea ) points . three millimeters from the tip of each point was cut and directly placed at the bottom of the culture wells . cultured cells were exposed to gutta percha ( groups g1 and g2 ) and resilon ( groups r1 and r2 ) segments . root canal filling materials were contaminated with 10 g / ml bacterial lps ( escherichia coli ; sigma , st . louis , mo , usa ) in groups g1 and r1 while being exposed to the cultured cells . positive control group included the bacterial lps , but no obturation material and the negative control comprised of the cells in culture medium only . viability of cells was tested in all groups after 24 , 48 , and 72 hours . regard , four hours prior to completion of each time period , the test and control groups were subjected to sterile 5mg / ml mtt powder ( sigma chemical co , st louis , mo , usa ) dissolved in pbs solution , according to the manufacturer s instructions . the samples were then incubated at 37 c in 5% co2 for another four hours . the results were read by elisa reader ( behring , marburg , germany ) at a wavelength of 570 nm considering 630 nm wavelength as the reference . data were analyzed using three - way anova and tukey s post hoc statistical test at 95% confidence level and spss 15.0 for windows ( spss inc . , chicago , il ) was used as the statistical tool . the mean and standard deviation for all groups , treatments and intervals are shown in table 1 . the mean and standard deviation values for all experimental groups ( g1= lps - contaminated gutta - percha , g2= non - treated gutta percha , r1= lps - treated resilon , r2= non - contaminated resilon , ctrl= control ) optical density ( od ) values for cell viability showed an almost constant time - dependent decrease in all experimental and control groups . mtt assay results indicated the highest value for cell viability in negative control group at all three time points . at 24 hours , wells containing non - treated gutta percha segments ( g2 ) indicated the lowest rate of cell viability . at 48 and 72 hours , the least number of viable cells were recorded in lps - contaminated gutta percha group ( g1 ) . the effect of three- and two - way interactions was not significant according to the statistical tests used . the p - value was calculated to be 0.995 for the three - way interactions . the interaction effect of time and the obturation materials was not significant either ( p=0.914 ) . also , the interaction between the obturation material and lps treatment was not significant ( p=0.942 ) . the interaction effect of time and lps treatment was not significant either ( p=0.750 ) . there was a significant difference among gutta percha , resilon and control groups ( p=0.031 ) . tukey s multiple comparisons showed that although resilon groups indicated more cytotoxic responses than gutta percha groups , the difference was not statistically significant ( p=0.105 ) . gutta percha groups showed significantly more cytotoxic responses in comparison to the control group ( p=0.034 ) . on the other hand , there was no significant difference between the cytotoxic responses of resilon groups and control , irrespective of the lps treatment ( p=0.866 ) . a significant decrease in cell viability was observed in all experimental groups ( irrespective of the material tested or lps contamination ) at 72 hours ( p<0.05 ) . cell viability in groups with lps treatment was lower than that in groups without lps treatment at all time points except for g1 and g2 groups in which almost equal results were obtained only at 24 hours . resilon groups showed higher cell viability than gutta percha groups , although statistically non - significant ( p>0.05 ) . cell viability values were lower in gutta percha than resilon groups when lps - treated and lps - untreated groups were compared independently at each time point . biocompatibility of endodontic materials is of particular interest , because incorporation of toxic ingredients may cause irritation or degeneration of host tissues . use of in vitro models offers the possibility of studying the effects of material components released on cultured cells . it has been well established that potentially toxic components released from a variety of endodontic materials may enter the blood circulation and cause systemic effects . use of cell lines is a common method for testing the cytotoxicity of dental materials because it is simple and yields reproducible and controllable results . in vitro testing also makes it possible to compare several materials using the same cell lines under the same conditions . macrophages are among the most important cells in inflammatory responses . in vitro evaluation of initiation of inflammatory and immune responses to dental biomaterials and their components , adverse reactions of macrophages to components of some dental materials at specific concentrations have been reported [ 9 , 13 , 14 ] . the aim of this study was to evaluate the effects of sterile and lps - contaminated resilon on viability of human monocyte / macrophage cell line ( i.e. , thp-1 ) in comparison with gutta percha under similar conditions . cytotoxicity evaluation in this study was performed at three time points of 24 , 48 , and 72 hours by the mtt assay in a triplicate fashion to obtain reproducible results . this technique is based on the reductive cleavage of yellow tetrazolium salt to a purple - colored formazan crystal by the dehydrogenase activity of intact mitochondria . therefore , this conversion can only be seen in living cells [ 1517 ] . although gutta percha is considered inert , our findings as well as those of several other studies showed that it may elicit moderate levels of toxicity [ 18 , 19 ] . some authors [ 20 , 21 ] have stated that such effect is attributable to the leakage of zinc ions . gutta - percha may be well tolerated , but severe tissue injuries as well as activation of complement components contributing to periapical inflammation have also been observed . resilon was introduced as a core root canal filling material to endodontic market for use in conjunction with resin sealers especially epiphany for root canal obturation to produce a so - called intraradicular monoblock . superiority of resilon - filled to conventionally filled root canals has been a matter of debate in terms of physical properties . on the other hand , few biological investigations have been conducted to evaluate the effects of resilon on human and animal cells , tissues and organs . studies about resilon convey that although this material is well tolerated by the periapical tissues of sub - human primates , some concerns exist regarding the risk of alkaline or enzymatic hydrolysis [ 2628 ] . our findings are comparable with those of key et al , who showed that cytotoxicity of resilon was equal to that of gutta percha , although cellular models differed . in addition , chang et al . did not show noticeable cytotoxicity of gutta percha on human gingival fibroblasts during 24 hours of incubation . similar results were also obtained for human immune cells and l929 fibroblasts derived from rats [ 31 , 32 ] . as stated previously , cytotoxic effects of gutta percha points may be attributable to leakage of some leachable toxic components , such as zinc oxide . this is difficult to establish , however , because the material composition is often proprietary [ 29 , 33 ] . it can be concluded that none of the root canal filling materials tested in the current study had toxic effects on cultured human monocyte cells whether in presence or absence of lps .

hemodialysis is one of the most prevalent treatments for these patients estimated as over 1 million worldwide . in iran , this estimate was about 11,250 patients in 2003 , 18,000 in 2007 , and 20,000 in 2012 . the patients undergoing hemodialysis usually have several complications which result from either the end stage of renal failure or the type of treatment . one of these complications is restless leg syndrome that is accompanied with sensory signs and motion disorders of limbs , especially legs . the signs of this syndrome are observed during rest or lack of activity and are reduced by moving the involved limb . although this syndrome signs are present during the day and night , they get worse at night , and it leads to patients ' sleep disorder and influences their function , especially in the evening and at night . this syndrome is more prevalent in chronic renal failure patients compared to normal population in such a way that primary estimation of its prevalence among hemodialysis patients showed it to be 2080% . the associated pathophysiology of restless leg syndrome is yet unknown , but as the syndrome is relieved by low dosage of levodopa consumption , dopaminergic system is considered to play the main role in causing such a syndrome . as medications have their own specific side effects , patients are always after non - medicational methods including complementary medicine , of which reflexology can be named . reflexology is a sort of pressure , which is often given on the feet . experts of reflexology believe that feet can be divided into several reflex points which are associated with and connected to all body organs and parts . the main mechanism of this treatment is through its effect on nervous system in such a way that the pressure to a specific part of skin stimulates nervous reflexes and their transmission to brain . on the other hand , reflexology provokes the chemical system of the nerves , balances the enzymes , and regulates endocrine function . there are other beneficial non - medicational methods to reduce the severity of restless leg syndrome . research showed that movement - based therapy methods have a positive effect on controlling some of the syndrome signs , and the patients ' daily function can be improved through physical activity strategies . movement therapy is among the effective strategies to improve motor function , prevent the complications , and lower the disabilities resulting from restless leg syndrome . among the movement therapy methods , stretching exercises are one of the oldest treatment methods , which improve muscles ' circulation and facilitate provision of nutrients to the cells . as poor circulation enhances manifestation of restless leg syndrome signs and activity modifies this condition , stretching exercises may be effective in reduction of this syndrome severity . based on the obtained results , majority of the activities of hemodialysis nurses are carried out in the absence of the physicians , such that their role in treatment of the patients has been estimated as 80% . nephrology nurses should be able to control the complications resulting from the disease , and administrate renal replacement treatment and proper non - medicational interventions for the patients . restless leg syndrome highly influences the physical and psychological aspects of hemodialysis patients and impairs their trend of life . on the other hand , the number of consumed medications is very high among these patients , and most of these medications are excreted through kidneys ; therefore , adding another medication to treatment can add up to patients ' problems , and thus , non - medicational methods seem essential for these patients . as research showed the effect of reflexology complementary medicine and stretching exercise on the level of dopamine , the researchers decided to investigate the effect of two methods of reflexology massage and stretching exercises on the severity of restless leg syndrome among patients undergoing hemodialysis . this is a three - group , two - stage ( before , after ) clinical trial ( nirct no . study population comprised all chronic end - stage renal failure patients who referred to selected hospitals in isfahan three times a week and underwent hemodialysis for 4 h in each session . inclusion criteria were patients aged 1865 years whose hemodialysis had started for at least 3 months prior to the study . those who had no idiopathic restless leg syndrome , not consuming medications to manage restless leg syndrome signs or medications worsening these signs ( three - cycle antidepressants , serotonin selective reuptake inhibitors , anti - nausea medications , antiepileptics , antipsychotics , dopamine antagonists ) , no infection , wound , and a serious complication in feet , and peripheral neuropathy or vascular problems in lower limbs were selected . whenever the subjects lost interest to remain in the study or any change occurred in the inclusion criteria at any stage , the subjects were excluded . in the first stage , after obtaining an informed written consent , all hemodialysis patients completed restless leg syndrome standard questionnaire . the clients who responded positive to all four questions of restless leg syndrome diagnostic questionnaire were considered as the clients with restless leg syndrome . next , through random numbers table , 90 subjects meeting the inclusion criteria were selected , and the goal of the study was explained to them by the researcher in their first meeting . demographic information form was completed through referring to patients ' medical files and questioning the clients . the subjects were notified that they could ask their questions and ambiguous points through telephone calls and receive the answers . in the second stage , the subjects were assigned to three groups through random allocation and use of sealed and plumed envelopes each patient randomly selected one of the envelopes and was assigned to the group mentioned in the envelope . then , through restless leg syndrome severity standard questionnaire , the score of restless leg syndrome was calculated for each patient separately and the total mean of each group was calculated . next , interventions of reflexology and stretching exercise were administrated for two groups as three sessions a week ( 12 sessions ) , each session lasting for 3040 min in the first 2 h of dialysis session in which there were no notable changes in bp respectively . the second questionnaire was restless leg syndrome severity standard measurement questionnaire in which the patients with scores less than 10 were categorized in minor ; 1120 as moderate ; 2130 as severe , and 31 or over as being in a very severe stage of the syndrome . restless leg syndrome severity questionnaires were completed by the researcher before and immediately after the interventions . validity and reliability of both questionnaires in iran were confirmed by habibzade et al . in 2011 . content validity and cronbach alpha were used to confirm the validity and reliability of the data collection tools . cronbach alphas were calculated to be 0.97 and 0.94 for restless leg syndrome diagnosis and restless leg syndrome severity investigation questionnaires , respectively . data were analyzed by paired t - test one - way analysis of variance ( anova ) and least significant difference ( lsd ) post hoc test , and the significance level was considered as p < 0.05 . findings of the present study showed that the subjects ' mean ( sd ) age was 55.45 ( 12.08 ) years , mean length of hemodialysis was 35.34 ( 29.01 ) months , and 50% of the subjects were women . statistical tests showed that frequency distribution of sex , mean age , and length of hemodialysis ( months ) were identical in the three groups and showed no significant difference . paired t - test showed a significant difference in the mean scores of restless leg syndrome severity before and immediately after intervention in reflexology and stretching exercises groups , respectively ( p < 0.001 ) , but showed no significant difference in the control group . one - way anova showed no significant difference in the mean score of restless leg syndrome severity in the three groups before intervention , but the difference was significant immediately after intervention ( p < 0.05 ) [ table 1 ] . mean score change of restless leg syndrome in three groups of reflexology , stretching exercises , and control immediately after intervention showed that the changes were significantly more in the two study groups compared to control ( p < 0.05 ) in such a way that both interventions reduced the severity of restless leg syndrome signs more , compared to the control group [ table 2 ] . comparison of restless leg syndrome mean scores in reflexology , stretching exercises , and control groups before and immediately after intervention restless leg syndrome mean score changes in reflexology , stretching exercises , and control groups immediately after intervention lsd post hoc test showed a significant difference in the mean scores of restless leg syndrome severity immediately after intervention in reflexology and control and in stretching exercises and control groups ( p < 0.05 ) [ table 3 ] , but there was no significant difference in the mean scores of restless leg syndrome severity immediately after intervention in reflexology and stretching exercises . comparison of the effects of reflexology and stretching exercises methods showed that both methods were effective , and there was no significant difference . the severity of restless leg syndrome signs was significantly reduced after reflexology therapy , compared to before intervention . stated that reflexology therapy reduced the severity of fatigue , pain , and muscular cramps of hemodialysis patients . wang et al . showed that reflexology reduced muscular cramps during the interval between two sessions of hemodialysis . ( 2011 ) reported that reflexology therapy reduced pain , controlled blood sugar , and improved nervous and temperature conductivity among diabetic nephropathy patients . lee et al . , in a systematic review study , showed the effect of reflexology therapy on fatigue , sleep disorders , and pain . the main mechanism of treatment in reflexology is its effect on the nervous system in such a way that the pressure given on a specific part of skin stimulates nervous reflexes and transmission of impulses to the brain . this mechanism seems to be effective on reduction of complications of various diseases including restless leg syndrome in hemodialysis patients . our obtained findings also showed that restless leg syndrome severity was significantly lowered after stretching exercises , compared to before intervention . giannaki et al . also showed that educating the patients with sports and administering low dosage of dopamine antagonist were effective on reduction of restless leg syndrome signs to 46% and 56% , respectively . ( 2006 ) showed that a sport program with low severity acted as an assistive treatment in improvement of physical function and efficiency of hemodialysis patients . showed that the fall risk of hemodialysis patients was reduced after a strength and balance exercises program . 's literature review study showed that sport had positive effects on reduction of cardiovascular diseases , increase in quality of life and physical function , reduction of depression and muscular disease , and reduction of mortality in patients with restless leg syndrome . smart and steele , in 15 studies , showed the effects of sports education on preservation of fat - free body mass , quadriceps muscle , prevention of knee deformation , and increase in strength of knee and pelvis joints in hemodialysis patients . reported the positive effect of aerobic sports on improvement of restless leg syndrome signs in hemodialysis patients . henrique et al . reported that regular aerobic exercises were effective on improvement of physical capacity and hypertension control of hemodialysis patients , which is consistent with the present study . although the mean changes of restless leg syndrome were higher in stretching exercises group compared to reflexology , they were not significant . therefore , stretching exercises seem to be more effective on reduction of disease complications as well as some of the problems of patients , such as restless leg syndrome . as learning these exercises is easy and almost possible for all patients and needs no special equipments and costs , the disturbing complications of this syndrome can be lowered through patients ' education of these exercises and supervising the patients ' proper practice during hemodialysis . in this way , the economic and psychological burden imposed on the patients and their families can be prevented . therefore , educating the patients and health care team concerning these two methods can be an effective action in reduction of this syndrome . the only limitation that was out of researcher 's control in the present study was subjects ' personal differences concerning social , cultural , psychological , and familial factors , which might have affected the evaluation and tolerance of the severity of signs in patients . in the end , the researchers suggest conducting a similar study with a bigger sample size and for a longer period of time , as well as investigating the severity of restless leg syndrome signs 2 and 6 months after intervention to declare the longevity of intervention and the mean score of restless leg syndrome signs ' severity . results showed that both reflexology and stretching exercises methods were effective on reduction of restless leg syndrome among the patients undergoing hemodialysis , and they are suggested in treatment of such patients .

choriocarcinoma is a highly aggressive malignant human chorionic gonadoprotein - producing neoplasm with usually an intrauterine location and can be gestational or non - gestational in origin . extrauterine choriocarcinomas are very rare and can be seen in other parts of the female genital tract like fallopian tube , ovary and cervix . the estimated incidence of gestational ovarian choriocarcinoma is one in 369 million pregnancies , associated with coincident or antecedent gestation . we report a case of primary gestational ovarian choriocarcinoma in a young lady where the diagnosis was rendered by intra - operative cytology and the proliferative potential was estimated by ki67 immunohistochemistry . a 25-year - old female presented with pain abdomen and bleeding per vaginum for 25 days . abdominal examination revealed a soft , non - tender palpable lump in the right iliac fossa . her urine tested negative for hcg and the serum level was slightly increased to 3080 miu . on laparotomy , the right ovary was replaced by a hemorrhagic , friable and necrotic mass . imprint cytosmears were prepared from the mass and fixed with absolute alcohol and stained with papanicolaou stain and hematoxylin and eosin ( h and e ) stain . two cytopathologists examined the smears , which showed small clusters of pleomorphic and hyperchromatic cells with clear to pale eosinophilic cytoplasm over a hemorrhagic and necrotic background [ figure 1a and b ] . we received the gross specimen as cut - opened uterus with cervix and a large hemorrhagic and friable mass . sections were taken from different areas of the ovarian mass along with uterine wall and cervix and stained with h&e stain after processing . histopathology revealed mostly areas of hemorrhage and necrosis with a biphasic tumor tissue comprising of pleomorphic cytotrophoblasts and syncytiotrophoblasts . the sections were also subjected toki67 immunostain and were found to be positive with diffuse and intense staining ( in more than 50% of cytotrophoblasts ) showing high proliferative index of the tumor cells [ figure 2b ] . in contrast , the syncytiotrophoblasts were negative for ki67 . ( a ) large cells with pale cytoplasm , centrally placed nuclei against a hemorrhagic and dirty background ( h and e , 100 ) . ( b ) cytotrophoblasts and syncytiotrophoblasts in the cytosmear , ( h and e , 400 ) ( a ) variegated friable mass with hemorrhage and necrosis and normal - appearing uterus . ( b ) diffuse and strong positivity of tumor cells with ki67 immunostain ( ihc , 400 ) ovarian choricarcinoma is a highly malignant tumor characterized by the presence of malignant trophoblastic cells histopathologically and a raised serum hcg level biochemically the distinction between the two is difficult , but necessary , as the non - gestational type has bad prognosis . the patients are 40 years or older , present with abnormal uterine bleeding and have a very high serum hcg level . the above - said patient was a young female of 25 years with a past history of ectopic gestation 1 year back , had a mild increase in the serum hcg level and had no abnormality in the uterus or fallopian tube . she was treated with methotrexate - based chemotherapy ( methotrexate , etoposide and actinmycin - d ) and her hcg level came down to normal after two cycles of chemotherapy , and is doing well till date . ki67 was applied here to study the proliferative index of choriocarcinoma , which was high ( more than 50% of cytotrophoblasts ) and also strong in intensity of staining . also , it can be used to determine the therapeutic response after treatment by comparing with the present index . but , in other situations where the diagnosis is not so straightforward , it can be used to differentiate from other gestational trophoblastic diseases like hydatidiform mole ( complete type ) and placental site trophoblastic reaction and tumor . there are no unique ultra - structural or immunohistochemical features to distinguish either of them . dna polymorphism analysis can be utilized to distinguish pure non - gestational choriocarcinoma of the ovary from gestational choriocarcinoma . genetic analysis showing whether a paternal contribution is present in the genome or not can be a useful modality in determining the origin of the choriocarcinoma . human leukocyte antigen typing for paternal antigen in trophoblastic elements can also be used to determine the gestational etiology . markedly elevated levels of serum human chorionic gonadoprotein levels are commonly found with gestational tumors but are not very reliable . the majority of patients with non - gestational ovarian choriocarcinoma are younger than 20 years . while most gestational choriocarcinomas show hematogenous spread , the non - gestational choriocarcinoma tend to follow the lymphatic system . the staging depends on the original site of the tumor , like for gestational ( metastatic or primary ) choriocarcinoma standard choriocarcinoma staging applies and for non - gestational choriocarcinoma staging is performed by ovarian cancer staging system . because this patient was a young female presenting with gestational choriocarcinoma of the ovary , she was treated with radical surgery with preservation of contralateral ovary and followed by methotraxate - based chemotherapy . we are reporting this case as it is rare and also because one should be aware of the cytologic findings of choriocarcinoma so as to give a correct diagnosis during operation to help the surgeons in the proper management of such lesions .

dental caries have been described as a social disease , as their formation is associated with biological , dietary , behavioral , and socioeconomic factors , as well as access to consumer goods and health services . although the prevalence and severity of dental caries has decreased among brazilian children and adolescents in the last two decades , expanding preventive resources to reach all communities and social strata remains challenging . numerous studies have addressed the correlation between oral health and social factors such as income , education , number of children , and other variables . the national surveys conducted in brazil in 2000 and 2010 showed that oral health is heterogenous , with differences found among regions and social groups . increasingly , women are considered as the reference person in the family . in this scenario , the mother assumes a central role in the formation , transmission , and environmental conservation and social development of other family members , especially her children . it is therefore likely that the health of both the mother and child are similar or are linked to social determinants , given that they live in the same social context . the purpose of the present study was to investigate the family social determinants associated with the caries history of children and the need for dental treatment ( ndt ) among their mothers . a cross - sectional study was performed among children ( age : 1 - 5 years ) enrolled in ten public schools in southern brazil and their mothers . the initial design of this study was approved by the ethics committee of the faculty of dentistry of araatuba ( foa case 2006 - 01460 ) in accordance with the declaration of helsinki . a sample size calculation was performed using epi info 6.0 considering a range of 95% and a 5% margin of error over the total population size for the study . because the numbers of children enrolled in each school proved to be very similar , the samples were not stratified according to this parameter . at the end of this process , data were collected by a single researcher , who was previously trained and standardized . the structured instrument used for this study was previously validated by a pilot study and it consists of three distinct parts . the first part addresses the family social characteristics , including the maternal education level , employment status , average monthly income , age , marital status , number of children , and frequency of visits to the dentist . the second part assesses the ndt of the mother ( maternal ndt ) , according to the presence of dental caries determined by oral examination . the third part assesses whether the child has a history of or has current untreated dental caries , based on oral examination . oral examinations of the mother - child pairs were performed at the ten schools . examinations were performed under artificial light , and included the use of exploratory probes , mirrors , clinical plans , wooden spatulas , and sterile pads . the initial group of mother - child pairs was subdivided into four groups based on the oral examination results . group 1 ( g1 ) included pairs with a maternal ndt and a child with caries history . group 2 ( g2 ) included pairs with no maternal ndt and a child with caries history . group 3 ( g3 ) included pairs with a maternal ndt and a caries - free child . group 4 ( g4 ) included pairs with no maternal ndt and a caries - free child . the diagnostic intra - rater concordance was determined by the kappa test ( k=0.98 ) . the association between categorical variables ( marital status , number of children , maternal education level , employment status , income , and frequency of visits to the dentist ) and the variables ( maternal ndt and child caries history ) was analyzed using the chi - square test ( ) . multivariate discriminant analysis ( mda ) was performed for the four groups by assessing the proximity - distance space between them , considering all of the social variables together . a cross - sectional study was performed among children ( age : 1 - 5 years ) enrolled in ten public schools in southern brazil and their mothers . the initial design of this study was approved by the ethics committee of the faculty of dentistry of araatuba ( foa case 2006 - 01460 ) in accordance with the declaration of helsinki . a sample size calculation was performed using epi info 6.0 considering a range of 95% and a 5% margin of error over the total population size for the study . because the numbers of children enrolled in each school proved to be very similar , the samples were not stratified according to this parameter . at the end of this process , data were collected by a single researcher , who was previously trained and standardized . the structured instrument used for this study was previously validated by a pilot study and it consists of three distinct parts . the first part addresses the family social characteristics , including the maternal education level , employment status , average monthly income , age , marital status , number of children , and frequency of visits to the dentist . the second part assesses the ndt of the mother ( maternal ndt ) , according to the presence of dental caries determined by oral examination . the third part assesses whether the child has a history of or has current untreated dental caries , based on oral examination . oral examinations of the mother - child pairs were performed at the ten schools . examinations were performed under artificial light , and included the use of exploratory probes , mirrors , clinical plans , wooden spatulas , and sterile pads . the initial group of mother - child pairs was subdivided into four groups based on the oral examination results . group 1 ( g1 ) included pairs with a maternal ndt and a child with caries history . group 2 ( g2 ) included pairs with no maternal ndt and a child with caries history . group 3 ( g3 ) included pairs with a maternal ndt and a caries - free child . group 4 ( g4 ) included pairs with no maternal ndt and a caries - free child . the diagnostic intra - rater concordance was determined by the kappa test ( k=0.98 ) . the association between categorical variables ( marital status , number of children , maternal education level , employment status , income , and frequency of visits to the dentist ) and the variables ( maternal ndt and child caries history ) was analyzed using the chi - square test ( ) . multivariate discriminant analysis ( mda ) was performed for the four groups by assessing the proximity - distance space between them , considering all of the social variables together . the loss of individuals from the sample was due to the refusal of mothers to participate ( n=67 ) or the absence of the child at the time of clinical examination ( n=57 ) . most of the mothers were married ( 79.8% , n=217 ) . in only 74 pairs ( 27.2% ) , the child was the firstborn . most mothers ( 50.7% , n=138 ) had only basic education , emphasizing the low education of the population considered . only 4 mothers ( 1.5% ) had completed higher education , 89 ( 32.7% ) had completed high school , and 41 ( 15.1% ) had completed primary education . the vast majority ( 95.6% ; n=260 ) were currently employed , although the average family income was low . of the 272 mother - child pairs , 148 ( 54.4% ) earned less than the brazilian minimum monthly wage , 73 ( 26.8% ) earned 1 - 3 the minimum wage , and only 37 ( 13.6% ) earned > 3 the minimum wage . mothers reported visiting a dental professional never ( 45.2% , n=123 ) , rarely ( 42.6% , n=116 ) , once a year ( 10.3% , n=28 ) , or more than once a year ( 1.8% , n=5 ) . we next examined the association between socioeconomic variables and the maternal ndt ( table 1 ) or the caries history of the child ( table 2 ) . the maternal ndt was positively associated with low maternal educational attainment , a monthly household income of less than the minimum wage , and infrequent ( never or rare ) visits to the dentist . the caries history of children with a maternal ndt was positively associated with a lower family income , lower maternal educational attainment , low frequency of visiting a dentist ( never or rarely ) , and a higher number of children in the family . association between socioeconomic status and need for dental treatment ( ndt ) in mothers of 272 children enrolled in public institutions of early childhood education in southern brazil brazilian minimum monthly wage ( bmmw ) ~ $ 465.00 brl ( u.s . $ 258.00 ) association between maternal socioeconomic variables and caries history of children enrolled in public institutions of early childhood education in southern brazil n=272 . $ 258.00 ) in order of frequency , the mother - child pairs largely belonged to g1 ( 49.9% ; maternal ndt and child caries history ) or g4 ( 29.2% ; no maternal ndt and caries - free child ) , followed by g3 ( 18.0% ; no maternal ndt and child caries history ) and distantly by g4 ( 3.6% ; maternal ndt and caries - free child ) ( table 3 ) . distribution of the children in each of the four ndt groups figure 1 shows the spatial relationship between the four groups . g1 and g2 were very close , g4 was more widespread , and g3 was substantially smaller and further away from the others . g1 and g2 have greater similarity in their variables and these are more concentric . g4 has greater range in the variables , while the variables in g3 are very different from the others . spatial discrimination analysis of groups g1 , g2 , g3 , and g4 according to the maternal need for dental treatment and the caries history of the child . population sample is children attending public institutions of early childhood education in southern brazil and their mothers in the present study , it was found that the maternal ndt was associated with lower family income , maternal education level , and fewer visits to the dentist . moreover , the caries history of the child was less dependent on the social variables than on the maternal ndt , as children with or without caries were part of families with closer behavior . the health conditions of a population , including oral health , are historically associated with social determinants . several studies have identified an association between oral health and socioeconomic variables such as family income , educational level , delay and disruption of the education of the childand social class , in various locations including australia , new zealand , jordan , japan , sweden , the uk and brazil . it was observed that the maternal ndt was positively associated with low family income , low maternal education level , and infrequent visits to the dentist . the maternal ndt was not correlated with the number of children or the maternal marital or employment status . interestingly , the child caries history was associated with the same variables as the maternal ndt , along with the number of children . the results of this study partly confirm the results of galindo , et al . ( 2005 ) , who demonstrated a relationship between the number of children and a higher rate of dental caries and maternal ndt . however , the number of children seems to be less important than the education level of the parents or the family income . because the maternal ndt and child caries history were associated with the same variables , a relationship seems to exist between the oral health statuses of the mother and child . ( 2008 ) also suggested a strong relationship between the maternal ndt and the caries history of children in a previous survey conducted in southern brazil . ( 2012 ) examined populations of 12 , 6 and 4 - 6 year old children , respectively , and showed that better family economic conditions and higher levels of maternal education were associated with fewer episodes of caries . ( 2008 ) found that low maternal educational attainment ( < 8 years of schooling ) and low family economic status were associated with increased incidence of dental caries in preschool children . other determinants of oral health may also exist in addition to those tested here . in addition , in some regions the social determinants are less pervasive . a longitudinal study of risk factors for dental caries in infants in the united states identified only previous colonization by streptococcus mutans and early consumption of sugars as risk factors . social variables did not increase the caries risk . among the mothers surveyed , 123 indicated that they had never consulted a dentist . the lack of access to dental care in this study was far greater than the brazilian average for adults . goettems , et al.(2012 ) , also in a cross - sectional study of 608 mother - child dyads , observed high association between irregular visits to a dentist by the mother and no dental visit by the child . the maternal oral health and number of visits to the dentist , which suggest barriers to oral health care access , are also reflections of socioeconomic conditions . ( 2006 ) found that the need for dental treatment , represented specifically by toothache , was associated with absent routine dental care . preventive visits to the dentist and local availability of dentists have been shown to be crucial to caries prevention . while numerous studies have revealed an association between socioeconomic status and caries history or ndt , virtually no studies have demonstrated this association considering all socioeconomic variables together . in this study , when analyzing all of the social variables , it was found that the theoretically worst oral care condition ( g1 ; maternal ndt and child with caries history ) was in close proximity to g2 and was further away from g3 and g4 . g2 was more inserted in the space of g1 and g3 , indicating that g1 and g2 have greater similarity of social variables than g1 and g3 . in other words , the ndt depended on larger social changes more than dental caries , because g3 ( maternal ndt with non - caries child ) was spatially further away . the ndt in mother was associated with lower family income , low maternal education level and fewer visits to the dentist . similarly , the variables income , maternal education and visits to the dentist were excessively associated with caries experience in this order . furthermore , the caries experience in child depends less from social oscillation than to maternal ndt , because the children with and without caries experience were part of families with closer social profiles .

the maxillofacial region occupies the most prominent position in the human body and rendering it vulnerable to injuries quite commonly . it is estimated that more than 50% of patients with these injuries have multiple trauma requiring coordinated management between emergency physicians and surgical specialists in otolaryngology , trauma surgery , plastic surgery , ophthalmology , and oral and maxillofacial surgery . maxillofacial injuries can occur as an isolated injury or may be associated with multiple injuries to the head , chest , abdominal , spinal and extremities . the causes of maxillofacial trauma vary and include road traffic accidents ( rtas ) , interpersonal violence , falls , sports and missile injuries . the relative contribution of each cause depends on such factors as geofigureical location , socio - economic factors and the seasons of the year . with regard to the anatomical sites , mandibular and zygomatic complex fractures account for the majority of all facial fractures and their occurrence varies according to the mechanism of injury and demofigureic factors , particularly , gender and age . the causes of maxillofacial injuries have changed over the past three to four decades and continue to do so . the main causes worldwide are assaults and traffic accidents , but the most frequent cause varies from one country to another . some studies have shown that assault is most common in developing countries , whereas , traffic accidents are more common in developed countries . the causes and pattern of maxillofacial injuries reflect trauma patterns within the community and , as such , can provide a guide to the design of programmes geared toward prevention and treatment . the coordinated and sequential collection of information concerning demofigureic patterns of maxillofacial injuries may assist health care providers to record detailed and regular data of facial trauma . consequently an understanding of the cause , severity , and chronological distribution of maxillofacial trauma permit clinical and research priorities to be established for effective treatment and prevention of these injuries . this study was developed because there is insufficient literary evidence from our region to accurately illustrate , analyze and document the etiologic factors and frequency responsible for maxillofacial trauma . the data for this study were obtained from the medical records of 689 cases reported to our centre during the period from 2006 - 2009 . records of patients who were either treated in the emergency room as outpatients or the department of oral and maxillofacial surgery as inpatients were analyzed retrospectively . age , gender , cause , site and type of injury and associated injuries were recorded . information relevant to the study was obtained from the patient directly ; when this was not possible , collateral history was obtained from the relatives attending to the patients . when each patient arrived at hospital for evaluation , a trauma form was filled and depending on the fracture pattern the appropriate treatment was given . data collected were analyzed using the statistical package for social sciences ( spss ) for windows version 17.0 . data was summarized in form of proportions and frequency tables for categorical variables and was subjected to chi - square test . a total of 689 patients were reported at our centre in that 523 ( 75.9% ) were male and 166 ( 24.1% ) were female [ figure 1 ] . a total of 293 ( 42.5% ) patients in our study fell in to the age group of 21 to 30 years [ figure 2 ] . road traffic accidents 512 ( 74.3% ) were seen as the main etiological factor resulting in maxillofacial injuries followed by assaults 106 ( 15% ) and 36 ( 5.2% ) falls [ figure 3 ] . among 689 maxillofacial fractures reported mandibular fractures were 347 ( 50.3% ) followed by 198 ( 28.7% ) maxillary , 57 ( 8.2% ) nasal , 49 ( 7.1% ) zygomatic complex , 34 ( 4.9% ) orbital and 4 ( 0.5% ) frontal fractures [ figure 4 ] . the most common site of fracture in the mandible was angle 135 ( 38.9% ) followed by parasymphysis 84 ( 24.2% ) , symphysis 47 ( 13.5% ) , condyle 39 ( 11.2% ) , body 33 ( 9.5% ) and ramus 9 ( 2.5% ) [ figure 5 ] . among the maxillary fractures the most common was lefort ii 107 ( 54% ) followed by lefort i 59 ( 29.7% ) and lefort iii 32 ( 16.1% ) [ figure 6 ] . associated injuries along with maxillofacial fractures were most commonly head and neck injuries 168 ( 53.8% ) followed by extremities 59 ( 18.9% ) , thoracic 46 ( 14.7% ) and abdominal 39 ( 12.5% ) [ figure 7 ] . p < 0.001 , statistically significant , chi - square test age wise distribution of maxillofacial injuries . p < 0.001 , statistically significant , chi - square test distribution of patients according to cause of injury . p < 0.001 , statistically significant , chi - square test distribution of maxillofacial fractures . p < 0.001 , statistically significant , chi - square test distribution of mandibular fractures . p < 0.001 , statistically significant , chi - square test distribution of maxillary fractures . p < 0.001 , statistically significant , chi - square test distribution of associated injuries . the etiological factors and pattern of maxillofacial injuries have been reported to vary from one geofigureical area to another depending upon the socioeconomic status , geofigureic condition and cultural characteristics . males are at greater risk due to their greater participation in high risk activities which increases their exposure to risk factors such as driving vehicles , sports that involve physical contact , an active social life and drug use , including alcohol . in agreement with other studies , the majorities of patients in the present study were young adult in their third decade ( 21 - 30 years ) . however , this observation in contrast to some studies , where the dominant age groups having a high incidence were 0 - 10 years and 11 - 20 years respectively . the possible reasons for the higher frequency of maxillofacial injuries in third decade may be attributed to the fact that people in this period of life are more active regarding sports , fights , violent activities , industry and high speed transportation . the low frequencies in the very young and old age groups are due to the low activities of these age groups . maxillofacial injuries are commonly caused by road traffic accidents , assaults , sports , fire arm / blast injuries and gunshot injuries . in this study road traffic accidents were the most common cause of trauma , comprising 74.3% of the etiology of injuries and also considering the fact that bangalore is the third most populated city in india . this figure was 40% in one study from the united states , 24.7% from england , 48% in a study from france , 55.2% in a study from jordan12 and 44% in a study from pakistan . the high number of maxillofacial injuries attributed to rta in our study is attributed to recklessness and negligence of the driver , often driving under the influence of alcohol or drugs and complete disregard of traffic laws , over speeding , overloading , underage driving and poor conditions of roads and vehicles . alcohol impairs judgment , brings out aggression , often leads to interpersonal violence , and is also a major factor in motor vehicle accident . in the present study , alcohol consumption prior to the injury was recorded in 41.6% of cases which is comparable to other studies . in a review of 230 cases of maxillofacial injuries in sharjah , united arab emirates reported no cases were associated with alcohol abuse . this discrepancy may be explained by differences between one country and another , in the strictness of laws governing the sale and consumption of alcohol which may be effective in preventing alcohol - related injuries . head injury ( 53.8% ) accounted for the greater majority of associated injuries and contributed significantly to missed maxillofacial injuries , similar to findings from other studies . the incidence of missed injuries has been reported to be higher in patients with associated severe head injuries . this is reflected in the high rate of missed maxillofacial injuries in our patients , the majority of them had associated severe head injuries . mandible was seen as the most commonly fractured bone in our study accounting for 50.3% of the fractures and the most common site was 38.9% . this finding is consistent in a similar study from bulgaria whereas the percentage of mandibular fracture in a study from pakistan and uae was 51% . among the maxillary fractures the most common was lefort ii 54% , similar to findings from other studies . the studies by al khateeb et al . have found zygomatic complex as the most common site of midface injury which is not coinciding with the results of this study . klenk et al . reported in their study that when considering midface fractures , zmc fractures predominate followed by le fort and dentoalveolar fractures respectively . fractures such as noe and isolated blowout fractures only accounted for less than 7% of the fractures . other authors have also reported that a zmc fracture has the highest incidence although there is variability in the frequency of the other midfacial bones . one reason for the high rate in the zmc fractures is that it is instinctive to turn the head when anticipating a blow to the midface in order to protect the globe . the involvement of nasal bone in most of the midface fractures may be attributed to its predominant location on the face and relative structural weakness as was reported in the studies of le et al . road traffic accidents ( rta ) were the major etiological factor of maxillofacial injuries in our centre and the young adult males were the main victims . public should be made aware of road safety legislations and the subsequent repercussions of failure in compliance . future studies should seek to understand the epidemiological factors influencing facial trauma in an effort to improve prevention and management of these injuries . in addition it is clear that trends are observed when analysing the data collated , however the limited numbers of patients do not reflect statistical significance . again , further research is required to encompass a larger sample size with adequate follow - up of clinical outcomes as to obtain more meaningful data with other criteria such as complication rates , sepsis rates and total hospitalization costs being incorporated .

malaria is a disease that is caused in humans by parasites of the plasmodium species through the bite of infected anopheles mosquitoes . about 3.3 billion people half of the world 's population are at risk of malaria . every year , this leads to about 250 million malaria cases and nearly one million deaths . nigeria is known for high prevalence of malaria [ 24 ] and it is a leading cause of morbidity and mortality in the country . who recently listed nigeria among high burden countries with limited evidence of decrease in malaria cases . this natural product was used by the inhabitants of peru to control malaria , and the jesuits introduced this practice to europe during the 1640s where it was rapidly accepted . however , it was not until 1820s that the active ingredient quinine was extracted from the bark and isolated . treatment of malaria involves supportive measures as well as specific antimalarial drugs . with early diagnosis and effective treatment chloroquine ( cq ) which was introduced in the 1940s and for many decades served as a cheap and reliable drug is no longer effective against p. falciparum due to the increasing emergence of chloroquine - resistant strains of the malaria parasite [ 8 , 9 ] . due to widespread reports of chloroquine ineffectiveness against the malaria parasite in endemic countries outside the african continent , the federal government of nigeria officially banned the use of chloroquine in 2004 . this policy was not effected until 2010 . however , chloroquine is still in circulation because it is still purchased over the counter in chemist stores . in the face of this challenge facing chemotherapy of malaria , artemisinin and its derivatives ( artesunate , artemether , arteether , and dihydroartemisinin ) have given renewed hope for combating resistant malaria [ 11 , 12 ] . artesunate ( art ) is part of the artemisinin group of drugs that treat malaria . clinical evidence of drug resistance to artesunate was first reported in western cambodia , where artemisinin monotherapy is common . over the past decade , a new group of antimalarials known as artemisinin - based combination therapies has brought new hope in the fight against malaria . in malaria endemic areas such as nigeria , self - medication is quite common and purchase of antimalarials in the open market is rampant . this study aims to assess the antimalarial efficacy of chloroquine and artesunate and their effects on pathology of malaria in vital organs of albino mice . a total of 20 adult albino mice weighing between 20 g to 22 g were obtained from the nigerian institute of medical research , yaba , lagos ( nigeria ) and randomly divided into four groups : a , b , c , and d. group a represented uninfected normal control . group b served as the infected but untreated control , while groups c and d received daily doses of 100 mg / kg of chloroquine and artesunate , respectively . malaria infection was established on the 5th day before commencement of treatment in the experimental mice . the mice were treated for ten days before sacrificing them for histological study . the chloroquine - sensitive plasmodium berghei nk65 strain used for this study was obtained from the biochemistry department , nigerian institute of medical research , yaba , lagos , nigeria . the infection of the recipient mice was initiated by needle passage of the parasite preparation from the donor to healthy test animals via an intraperitoneal route as described by david et al . and peter and anatoli . briefly , p. berghei - infected red blood cells obtained from the tail vein of infected mice was diluted with phosphate buffered saline ( pbs ) so that each 0.2 ml that was subsequently injected contained approximately 10 infected red cells ( parasite ) per kilogram of body weight . this study was conducted in the animal house of the biological sciences department , college of natural sciences , university of agriculture , abeokuta , nigeria . animal acclimatization , drug administration , histological studies , and evaluation of results lasted for four months ( november 2009 to february 2010 ) . the test drugs ( artesunate and chloroquine phosphate ) used for this experiment were prepared in aqueous solution and administered orally at a dose level of 100 mg / kg body wt for ten days . the dose of the treatment was chosen based on our previous data where a dose of 100 mg / kg produced parasite clearance . histological studies were carried out in the pathology laboratory , college of veterinary sciences , university of agriculture , abeokuta . the animals were sacrificed by cervical dislocation 24 hour after the last dose on the 10th day of the respective treatment and the organs were harvested . primarily , pathological changes were observed in six main organs liver , kidney , spleen , lungs , brain , and the heart of the different groups of experimental mice . for light microscopic examination , after routine processing , paraffin sections of each tissue were cut into 5 m thickness and stained with haematoxylin and eosin . the photomicrographs of the relevant stained sections were taken with the aid of a light microscope . the following scores were used to grade the degree of histopathological changes or lesions observed in the organs : not present ( ) , very mild ( + ) , mild ( + + ) , moderate ( + + + ) , and severe ( + + + + ) . microscopic examination of blood films prepared from blood sample collected from the tail showed that the inoculums produced a mean parasitaemia of not less than 2% in 5 days before starting treatment . no parasitaemia was observed in the artesunate group on the second day of treatment while cq cleared all parasitaemia on the third day of treatment . physical examination of the internal organs after dissection of experimental mice showed mild enlargement of the spleen ( splenomegaly ) in the group treated with artesunate . the liver , lungs , and kidneys of the two groups treated with chloroquine and artesunate , respectively , were without macroscopic changes . histological studies revealed some damages caused by the malaria parasite and extent of reduction of these damages by the administered drugs , for example , the accumulation of iron in the liver and spleen ( haemosiderosis ) , death of cells of the liver ( hepatic necrosis ) , marked damage of the nephron in the kidney ( tubular nephrosis ) ( tables 1 and 3 ) , and gradual breakdown of the alveoli walls ( emphysema ) in the lungs ( table 4 ) . the degrees of damages caused in the organs were scored or assigned as follows : not present ( ) , 1 to 4 foci / section examined ( + ) , 5 to 8 foci / section examined ( + + ) , > 9 foci / section examined ( + + + + ) . tubular nephrosis was graded ( + + ) when scattered cells were detected with pyknotic , karyorrhexis , karyolysis nuclei , or loss of polarity , ( + + + ) when these changes were present in larger sections of a tubule , and ( + + + + ) when multiple tubules in an area were affected . the effect of artesunate at reducing the pathologies caused by the malaria parasite was more noticeable compared to that of chloroquine . haemosiderosis and kupffer cell hyperplasia were not observed in the artesunate group but these changes were mildly observed in the chloroquine group . the two drugs also mildly hepatic necrosis was mildly seen in the two treatment groups . however , all these pathological changes were severe in the control group ( table 1 ) . chloroquine and artesunate prevented the infiltration of perivascular interstitial mononuclear cells in treated mice but mildly reduced tubular nephrosis unlike the control group which was severe ( table 2 ) . cq and art treatment showed mild reduction of these pathologies caused by the parasite in the tissues of the spleen compared to the control which was severe ( table 3 ) . art totally reduced the thickening of the alveolar wall , but cq mildly reduced it . the two drugs completely reduced the infiltration of interstitial mononuclear cells when compared to the control ( table 4 ) . moderate lymphoid hypoplasia and severe megakaryoblast hyperplasia were observed in the spleens of groups treated with chloroquine and artesunate , respectively . likewise , mild periportal mononuclear cells infiltration was observed in the liver , but these side effects were not observed in the untreated infected control ( tables 1 and 3 ) . the total clearance of parasitaemia in the experimental mice by artesunate and chloroquine on the third and fifth days , respectively , showed that the drugs are still effective against malaria parasites that have not developed resistance yet . the severe damages observed in the liver of the control mice were as a result of parasite life cycle during erythrocytic stages in the blood stream . at the early stage of the exoerythrocytic phase , a parasite infects the hepatocyte and multiplies asexually releasing merozoites which rupture their host cells . the liberated merozoites leave the liver and enter the blood stream where they infect the red blood cells . in response to the damage caused , macrophages ( kupffer cells ) in the liver actively proliferate ( kupffer cell hyperplasia ) breaking down ruptured red blood cells by phagocytic action and splitting the haemoglobin molecules . this results into pathological effects like accumulation of iron in the liver ( haemosiderosis ) , which is usually linked with anaemia and could sometimes lead to liver cirrhosis , a condition of decreased liver function if not treated effectively . extensive and rapid death of parenchyma cells of the liver ( hepatic necrosis ) also occurs . hemozoin ( malaria pigment ) is a disposal product formed from the digestion of red blood cells by malaria parasites , and it is observed in either the cytoplasm or outside hepatocyte and kupffer cells as black or brownish granules . on the other hand , haemosiderin is a granular brown substance composed of ferric oxide left from the breakdown of haemoglobin . both haemosiderosis and hemozoin were observed in the liver , but hemozoin was more in the spleen . in the liver , the absence of kupffer cell hyperplasia and haemosiderosis in the artesunate group is partly due to early clearance of parasitaemia on the second day of treatment , but mild hepatic necrosis was still observed . chloroquine , on the other hand , mildly reduced kupffer cell hyperplasia , haemosiderosis , and hepatic necrosis which in part may be due to the brief delay before parasite clearance ( figure 1 ) . in the kidney , chloroquine and artesunate totally prevented the infiltration of perivascular interstitial mononuclear cells which are located between tubules of the nephron . this may be due to the activities of these drugs to mildly reduce destruction of the nephron ( tubular nephrosis ) by the malaria parasites in the kidney of treated mice ( figure 3 ) . in the spleen , haemosiderosis was also observed . this organ is the site for the breakdown of worn - out red blood cells and stores the iron they contain . therefore , the spleen also contributed to the accumulation of these iron molecules which was also noticed in the liver of the group treated with artesunate . lymphoid hypoplasia ( underdevelopment or reduced size in lymphatic tissue ) observed in the spleen of mice treated with chloroquine and severe megakaryoblast hyperplasia caused by artesunate may likely be an indication of the toxicity of this antimalarial drugs . megakaryoblast is a precursor cell to a megakaryocyte during haematopoiesis ( the process involved in the formation of blood cellular components ) . however , previous studies had shown that chronic administration of chloroquine increased megakaryocytes , blast cells , and mast cells in the spleen of albino mice which indirectly indicate possible alteration in haemopoiesis [ 19 , 20 ] ( figure 2 ) . in the lungs , the gradual breakdown of the thin walls of the alveoli ( emphysema ) was absent in the group treated with chloroquine whereas mild emphysema was observed in mice treated with artesunate . elastase is an enzyme produced by some white blood cells which destroys elastic fibres in the lungs as part of the normal turnover of elastic tissue . naturally , inhibitors such as aat ( -1-antitrypsin ) which is produced in the liver and released into the blood prevents the breakdown of elastic tissue in the walls of the alveoli . these inhibitors may become deficient since the malaria parasites had altered the normal functioning of the liver by destroying the liver cells . therefore , the alveolar thin wall may lose its elasticity and lead to the development of emphysema in the lungs . thickened alveolar wall was not observed in the lungs of mice treated with artesunate but mildly present in mice treated with chloroquine . in all the organs assessed in this study except the liver , infiltration of interstitial mononuclear cells was not observed in mice treated with the drugs of study but observed only in the untreated infected control . in the liver , this observation was very mild in the treated groups but absent in the untreated infected control . from this present result , reasons for these observations can not be stated , as well as if this observation leads to liver pathology . further experiments such as those aimed at measuring liver enzymes in the plasma of the treated groups may be considered . this study was sponsored by the authors because we could not get a research grant as at the time the study was conducted . this present study contributes to previous studies on pathological damages caused by the malaria parasite on vital organs . it therefore compares the effect of chloroquine and artesunate on the pathological damages of malaria in mice infected with p. berghei . the parasitaemia result shows that artesunate is more effective than chloroquine as observed in the number of days of parasite clearance . the observed effect of artesunate on the pathological damages caused by the malaria parasite was remarkable in the liver than the effect of chloroquine . however , this is a pilot study to future research where act ( artemisinin combination therapy ) will be assessed .

gait stability can be defined as the ability to maintain functional locomotion despite perturbations ( england and granata , 2007 ) . as a minimum , this ability is provided by position- and velocity - dependent resistance to perturbation - elicited deviations from the ongoing locomotion ( krasovsky et al . , 2012 ) . gait stability depends on the interplay between central and peripheral factors , which include the central pattern generator , muscle - reflex properties , and the mechanical interaction of the body with the environment ( taga et al . , 1991 ) . as of the former , locomotion on level have no difficulty due to improved function of gait stability , but often cause falling injuries by irregular surface and nonperceived visually , thus may cause serious problem in that an unexpected burden of medical expense and a lowering of living quality cause ( smeesters et al . , 2001 ) . mostly falling injuries occur in pathological gait or the old age , but performed preceded studies on the recovery response of posture after inducing tripping with young adult ( eng et al . , 1994 ; forner cordero et al . , 2000 ; smeesters et al . , 2001 ) because tripping is one of the source of the falling injuries during gait ( berg et al . , 1997 ; blake et al . , 1988 ; nevitt et al . , 1991 ) . when summarizing the above studies , preventing of falling injuries after tripping was to reduce angular momentum generated from impact between obstacle and body . particularly one of how to reduce angular momentum was to protract lower - leg more forwardly than center of gravity ( cog ) position in order to generate muscle power forward direction of gait ( grabiner et al . pavol et al . , 2001 ) , and thus trip leg is necessary to perform a delaying role in case of stance limb at time of tripping until the trip leg touch - down ( pijnappels et al . , 2004 ) . but afterwards studies considered irregular conditions of ground performing gait ground and intended manipulation of the obstacle is necessary and particularly on posture recovery by inducing tripping and response . because moment of hip , knee , and ankle joint increases relative to faster locomotion velocity in case of young adult and juvenile than the old age ( schultz , 1995 ) , these tripping may resulted in unrecoverable motor ability and serious handicapped situation . therefore it is necessary to diagnosis an injury part through epidemiologic survey helpful for rehabilitation and treatment relative to types of tripping during gait , but rare materials . the aim of this study was to analyze quantitatively correlation among variables of gait characteristics and incline angle of whole body by inducing tripping with young female . the subjects participated in the study consisted of adult female ( n=6 ; mean age , 21.830.75 years ; mean height , 159.914.16 cm ; mean weight ; 56.058.82 kg ) . floor and set up 4 camera ( hdr / hdv 1080i , sony corp . , control object box ( 2 m2 m1 m ) attached with 36 points for real spatial coordinates was filmed for 5 sec before filming for subjects . body segment parameters for film analysis was attached on each joint of the subject with the 31 reflexive markers ( rightleft toe , rightleft heel , rightleft lateralmedial malleolus , rightleft shank , rightleft lateralmedial epicondyle , rightleft thigh , rightleft anterior superior iliac spine , sacrum , rightleft lateralmedial wrist , rightleft lateralmedial elbow , rightleft shoulder , chin , nose ) on the basis of ( plagenhoef et al . , 1983 ) . as of fig . 1 to prevent a risk of falling and other s injuries , safety mat was spread out in front of gait direction and in right and left lateral direction . in particular subjects were blinded visual information with an eye patch in order not to predict a point of tripping time and controlled not to perceive the distance information after randomly performed the locomotion forward anteriorposterior direction . that is , tripping situation was not being fixed on gait path , obstacle was positioned in front of right foot after confirming a point of left foot supporting phase of subjects ( fig . then , gait direction was defined as y - axis of forward , x - axis of laterals and y - axis of vertical direction to y - x axis direction . supporting phase of one leg was set up on the basis of left foot , and then velocity of cog was analyzed from take - off after supporting phase of left foot , incline angle of whole body in anteriorposterior direction ( fig . 2 ) was set up as range of motion ( rom ) in anteriorposterior direction for projected vector of left foot - toe and chin to vertical line in sagittal plane . kwon3d xp v4.0 ( visol inc . , gwangmyeong , korea , 2007 ) was used to process obtain kinetic variables during gait and tripping . the kinetic variables were calculated using a control point , body - joint coordinate transformation and tuning , while the 3d coordinates were calculated using the direct linear transformation method . the cutoff frequencies obtained using the butterworth low - pass filtering method were smoothed at 6 hz to remove the noise before using them as data . the average and the standard deviation of the calculated variables were obtained using ibm spss statistics ver . 22.0 ( ibm co. , armonk , ny , usa ) and paired t - test was to draw comparisons between the two gaits ( =0.05 ) . the subjects participated in the study consisted of adult female ( n=6 ; mean age , 21.830.75 years ; mean height , 159.914.16 cm ; mean weight ; 56.058.82 kg ) . floor and set up 4 camera ( hdr / hdv 1080i , sony corp . , control object box ( 2 m2 m1 m ) attached with 36 points for real spatial coordinates was filmed for 5 sec before filming for subjects . body segment parameters for film analysis was attached on each joint of the subject with the 31 reflexive markers ( rightleft toe , rightleft heel , rightleft lateralmedial malleolus , rightleft shank , rightleft lateralmedial epicondyle , rightleft thigh , rightleft anterior superior iliac spine , sacrum , rightleft lateralmedial wrist , rightleft lateralmedial elbow , rightleft shoulder , chin , nose ) on the basis of ( plagenhoef et al . , 1983 ) . as of fig . 1 to prevent a risk of falling and other s injuries , safety mat was spread out in front of gait direction and in right and left lateral direction . in particular subjects were blinded visual information with an eye patch in order not to predict a point of tripping time and controlled not to perceive the distance information after randomly performed the locomotion forward anteriorposterior direction . that is , tripping situation was not being fixed on gait path , obstacle was positioned in front of right foot after confirming a point of left foot supporting phase of subjects ( fig . then , gait direction was defined as y - axis of forward , x - axis of laterals and y - axis of vertical direction to y - x axis direction . supporting phase of one leg was set up on the basis of left foot , and then velocity of cog was analyzed from take - off after supporting phase of left foot , incline angle of whole body in anteriorposterior direction ( fig . 2 ) was set up as range of motion ( rom ) in anteriorposterior direction for projected vector of left foot - toe and chin to vertical line in sagittal plane . , gwangmyeong , korea , 2007 ) was used to process obtain kinetic variables during gait and tripping . the kinetic variables were calculated using a control point , body - joint coordinate transformation and tuning , while the 3d coordinates were calculated using the direct linear transformation method . the cutoff frequencies obtained using the butterworth low - pass filtering method were smoothed at 6 hz to remove the noise before using them as data . the average and the standard deviation of the calculated variables were obtained using ibm spss statistics ver . 22.0 ( ibm co. , armonk , ny , usa ) and paired t - test was to draw comparisons between the two gaits ( =0.05 ) . the gait characteristics relative to tripping showed as table 1 . supporting phase and 1-stride time elapsed showed more delayed in tripping than that of normal gait and showed significant ( p<0.001 ) . length of one stride in tripping showed longer than that of normal gait , and showed significant ( p<0.001 ) . max change of velocity of cog during left supporting phase showed faster in tripping than that of normal gait and showed significant ( p<0.001 ) . incline angle of anteriorposterior direction of whole body showed more rom in tripping than that of normal gait and showed significant ( p<0.01 ) . supporting time and 1-stride time elapsed showed high correlation ( r=0.973 ) , and showed significant ( p<0.01 ) . relation between supporting time and change of cog velocity showed high positive correlation ( r=0.920 ) , showed significant ( p<0.05 ) . change of cog velocity and incline angle in anteriorposterior direction showed high positive correlation ( r=0.784 ) , and showed significant . if steady - state walking is not regained following perturbation , a fall may occur ( krasovsky et al . , 2013 ) . the study analyzed kinematic variables and its correlation on the change of posture before falling by inducing the tripping of right foot during gait . analysis of supporting time elapsed and 1-stride time elapsed of left foot showed more delayed in tripping than that of normal gait as of 37.35% and 61.74% respectively and gait length showed increased tendency of 36.37% in tripping than that of normal . ( 2004 ) shown to be more supporting time and 1-stride length in tripping than that of normal gait . also pijnappels et al . ( 2005 ) reported that only sufficient power from supporting leg at push - up phase during gait cycle may cause posture recovery and decrease angular momentum of body . when compared with this study , time delay of supporting leg provides a room of time deciding touch - down position of recovery leg and this mechanism are helpful for dynamic stability and control of leg position . particularly change of cog velocity showed faster ( 36.62% ) in tripping than that of normal gait and correlation between supporting time and 1-stride time elapsed ( r=0.973 , r=0.947 , p<0.001 ) showed positive correlation , and r=0.920 ( r=0.846 , p<0.001 ) with change of cog velocity and r=0.970 ( r=0.941 , p<0.001 ) with 1-stride time elapsed . that is , when tripped during gait , it may prevent falling injuries by counteraction of angular momentum and recovery of dynamic pattern of the faster forward velocity of cog . forward incline angle showed more rom in tripping than that of normal as of increase of 105% ( r=0.833 , r=0.613 , p<0.05 ) . the result shown due to fast forward incline angle of whole body as a result of failure of forward locomotion of supporting foot during tripping . thus because of instantaneous stoppage is impossible during tripping , it may bring about effective prevention of falling injury and decrease of angular momentum by protraction of trunk in front of cog position . that is , when tripped at one leg supporting phase during gait , instantaneous stoppage may be impossible due to forward velocity of cog and angular momentum . in case of sufficient time and the faster forward velocity of cog , it may recover an imbalance of tripping , but may increase possibility of falling injury due to difficulty to counteract an angular momentum if increased velocity of cog , wearing of bag and carrying of bag before tripping .

in this work , we demonstrate the concept of a sers - nanoreactor cluster using the capability of cb[n ] vessels to bind aunps creating precisely separated hot - spot nanojunctions for sensitive sers . the cb[n ] sequesters reactants in its internal cavity and their optimal location within these sers - active cluster nanojunctions enables photochemical reactions in the cavity to be tracked both in situ and in real - time . through choice of the smaller and larger homologues cb and cb , respectively , the internal volume of the cb[n ] nanoreactors allows us to obtain direct evidence for photoisomerization and photodimerization using sers . in addition , we are able to exploit the sers - nanoreactor motif to explore the role of ph on das cb host guest complexation and resultant photoreactions . the flexibility and versatility of the sers - nanoreactor concept is likely to provide great utility for studying and quantifying different chemical reactions that complex with cb[n ] and possess distinct raman spectra .

but the combination of nasogastric tube and double aortic arch ( daa ) , a rare congenital vascular abnormality , is known to cause fatal aortoesophageal fistula ( aef ) . moreover , daa had never been reported as a cause of superior mesenteric artery ( sma ) syndrome . a 19-year - old woman was admitted to our hospital for nausea and vomiting in april , 2009 . she had a past history of bronchial asthma in her childhood . as she was emaciated due to dysphagia and her unbalanced diet for 1 year , her body indices became low as follows : body height 153 cm , body weight 29.8 kg and body mass index 12.7 . computed tomography ( ct ) revealed that her stomach and duodenum were expanded to their limit , being filled with food , and that the third portion of the duodenum was stenotic between the sma and the abdominal aorta . a nasogastric tube was immediately inserted and over 5,600 ml of gastric contents were drained during the consecutive 4 days , and she became symptomless . esophagogastroduodenoscopy on the 5th hospital day disclosed extramural compression , smooth - surfaced stenosis with top ulceration that might be caused by the physical pressure of the nasogastric tube at the upper esophagus ( fig . a liquid diet was started on the 6th hospital day and the patient became better . suddenly , massive hematemesis appeared and she fell into shock on the 12th hospital day . a retrospective detailed clinical interview disclosed the fact that daa was pointed out in her childhood , although she quitted visiting a clinic since she had no symptoms . daa is a rare vascular congenital abnormality and forms a complete vascular ring tightly surrounding the trachea and esophagus . complete vascular ring is composed of a double aortic arch comprising the right aortic arch with the left ductus arteriosus and the aberrant left subclavian artery . most cases are diagnosed in infancy with only a few exceptions [ 1 , 10 , 11 ] . as symptoms are usually due to compression pressure to the surrounding organs such as the bronchus and esophagus , wheezing , stridor , noisy breathing and dysphasia are usual symptoms . thus , daa tends to be misdiagnosed as bronchial asthma or bronchitis in connection with wheezing and stridor , and as psychosomatic disorders in connection with dysphagia . our case is just such an example , as she was diagnosed with bronchial asthma in childhood and was strongly suspected of anorexia nervosa according to the diagnostic and statistical manual iv ( dsm - iv ) criteria at admission . tracheal development when growing up had gradually hidden the symptoms due to the compression of the trachea and bronchus . actually , adult cases of daa complain of swallowing difficulty rather than respiratory symptoms . both dysphasia due to daa and anorexia nervosa recently , 3d - ct is reported to be more useful for the diagnosis of daa than other modalities . the only therapy for daa is operation , decompressing bronchus and esophagus by dividing the non - dominant arch . the outcome of operation is good and it provides symptomless status in almost all cases . aef that is caused by intubation into the esophagus is reported to be a fatal complication in daa patients , although only 1 case has been reported in adults . as both an inserted tube and the vascular ring together compress the esophageal wall , necrosis and ulceration develop and often result in aef accompanied with massive hematemesis . once exsanguine hemorrhage occurs in daa patients under intubation , aef is a most probable complication , and a sengstaken - blakemore tube ( sb tube ) is only a choice of emergency actions in such instances , controlling hematostasis through its balloon effects , and emergency operation should immediately be taken into consideration . thirteen cases of aef caused by nasogastric tube insertion in daa have been reported so far ( table 1 ) , in which all but 1 case were non - adults . almost all cases had respiratory symptoms that might be related to weakness and immaturity in the bronchus . the duration of intubation with nasogastric tubes spanned 4 to 60 days , which means that intubation periods have no relation to an occurrence of aef . it is crucial to start the treatment quickly , since 3 cases in whom the sb tubes were inserted immediately after the diagnosis of aef were alive . endoscopic observation was performed in 4 cases , but the bleeding point of aef could be revealed in only 1 patient . based on these observations , it may be concluded that endoscopy is less effective in both diagnosis and therapy of aef . the compression of the third portion of the duodenum by the aorta and sma causes the syndrome . as our case showed an extremely dilated full stomach , a nasogastric tube was inserted to drain the gastric content . the patient immediately became symptomless ; thus , the nasogastric tube was removed after 4 days . as a result , aef was abruptly revealed . in this context , our case is extremely special because daa indirectly causes sma syndrome in adults , since dysphagia due to daa may decrease the amount of oral intake and it may in turn result in a marked emaciation that is a major cause of the syndrome . firstly , drainage is apparently needed for sma syndrome , although intubation is a contraindication in daa . secondly , an emergency operation should always be considered whenever aef due to esophageal ulceration is found because endoscopic hemostasis is very difficult , or even impossible . only the sb tube insertion may temporally be effective for a short duration . in our case , operation would have been indicated immediately after the esophageal ulceration was found by esophagogastroduodenoscopy , although in fact we could not imagine in how far it was important . in this context , our alternative proposal is to perform intubation and drainage of the gastric content as soon as possible , and thereafter the repair operation for daa should be indicated , since the operation is inevitable because the symptoms due to daa were already present , one of which was sma syndrome , which must be treated by nasogastric tube insertion . in conclusion , all of us should pay attention to the fact that nasogastric tube insertion is a contraindication in sma syndrome that is caused by daa , since it may cause very serious complications such as aef . whenever massive hematemesis occurs in daa patients under nasogastric tube intubation , sb tube is a possible temporary choice of action , and following emergency operation is mandatory . thus , we believe that before intubation for sma syndrome a detailed clinical interview clarifying whether a past history of daa exists is mandatory to avoid aef .

among the currently investigated metal - based chemotherapeutic agents , ruthenium ( ru ) and its compounds hold a promising future as these compounds exhibit different oxidation states and lower toxicity compared to other metal - based drugs.1 in general , ru compounds with oxidation states ii and iii have potential applications in the field of medicine , particularly as chemotherapeutic agents for various cancers.2 as illustrated in figure 1 , ru possesses unique properties that make it an attractive anticancer chemotherapeutic agent . in addition , ru forms various structural dimensions when combined with other compounds , and these combined compounds exhibit various modes of action such as interaction with dna and activation of dna damage mechanisms , as well as inhibition of the activities of several enzymes , including extracellular met - alloproteases.3 the relationship between function and composition of ru complexes is summarized in table 1 . therefore , ru complexes are considered more efficient , less toxic , and target - specific noncovalent dna - binding anticancer drugs.4 another property of ru compounds that makes them ideal candidates for medicinal applications is their ability to mimic iron binding to certain biomolecules such as serum transferrin and albumin , which are utilized by mammals to solubilize and transport iron , thus reducing cellular toxicity.4,5 in vivo studies demonstrated that rapidly multiplying cells have increased number of transferrin receptors to sequester more metal - loaded transferrin , thus attracting more ru.5,6 there is confirmation to suggest that ru complexes might directly interfere with specific proteins involved in signal transduction pathways , compared to any other metal complexes including iron complexes , and alter cell adhesion and migration processes.7 in addition , the redox potential between the different oxidation states of ru facilitates the body to catalyze oxidation and reduction reactions depending on the physiological environment . among the other metal complexes , ru complexes have different geometries , which allow for the design of compounds with a specific cellular target.8 the rigid and well - defined spatial arrangement of a series of ru complexes has enabled the preparation of highly potent and selective enzyme inhibitors , which is a very helpful characteristic feature in cancer therapy . moreover , the investigations of ru complexes in clinical trials attest to the possibility of utilizing non - platinum metal - based compounds in the treatment of cancer.46 because of these unique properties , ru is the most promising metal to replace platinum in future medicinal and pharmaceutical applications . however , application of these drugs in cancer treatment has been hampered by the low transport efficiency of their active species to biological targets . this has led to the design and development of new technologies based on nanostructured materials . the nanostructured materials generally act as vectors for metallodrug delivery or simply as protectors of active species of the complexes to enhance their activities and reduce their degradation.9 this review focuses on the recent developments in the nanostructured materials functionalized with ru complexes for tumor - targeted drug delivery . cancer is one of the leading causes of death in the world and is becoming an important topic in scientific research . cancer cells accumulate multiple mutations that confer them a selective growth advantage over other cells , causing them to grow uncontrollably , become invasive , and eventually spread to other parts of the body . in addition , cancer cells typically exhibit aberrations in signaling pathways that regulate various cellular processes , such as cell growth and division , and cell - to - cell communication.10 therefore , the genetic and phenotypic heterogeneities of cancer cells make cancer a disease that is difficult to treat . among the effective anticancer treatments , transition metal complexes play important roles in all living systems and have various therapeutic applications.11 these metals form positively charged ions in aqueous solution that can bind to negatively charged biological molecules . consequently , the charge can be modified depending on the coordination surroundings involved , leading to the generation of a species that can be anionic , cationic , or neutral . in addition , metal ions with high affinity for electrons can polarize groups that are coordinated to them , fostering the generation of hydrolysis reactions.12 the oxidation state of a metal is also an imperative consideration in the design of coordination compounds , as it allows the participation in biological redox chemistry and plays a crucial role in determining the optimal dose and bioavailability of the agent administered.13 furthermore , transition metals can undergo ligand exchange reaction , and this property allows them to interact with and coordinate to biological molecules . among the transition metal complexes , ru - based complexes are regarded as highly promising drug candidates . therefore , the field of coordination and organometallic chemistry of ru has expanded considerably over the past few years.14,15 as previously discussed , the unique properties of ru - based complexes , including their high ability to bind nucleic acids and proteins and undergo ligand exchange reaction , the prevalence of the two main oxidation states , ru(ii ) and ru(iii ) , as well as the iron - mimicking property when bound to biological molecules have made them potential drug candidates for various medical applications.16 in addition , these complexes have reduced toxicity and can be well tolerated in vivo.17 studies have also shown that most ru complexes have low systemic toxicity , exhibit slow ligand exchange rates similar to that of platinum compounds , and can accumulate in cancer cells more effectively than platinum . the anticancer mechanisms of ru complexes have been extensively investigated as illustrated in table 2 . the various mechanisms that mediate the anticancer activities of ru complexes include inhibition of topoisomerase activity , inhibition of metastasis , production of reactive oxygen species ( ros ) , interaction with proteins and dna , as well as induction of apoptosis . in addition , the anticancer effects of ru complexes depend largely on the ligand involved , characteristics of the complexes , and the presence of uncoordinated sites in the coordination sphere of the metal center.7,18 as such , much effort has been directed toward exploring the tumor - inhibiting properties of various ru complexes.18 as listed in table 2 , although a large number of ru complexes have shown great promise as potential antitumor agents , only three complexes , nami - a , kp1019 , and nkp1339 ( table 3 ) , have entered clinical trials.16,19,20 clinically , all the three complexes exhibited promising anticancer activities with minimal side effects ; an ideal property of a good anticancer drug ( table 3 ) . in addition , these complexes have octahedral coordination geometry with respect to the ru(iii ) metal center ; however , they exhibit different biological activities despite their structural and chemical similarities.19 recently , 2,2,6,6-tetramethylpiperidine-1-oxyl radical ( tempo)-functionalized ru(ii ) polypyridyl complexes were screened for their efficiency as theranostic photosensitizers for cancer treatment.21 the presence of a redox - sensitive tempo moiety enhanced the intracellular fluorescence of tempo - functionalized ru(ii ) complexes in photodynamic treatment , which was observed using confocal microscopy and flow cytometry . hence , the combination of tempo with photosensitizers is a promising strategy for the development of novel photosensitizer - based theranostic platforms , which can induce and monitor photodynamic therapy responses simultaneously . in another study , gill et al demonstrated that the ru(ii ) polypyridyl complex [ ru(dppz)2(pip ) ] intercalates dna and acts to stall replication fork progression in human cancer cells , leading to the activation of dna replication stress signaling responses and inhibition of cell growth by cell - cycle deregulation . combination of the ru complex with a pathway - specific dna damage response inhibitor resulted in synergistic cancer cell killing . in addition , the ru complex functions as a radiosensitizer in combination with external beam ionizing radiation . these findings indicate that ru polypyridyl complexes ( rupops ) are useful tools to study the molecular consequences of dna intercalation . hence , the potential benefits and application of these complexes in combination therapy for cancer treatment warrants further investigation.22 recently , koceva - chya et al evaluated the in vitro anticancer activity of dinuclear trithiolato - bridged arene ruthenium complex diruthenium-1 ( diru-1 ) against a panel of human cancer cell lines including hepg2 ( hepatocellular carcinoma ) , mcf-7 ( estrogen - responsive breast adenocarcinoma ) , and mda - mb-231 ( triple - negative breast adenocarcinoma ) cells . diru-1 was found to be extremely toxic to these cell lines , with half - maximal inhibitory concentration ( ic50 ) values in the low - nanomolar range . the cytotoxic and proapoptotic effects of diru-1 were attributed to the increase in intracellular levels of ros , while the anticancer effect was due to the induction of dna lesions caused by apoptotic dna fragmentation and cell - cycle arrest at the g2/m checkpoint.23 in summary , the diverse modes of action of ru anticancer drugs likely enhanced their anticancer activities and minimized the potential for cancer cells to develop resistance against these drugs.24 novel approaches to metabolic studies are required to give additional mechanistic insights , which could lead to the rational design of better ru anticancer drugs . despite solving many key issues encountered with conventional anticancer therapies such as chemotherapy , systemic delivery of nanoparticles faces several of its own challenges , one of which is the lack of successful tumor localization . among the various strategies , nanostructured materials functionalized with metal complexes have attracted the most interest among oncologists and medicinal chemists as a better alternative for administration of metallodrugs in targeted anticancer therapy.9 in addition , the encapsulation and delivery of metallodrugs in nanostructures have overcome the problems of poor bioavailability , degradation , and side effects.25 nanotechnology currently allows for a unique control of the material world , and any material comprising an average phase or grain size in the order of a nanometer ( 10 m ) is defined as a nanostructured material . at this scale , man - made objects are able to gain access to cells as nanostructured materials have enhanced mechanical properties compared to conventional materials due to their ultrafine microstructure and ability to interface with living cells . nanostructured materials of varying compositions play important roles in cancer therapeutics due to their passive tumor targeting property.26,27 in this context , inorganic metal complexes occupy a pioneer niche as they offer a multipurpose platform for drug design and development , and the nanostructures used in the function - alization of metallodrugs act as transport vehicles to deliver the metallodrugs to the biological target . nanostructured systems , such as macromolecular systems , carbon nanotubes , dendrimers , metallacages , ceramic materials , liposomes , and lipid nanocapsules , as well as metal and polymeric nanoparticles are commonly used for drug delivery . in addition , the optimum loading and sustained release of metallodrugs from nanostructures are essential requirements for every drug delivery system . therefore , it is important to ensure that metallodrugs are attached to the surface of nanoparticles with adequate strength to withstand the wear and tear during the passage to the target site . however , it is also crucial to ensure that the bond is weak enough to release the metal - lodrugs at the target site.9 in a study conducted by wu et al , gold nanoparticles ( aunps ) were shown to be efficient carriers for small interfering rna ( sirna ) delivery into cancer cells . in this study , cysteamine - functionalized aunps were found to effectively deliver transforming growth factor beta 1 ( tgf-1 ) sirna into hepatoma hepg2 cells , and compared with aunp - mediated nano carrier ( nc ) sirna ( aunp sinc ) , aunp - delivered tgf-1 sirna ( aunp sitgf1 ) successfully decreased the level of tgf-1 , increased cell apoptosis , and significantly inhibited the proliferation of recipient tumor cells . likewise , systemic administration of aunp sitgf1 complexes into human hepg2 xenografted mice reduced tgf-1 expression and its downstream signaling . sitgf1 strongly inhibited tumor growth and improved the survival rate of tumor - bearing mice compared with the control group . these results suggest that aunp is a highly effective method for delivering rna interference therapeutics into tumor cells.28 in another study , titanium dioxide ( tio2 ) was functionally modified with amine , sulfate , and phosphate ions into the biocompatible titania ( f - tio2 ) . due to the porous nature of f - tio2 , it can trap drugs into its cavity , allowing it to be used as a nanocarrier . f - tio2 showed little to no toxicity when tested at various concentrations in several cancer cell lines , with < 10% cell death . these results suggest that f - tio2 can be used as a nanostructured carrier to deliver drugs effectively and safely by functionally modifying the surface to increase its affinity toward acidic environment ( tumor ) , thus minimizing toxicity and accumulation in normal cells and tissues.29 in recent years , ru complexes have attracted much attention as an alternative to existing platinum drugs , showing remarkable antitumoral and antimetastatic activities with low toxicity.30,31 however , one of the foremost drawbacks of ru complexes is their rather limited stability in aqueous solutions . therefore , designing long - life ru - based antineoplastic agents is the primary goal to overcome this limitation , and nanostructured materials functionalized with ru complexes serve as one of the alternatives for targeted drug delivery.3235 a recent study by huang et al showed that the antitumor efficacy of rupops could be enhanced by using cancer - targeted dna origami as a biocompatible nanocarrier . under this condition , the formation of unique tetrahedral nanostructure of dna cages effectively enhanced the partial intercalation of rupop , thus increasing the drug - loading efficacy . in addition , conjugation of biotin to the dna - based nanosystem ( bio - cage@ru ) enhanced its specific cellular uptake , drug retention , and cytotoxicity against hepg2 cells . unlike free rupop and the cage itself , bio - cage@ru is translocated to the cell nucleus after internalization , where it undergoes self - immolative cleavage in response to dnases , triggering drug release , and inducing ros - mediated cell apoptosis . in the nude mouse model , this nanosystem exhibited satisfactory in vivo antitumor efficacy as it specifically accumulated in tumor sites and alleviated rupop and tumor xenograft - induced damages to the liver , kidney , lung , and heart function . therefore , this study demonstrates a strategy for the development of biocompatible and cancer - targeted dna origami with enhanced anticancer efficacy and reduced toxicity for next - generation cancer therapy.32 a novel ph - sensitive nanocapsule was recently shown to effectively deliver tris(1,10-phenanthroline ) ru(ii ) complex ( 3p - ru ) into glioma cells . in this study , delivery of 3p - ru using the ph - sensitive nanocarrier significantly inhibited cell growth in both in vitro and in vivo models . the nanocapsule - induced cell death was mediated in part through the induction of apoptosis . as such , this novel tumor targeting approach holds great potential for theranostics applications.33 in another study , a ph - sensitive mesoporous silica nanocarrier , runhc@msns chitosan biotin , was synthesized for the targeted delivery and controlled release of a ru(ii ) n - heterocyclic carbene ( runhc ) complex . the authors demonstrated that the complex is selectively internalized by cancer cells through biotin receptor - mediated endocytosis , which enhanced its anticancer activities . hence , this approach offers a platform for the development of accurately controlled cancer therapy.34 in another study , a dual - fluorescent nanocarrier was synthesized by coating human serum albumin on ru complex - loaded lanthanide - doped upconversion nanoparticles to generate ru human serum albumin upconversion nanoparticles . interestingly , this conjugate showed very low inhibitory effect on cell proliferation in the dark , whereas light irradiation considerably improved its cytotoxicity toward cancer cells . in addition , irradiation activated this ru complex and the product became highly reactive to dna . the obtained results suggest the potential application of this conjugate in the controlled release of active anticancer agents in tumor sites.35 recently , two novel ru metalloarenes were synthesized based on anthracene units , and both compounds showed high cytotoxicity against hl-60 cancer cell line , mediated by the induction of apoptosis . in addition , these ru metallodrugs were successfully encapsulated into two well - known biocompatible and stable mesoporous silicas ( mcm-41 and sba-15 ) using impregnation and grinding strategies.36 in a study , xue et al developed a supramolecular approach that assembles a -cyclodextrin ( cd)-functionalized ru(ii ) polypyridyl complex with an adamantane - appended tumor - targeting cyclic arg - gly - asp c(rgdyk ) peptide . in this study , the host consequently , the formed nanoparticles ru - cd - rgd display high selectivity for integrin v3-rich human glioblastoma ( u87 mg ) cancer cells over integrin v3-deficient cancer cells . mechanistic studies show that ru - cd - rgd can induce apoptotic cell death through lysosomal damage , ros elevation , and caspase activation.37 in another study , ru(ii ) complex - functionalized single - walled carbon nanotubes were synthesized as nanotemplates for bimodal photothermal and two - photon photodynamic therapy . here , the synthesized single - walled carbon nanotubes have the ability to load a large quantity of ru(ii ) complexes via noncovalent interactions . in addition , the loaded ru(ii ) complexes are competently released by the photothermal effect of irradiation . the released ru(ii ) complexes produce singlet oxygen species ( o2 ) upon two - photon laser irradiation and can be used as a two - photon photodynamic therapy agent . based on the combination of photothermal therapy and two - photon photodynamic therapy , ru@ single - walled carbon nanotubes have greater anticancer efficacies than either photodynamic therapy using ru(ii ) complexes or photothermal therapy using single - walled carbon nanotubes in two - dimensional cancer cell and three - dimensional multicellular tumor spheroid models.38 in a study by wang et al , a carbon nanotube - based radiosensitive drug delivery system was developed to provoke the multidrug resistance in hepatocellular carcinoma . here , the nanosystem was loaded with rupop via interaction and the formation of a hydrogen bond . the functionalized nanosystem enhanced the cellular uptake of rupop in liver cancer cells , particularly drug - resistant human hepatoma cell line , through endocytosis.39 a list of nanostructured materials functionalized with ru complexes for targeted drug delivery are summarized in table 4 . in addition to the above - mentioned findings , the miniaturization of metal organic frameworks ( mofs ) has generated great interest among researchers to integrate these materials into various applications such as sensing or drug delivery . zhang et al demonstrated that ru complexes combined with mofs exhibited effective two - photon adsorption for singlet oxygen ( o2 ) generation . they successfully synthesized a novel heterogeneous o2 generation system , which may prove helpful in photodynamic therapy and chemotherapy integrative collaboration for cancer treatment.40 functionalization of metallodrugs into nanostructures is an increasing area of research , and more groundbreaking advances are expected to be made in the future . nanostructured ru complexes can be used to enhance the efficacy of cancer treatment by targeted delivery of drugs or genes into cancer cells and reduce the adverse effects and systemic toxicity by optimization of the pharmacokinetics property of these complexes . although nanostructured materials functionalized with ru complexes have shown great potential in the treatment of cancer , there are still many challenges in translating basic research into clinical applications . therefore , the most promising area in this field is the development of functionalized nanostructured metal complex drug delivery systems for controlled release , which can greatly improve the activity of complexes without causing any severe adverse effects . in spite of their wide application in cancer chemotherapy , , there is an urgent need to thoroughly study the pharmacokinetic behaviors of different types of nanoparticles both in vivo and in vitro .

paracoccidioidomycosis ( pcm ) , also known as south american blastomycosis , is a chronic mycosis caused by paracoccidiooides brasiliensis . pcm more commonly affects farm workers and/or people who live in rural areas , people between 30 and 50 years of age , particularly males . the disease is more prevalent in south and central americas , especially in regions with wet climate , high rainfall , and acid soils . in general , pcm manifests firstly in lungs , and later may spread systemically , affecting lymph nodes , skin , oral and nasal mucosae , adrenal glands , gastrointestinal tract and central nervous system through lymphatic and blood vessels . expectoration , cold , dyspnea , thoracic pain , fever , hemoptysis , anorexia , and weight loss are observed in most cases . this study presents a case report of pcm with lung , skin and oral manifestations whose diagnosis was reached based mainly on oral lesions , by the dentist . a 41-year - old mulatto , male , bricklayer , reported to our institution with a chief complain of an ulcer located in the palate with 5 months of duration that , in his own words , caused a nuisance during meals . he reported that he smoked 20 cigarettes a day for 20 years and that lived at rural area of the city . the patient also reported having lost weight ( 4 kg in the previous month ) , nocturnal sudoresis and dry cough . general clinical examination revealed an ulcer and a nodule , on the right shoulder skin [ figure 1a ] . intraoral examination revealed an ulcerative lesion with punctuate and hemorrhagic appearance involving gum and palate [ figure 1b and c ] and poor oral hygiene habits . ( a ) cutaneous lesions presenting as a sessile papule , covered by apparently normal skin and as an ulcer with punctuate and hemorrhagic appearance . ( c ) palatal view showing an extensive ulceration with necrotic areas extending from midline to gingival margin . a sample was retrieved from the posterior portion of the palate , near midline [ figure 1c ] . these cells presented small round birefringent structures indicating presence of fungi , whose size and morphology were suggestive of pmc [ figure 2 ] . microscopic view presenting granulomatous inflammation characterized by collections of epithelioid macrophages and multinucleated giant cells with small round structures indicating presence of fungi ( detail ) blood test indicated leukocytosis and neutrophilia , suggesting the presence of infection . radiographs of the thorax revealed reticulonodular infiltrate distributed in lungs , reinforcing the presence of infection . taken together , clinical , histopathological , laboratory , and radiographic findings led to a final diagnosis of pcm . the patient was referred to pneumology treatment , which included itraconazole ( 200 mg / day for 6 months ) . the patient was also referred to dental practitioner to perform scaling and root planning procedures , but he did not follow the recommendation . after 5 months , the follow - up consultations were recover , and patient showed regression of skin [ figure 3a ] and oral [ figure 3b and c ] lesions , which leaved scar areas . pcm is a systemic mycosis , noncontagious , acquired from soils contaminated with the microorganisms . therefore dentists must be capable to recognize its clinical features , in order to establish an accurate diagnosis . our patient presented the disease for five months prior to diagnosis , and had attended to some health professionals who did not manage the disease properly , even though south brazil is an endemic area for pcm . the presence of cough , tiredness and weight loss raise the hypothesis of a chronic infectious lung disease followed by the emergence of skin and oral lesions . the most common oral manifestations of these diseases are ulcerative lesions , especially in the palate and gums . another possibility is that the patient had a lung disease unrelated to these mucocutaneous lesions , such as recurrent pneumonia . however , it is well - known that the radiological changes observed in pcm are diverse and unspecific . regardless , it should be emphasized that multidisciplinary efforts become evident , in order to achieve the definitive diagnosis . skin lesions associated with pcm are polymorphic , presenting as papulae , plaques or nodules , which sometimes become verrucous or ulcerative . the diverse clinical presentation regarding frequency , number , distribution , and morphology of the skin lesions is related to the interaction between agent and host . about 60% of cases , presents concomitantly in oral mucosa and skin . the present case is unusual , because the patient had skin and oral lesions , simultaneously . interestingly , these lesions were located on upper limbs , which are reported in only 15% of cases . this case attempts to highlight the importance to include a strict evaluation of general health conditions during clinical examination , which is sometimes neglected by a significant number of the general dentists . particularly in our case , the observation of the skin lesion manifested as fine granular ulcers and pinpoint hemorrhages was strongly contributory , since these may be considered as pathognomonic sign of pcm . since our patient showed multiple oral lesions , squamous cell carcinoma ( often included in differential diagnosis of pmc ) was not considered , since it presents typically as a single lesion . on the other hand , the occurrence of an extensive oral ulcer includes leishmaniasis , cryptococcosis , histoplasmosis , tuberculosis and aspergillosis as differential diagnosis . the observation of morifom lesions in the gums was helpful , since these characteristics are described in 76% and 50% of pcm cases , respectively . in spite of that , the diagnosis could only be confirmed based on the biopsy and histological findings . this specific evaluation addresses morphological characteristics of the pathogen as to spore size , type of gemulation , and presence of septate hyphae . exfoliative cytology exams may be useful in diagnosis , but it requires experienced and training of professionals . serological investigation , besides potentially contributory , is not routinely used in oral medicine in these cases . several serological and molecular assays , like enzyme - linked immunosorbent assay and polymerase chain reaction , may also be useful for that purpose . sensitivity and specificity of these assays may vary between 73.4 - 100% and 87.5 - 100% , respectively . although sensitive and specific , serological assays are not reliable as a cure parameter , since high titers may be found even after cure . in this sense , definition of clinical criteria for resolution is difficult , and should be based on careful clinical evaluation . although pcm is a well - known disease , there are reports of cases whose diagnosis was difficult . in most cases addressed correctly , diagnosis is reached in approximately 3 months . in the present case report , the time interval elapsed between emergence of lesions on skin and definition of diagnosis based on oral lesions was 5 months . this delay may be explained in the light of the fact that symptoms of lung manifestations were unspecific , and that the clinical picture progressed slowly , since skin lesions were not appropriately diagnosed . the delay in reaching an accurate diagnosis may lead to disease spreading to abdominal lymph nodes , spleen , liver , suprarenal glands , urogenital tract , bones , gastrointestinal tract or brain , and eventually life risk . one possible reason is acrolein , a cigarette component , which may cause inflammation , bronchoconstriction , increase of mucous secretion favoring respiratory infections like pcm . the drug choice is based on the severity and degree of dissemination of the disease , as well as on the immune status of the host . itraconazole , amphotericin b and sulfonamides are the most commonly drugs prescribed . in our case , reported here , the infection subsided after 7 months , and the patient remained under follow - up . since pcm is a systemic disease , the treatment is conducted by the physician and the regression of oral lesion must be followed by dentist . multidisciplinary management allows the adoption of the best supporting measures to address the systemic changes associated to disease , like malnutrition and other comorbidities , as well as dental treatment . in addition , it should be emphasized that follow - up for extended periods is mandatory . the present case report shows that the dentist has an important role in the patient 's general health evaluation . the adoption of the all steps of clinical examination as routine decreases the chances of misdiagnosis and wrong therapeutic approaches . early diagnosis is extremely important to prevent the more severe manifestations of the disease , which may lead to higher morbidity or even mortality .

nonalcoholic fatty liver disease ( nafld ) represents the most common and emerging form of chronic liver disease worldwide . indeed , nafld reached epidemic proportions and the general prevalence of this condition is reported to be ranging between 2030% and 7090% in patients with severe obesity or with type 2 diabetes mellitus [ 14 ] . nafld includes a wide spectrum of liver diseases ranging from simple fatty liver to nonalcoholic steatohepatitis ( nash ) , which may progress to fibrosis and even cirrhosis and hepatocellular carcinoma . simple steatosis generally represents a benign condition following a nonprogressive clinical course . on the contrary , a subset of patients with nash , in particular those with a more severe fibrosis , is at higher risk for progressing to liver disease complications such as decompensated cirrhosis and hepatocellular carcinoma . several lines of evidence suggest that chronic oxidative stress is one of the key mechanisms responsible for liver damage and disease progression in nafld . in particular , according to the two - hit theory , oxidative stress is a major player triggering the progression of steatosis to nash as the result of an imbalance between pro - oxidant and antioxidant chemicals that lead to liver cell damage . consistent with the above theory , in a previous study , we demonstrated an increased systemic oxidative stress in subjects with nafld , which was associated with severity of liver steatosis ( submitted ) . recently , it has been reported that blood levels of cytokeratin-18 ( ck-18 ) fragments , the major intermediate filament protein in the liver , may predict histological nash and severity of liver damage in patients with nafld , representing a marker of hepatocyte apoptosis . indeed , cell repair , inflammation , regeneration , and fibrosis typical of nash may be triggered by hepatocyte apoptosis . a link between hepatocyte apoptosis and liver fibrogenesis in fact is supported by both experimental and human studies , and data suggest that apoptosis is prominent in nash but not in simple steatosis . however , very limited information is available regarding the possible role of oxidative stress in triggering hepatocyte apoptosis in this setting . notably , no study has documented the relationship between novel and validated markers of oxidative stress such as urinary 8-iso - prostaglandin f2 ( 8-iso - pgf2 ) or soluble nox2-derived peptide ( snox2-dp ) and ck-18 serum levels . the aim of this study was to determine the predictors of ck-18 levels to elucidate the possible role of oxidative stress in the severity of liver disease in a population of nafld patients . the study was performed in 209 consecutive patients referred to our metabolic outpatient clinic for suspected metabolic disease , who had a liver ultrasonographic scanning ( us ) positive for nafld performed as part of routine clinical examination . to be eligible for the study , patients had to have fulfilled the following criteria : no history of current or past excessive alcohol drinking as defined by an average daily consumption of alcohol > 20 g ; negative tests for the presence of hepatitis b surface antigen and antibody to hepatitis c virus ; absence of history and clinical , biochemical , and us findings consistent with cirrhosis and other chronic liver diseases . none of the subjects was taking amiodarone and other drugs known to promote fatty liver disease . waist circumference , height , and weight were recorded and body mass index ( bmi ) was calculated as weight ( kg ) divided by height . subjects taking insulin or oral antidiabetic drugs were considered to have diabetes . according to the modified criteria of the atp iii expert panel of the us national cholesterol education program , mets was diagnosed on the concomitant presence of at least three of the following five clinical features : waist circumference ( central obesity ) 102 cm in men and 88 cm in women , fasting blood glucose 100 mg / dl , triglycerides 150 mg / dl , hdl - cholesterol < 40 mg / dl in men and < 50 mg / dl in women , and arterial systolic / diastolic blood pressure 130/85 mm / hg . a metabolic score was calculated for each patient based on the number of the discrete components of mets . nafld fibrosis score ( nfs ) based on age , bmi , hyperglycemia , platelet count , albumin , and ast / alt ratio , was calculated for each patient according to angulo et al . . the study was approved by the hospital ethics committee and conforms to the ethical guidelines of the 1975 declaration of helsinki . all us were performed by the same operator who was blinded to laboratory values using a ge vivids6 apparatus equipped with a convex 3 , 5 mhz probe . liver steatosis was defined according to hamaguchi criteria based on the presence of abnormally intense , high level echoes arising from the hepatic parenchyma , liver - kidney difference in echo amplitude , echo penetration into deep portion of the liver , and clarity of liver blood vessel structure [ 13 , 14 ] . steatosis was assessed semiquantitatively on a scale of 06 : 0 , absent ; 1 , 2 mild ; 3 , 4 moderate ; and 5 , 6 severe . the splenic diameter was calculated as the maximum length of the spleen after visualizing the organ in a plane passing through the splenic hilum . serum total cholesterol , hdl - cholesterol , and triglycerides were measured by an olympus an 560 apparatus using an enzymatic colorimetric method . the homeostasis model of insulin resistance ( homa - ir ) , based on serum fasting glucose and insulin levels , was used as a measure of insulin resistance . serum levels of cytokeratin 18-m30 ( ck-18 ) were measured as markers of liver damage with a commercial immunoassay ( tema ricerca , italy ) and expressed as ml u / ml . intraassay and inter - assay coefficients were 6% and 7% , respectively . urinary 8-iso - prostaglandin f2 ( 8-iso - pgf2 ) , as marker of whole body oxidative stress , was measured by a previously described and validated enzyme immunoassay method . intra - assay and inter - assay coefficients of variation were 2.1% and 4.5% , respectively . serum levels of soluble nox2-derived peptide ( snox2-dp ) were detected by elisa method as previously described ; intra - assay and inter - assay coefficients of variation were 5.2% and 6% , respectively . adiponectin ( apn ) serum levels were measured with a commercial immunoassay ( tema ricerca , italy ) . intra - assay and inter - assay coefficients of variation were 6 and 8% , respectively . statistical analysis was performed by using the spss statistical software version 20.0 for windows ( spss , inc . , proportions and categorical variables were tested by the -test and by the 2-tailed fisher 's exact method when appropriate . data are expressed as median followed by 25th and 75th centiles for nonnormally distributed data and as mean sd for normally distributed variables . group comparisons for nonnormally distributed variables were tested by the mann - whitney test and kruskall - wallis test . instead , normally distributed variables were analyzed by the use of analysis of variance ( anova ) and unpaired student 's t - test when appropriate . all p values are two - tailed ; a p value of less than 0.05 was considered to indicate statistical significance . stepwise , multivariate , regression analysis was performed to assess the independent predictors of serum ck-18 values . the predictor variables entered were age , bmi , homa - ir , serum triglycerides , alt and apn , urinary 8-iso - pgf2 , snox2-dp , hamaguchi score , and spleen diameter . the study was performed in 209 patients with nafld ( 43 with mild , 87 with moderate , and 79 with severe steatosis , resp . ) . median serum ck-18 values progressively increased with nafld severity ( from 169.5 ( 129.3/183.8 ) to 176 ( 140/190 ) and 180 ( 169.5/192.5 ) iu / ml in mild , moderate , and severe steatosis , resp . ; p < 0.01 ) . after stratification of subjects in serum ck-18 tertiles , significant differences of some variables were found . bmi ( 27.2 3.9 versus 31.6 3.3 versus 36.1 5.4 kg / m ; p < 0.001 ) , homa - ir ( 2.8 ( 1.9/4.0 ) versus 3.2 ( 2.4/5.7 ) versus 5.3 ( 3.4/7.3 ) ; p < 0.001 ) , serum triglycerides ( 125.5 ( 85.5/160.3 ) versus 138 ( 102.0/180.5 ) versus 165.0 ( 125.0/206.0 ) mg / dl ; p < 0.01 ) , prevalence of diabetes ( 20.3% versus 30.1 versus 43.3 ; p < 0.05 ) , prevalence of severe nafld ( 24.6 versus 42.5 versus 46.3 ; p < 0.05 ) , urinary 8-iso - pgf2 ( 616.7 122.9 versus 727.5 82.1 versus 800.9 82.1 pg / mg creatinine ; p < 0.001 ) , and snox2-dp ( 49 ( 40/58.3 ) versus 60 ( 50/66 ) versus 67 ( 64/71 ) pg / ml ; p < 0.001 ) increased significantly from first to third ck-18 tertile . instead , serum hdl - cholesterol ( 50 ( 43/61 ) versus 47 ( 38/54 ) versus 43 ( 39/51 ) mg / dl ; p < 0.05 ) and apn ( 12 ( 10.4/14.1 ) , versus 7.5 ( 5.5/10.5 ) versus 5 ( 4/7 ) ng / ml ; p < linear bivariate regression showed a positive correlation between serum ck-18 and bmi ( r = 0.58 ; p < 0.001 ) , homa - ir ( r = 0.19 ; p < 0.01 ) , hamaguchi 's score ( r = 0.19 ; p < 0.01 ) , urinary 8-iso - pgf2 ( r = 0.61 ; p < 0.001 ) , snox2dp ( r = 0.45 ; p < 0.001 ) , and nfs ( 0.30 ; p < 0.01 ) ; conversely , a negative correlation between serum ck-18 and serum hdl - cholesterol ( r = 0.15 ; p < 0.05 ) and apn ( r = 0.45 ; p < 0.001 ) was observed ( table 3 ) . stepwise , multiple regression analysis was performed to assess the independent contributors to serum ck-18 levels . in the final equation , bmi , serum apn , and snox2-dp were independent predictors of serum ck-18 levels , after controlling for age , homa - ir , serum triglycerides , alt , urinary 8-iso - pgf2 , hamaguchi score , and spleen diameter ( adjusted r = 0.36 ) ( table 4 ) . in our study , performed in a large series of subjects with documented nafld at us scanning , we have demonstrated a strong and independent association between snox2-dp and ck-18 serum levels . moreover , at univariate analysis , also urinary 8-iso - pgf2 was positively correlated with ck-18 values . to our knowledge , this is the first evidence of an association between two markers of systemic oxidative stress and a marker of apoptosis and liver disease severity in subjects with nafld . to assess oxidative stress in vivo , we measured urinary 8-iso - pgf2 and serum levels of snox2-dp . measurement of urinary 8-iso - pgf2 is widely accepted as a reliable indicator of oxidative stress in vivo [ 18 , 19 ] . soluble nox2-dp is a marker of nox2 activation by blood cells , which is a member of the nadph oxidase family which plays an important role in ros generation [ 20 , 21 ] . in previous studies , we have described elevated urinary 8-iso - pgf2 and serum snox2-dp levels in a number of chronic inflammatory and metabolic diseases such as metabolic syndrome , hypercholesterolemia , obstructive sleep apnoea syndrome , and obesity [ 2225 ] . moreover , in a recent study , we demonstrated an increased nox2 generated oxidative stress also in subjects with nafld ( submitted ) ; in this clinical setting , oxidative stress was independent from obesity , diabetes , and mets and increased with the severity of liver steatosis at us . to assess liver damage and hepatocyte apoptosis , we measured serum ck-18 levels , which have been recently reported to predict histological nash and severity of liver disease in patients with nafld [ 7 , 26 ] . indeed , some recent studies investigated circulating levels of ck-18 fragments as novel biomarkers for the presence of nash in patients with nafld , and suggested the potential usefulness of this test in clinical practice . in fact , in a study performed in 44 consecutive patients with suspected nafld at the time of liver biopsy , plasma ck-18 fragments were markedly increased in patients with nash compared with patients with simple steatosis or normal biopsies and independently predicted nash ( or 1.95 ; 95% ci 1.183.22 for every 50 u / l increase ) . moreover , ck-18 fragment levels were validated as noninvasive biomarkers for nash also in a multicenter study performed in a large , diverse population of patients with biopsy - proven nafld . consistent with this theory , we found a significant correlation between ck-18 serum levels and nfs , an accurate , noninvasive scoring system based on routinely measured and readily available clinical and laboratory data , that identifies advanced liver fibrosis in patients with nafld . recently , nfs has been validated for predicting death or liver complications in nafld patients over long - term follow - up . to our knowledge , this is the first time that the association between serum ck-18 and nfs has been described . the finding of a strong independent positive association of two reliable markers of oxidative stress with a marker of hepatocyte apoptosis is consistent with the two - hit theory based on the prominent role of oxidative stress as a major player triggering the progression of steatosis to nash . in fact , according to the two hits hypothesis , the development of nash requires two hits to become manifested . the first one is represented by the development of steatosis , while the second hit is induced by a disbalance between oxidative stress and antioxidant systems , leading to cell injury and inflammation ( i.e. , steatohepatitis ) and lipid peroxidation . in keeping with this theory , hepatocyte apoptosis is likely to be considered a component of the second hit . accordingly , a working model in which apoptosis and formation of reactive oxygen species are caspase dependent , with final release of ck-18 fragments , has been proposed . indeed , cell repair , inflammation , regeneration , and fibrosis typical of nash may be triggered by hepatocyte apoptosis . a link between hepatocyte apoptosis and liver fibrogenesis is supported by both experimental and human studies . high serum ck-18 values were associated with high homa - ir , high fasting blood glucose and triglycerides , and low hdl cholesterol , that is , the metabolic features of mets , whose prevalence in patients belonging to the top ck-18 tertile reached 85% . at multivariate analysis , an independent association between low serum apn and increased ck-18 was also observed . apn is lower in central obesity and has powerful antioxidant properties . in a previous study , we have shown that higher apn serum levels are associated with nadph oxidase down - regulation . higher levels of apn have been also associated with a more beneficial oxidative stress profile , and higher levels of antioxidants together with lower levels of lipid peroxidation . moreover , serum apn negatively correlated with urinary isoprostanes raising the possibility that apn may modulate oxidative stress , resulting in a less proinflammatory state . this is in keeping with the observation that lower serum apn levels are associated with more extensive necroinflammation in nafld and that they may contribute and even be a potential indicator of the progression from simple steatosis to nash [ 3234 ] . first , we detected fatty liver by ultrasound , which is a qualitative method inadequate to quantify less than 30% liver fat content . the gold standard for the diagnosis of nafld is liver biopsy , but this is an invasive procedure with potentially serious complications and is therefore not acceptable without clinical indication . we acknowledge that grades of steatosis could have been better determined by magnetic resonance spectroscopy . however , the hamaguchi score showed 100% specificity and 91.7% sensitivity when compared with liver biopsy in nafld patients . second , although performed in a large series of patients , the study has been carried out in patients recruited in a hospital - based setting and the study design did not contemplate controls . finally , our study has a cross - sectional design and prospective interventions with antioxidants are needed to demonstrate the causal role of oxidative stress on nafld to nash progression . in summary , our findings support the concept of an association between oxidative stress and severity of liver damage in patients with nafld . moreover , to the best of our knowledge , this is the first association study that considered urinary 8-iso - pgf2 and serum snox2-dp as markers of systemic oxidative stress , and ck-18 for the assessment of hepatocyte apoptosis .