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We assumed a monopolar spherical electrode in an infinite homogeneous extracellular medium with a resistivity of e=300 cm . Under quasi - static conditions the extracellular potentials ve along the rectified neuron was calculated by ve=eiel/4r, where r is the distance from a compartment to the electrode radiating a monophasic stimulating current pulse with amplitude iel . The current to the center of the n - th compartment of the model neuron consists of the following components: capacitive current, ion currents across the membrane and ohmic currents to the left and right neighbors . Applying kirchhoff's law for compartment n results in(1)d(vi, nve, n)dtcm, n+iion, n+vi, nvi, n1rn/2+rn1/2+vi, nvi, n+1rn/2+rn+1/2=0 with vi, r and cm denoting the intracellular potential, axial resistance and membrane capacity, respectively . Intracellular resistivity was 150 cm, membrane capacity 1 f / cm . The following system of differential equations is deduced by introducing the transmembrane voltage v = vive to compute the time courses of vn in every compartment (rattay, 1999):(2)dvndt=[iion, n+vn1vnrn1/2+rn/2+vn+1vnrn+1/2+rn/2+ve, n1ve, nrn1/2+rn/2+ve, n+1ve, nrn+1/2+rn/2]/cm, n the direct stimulating influence of the extracellular potential on compartment n is defined by the activating function(3)fn=[ve, n1ve, nrn1/2+rn/2+ve, n+1ve, nrn+1/2+rn/2]/cm, n the physical dimension of fn is [v / s]. If the neuron is in the resting state before a stimulating current impulse is applied, fn is the slope of membrane voltage vn at stimulus onset (fig . As r decreases with increasing compartment diameters, f increases with diameter and consequently processes with larger diameters are generally easier to excite . The excitability of the rectified model neuron was initially tested with the original model data obtained from the neuron modeldb corresponding to the model of a cortical pyramidal cell by hu et al . This model incorporates tapering diameters, uneven channel distribution within compartments of one type as well as a reduced membrane capacity of 0.02 f / cm in order to simulate myelination . High threshold sodium nav1.2 and low threshold nav1.6, fast voltage - gated k, slow non - inactivating potassium current, high - voltage activated ca and calcium dependent k currents are non - uniformly integrated and combined with a linear leakage current throughout the cell . During single pulse experiments for a reduced model we obtained quite similar threshold characteristics in spite of either assuming constant channel densities within every main segment or including only nav1.2, nav1.6, fast voltage - gated k channels and a linear leakage current with eleak=70 mv and gleak=0.033 ms / cm throughout the whole neuron . In detail, the reduced model distinguishes soma and dendrites compartment, both with maximum conductances of gnav1.2=8, gnav1.6=0, gkv=10 ms / cm, the axon hillock with gnav1.2=320, gnav1.6=0, gkv=100 ms / cm, the ais with gnav1.2=100, gnav1.6=320, gkv=100 ms / cm, the unmyelinated axon with gnav1.2=0, gnav1.6=300, gkv=150 ms / cm and the nodes of ranvier with gnav1.2=0, gnav1.6=160, gkv=20 ms / cm . Sodium current kinetics are calculated via inav1.j = gnav1.j mh (v ena) with j equals either 2 or 6 and a reversal potential of ena=60 mv . Details on the differential equations of the different variables have been presented for example by mainen et al . The values for the half (in)activation voltages v1/2, the slopes k and the coefficients a were obtained from a previously published model in the neuron model db (hu et al ., 2009) after subtracting the corresponding value for the shift of voltage dependence of the kinetics . Therefore the sodium currents inav1.j have the same values for a, that is a(m)=0.182, a(m)=0.124, a(h)=0.024, a(h)=0.0091, and the slope of inactivation, that is k(h)=5 and k(h)=6.2, in contrast to altered slope of activation, that is k(m)=k(m)=7 for nav1.2 but k(m)=k(m)=6 for nav1.6 . To account for the reduced threshold of nav1.6 channels v1/2 (m) is decreased to 41 mv compared to the calculated value of 28 mv for activation of nav1.2 channels . The corresponding values in mv for the inactivation of nav1.2/nav1.6 channels are v1/2(h)=35/41, v1/2 (h)=60/73, v1/2 (h)=57/70 . The potassium currents are determined by ik = gk n (v ek) with ek=90 mv . To be consistent the corresponding values of a()=0.02, a()=0.002, v1/2()=v1/2()=25 mv and k()=k()=9 which were used for the reduced model were also obtained from the neuron model db (hu et al ., 2009). Since the presented results are simulated for 37 c, a temperature coefficient of 3.209 has to be applied when calculating the conductances and the channel kinetics to account for the original model temperature of 23 c and a q10 of 2.3 . The internodes are simulated with 17 sheets of membrane with a conductance of 1 ms / cm and c=1 f / cm per sheet (rattay, 1999). Dendritic excitation with spike propagation across the soma into the axon was analyzed with a simple compartment model with a straight axis (fig . The model neuron consists of a single non - branching dendrite (500 m, d=5 m), soma (20 m), axon hillock (10 m, d=3.1 m), ais (50 m, d=1.22 m), unmyelinated axon (200 m, d=1 m), myelinated axon (500 m, d=1 m) and unmyelinated terminal (50 m, d=1 m). Also assumptions for ion channel distribution are quite similar as modeled by hu et al . (2009): the same constant nav1.2 channel density for dendrite and soma, but 40 times higher sodium channel density in hillock and ais with a change to the low threshold type nav1.6 in the axon (fig . 1b). In a distance of 80 m from the dendrite axis a propagating spike is generated by a 100 s cathodic pulse from the tip of an electrode which is modeled as point source in an infinite homogeneous medium (fig . According to the activating function concept (rattay, 1986, 1999) the slopes of membrane voltage at the beginning of the stimulus pulse reflect the first response of the applied electric field and these slopes are proportional to fictive current pulses injected individually in each of the compartments . Therefore spike initiation is expected in a region with positive activating function, that is, where the slopes at stimulus onset are positive (fig . Assuming a homogeneous external medium and a straight long fiber with constant diameter, the activated region is limited by an angle of 70 (fig . This initial excitation pattern is independent from ion channel composition as well as internal and external resistivities . In order to obtain a propagating spike, inward sodium current has to be large enough to supply the neighboring region with enough intracellular axial current flow to reach threshold . This fact demands for a minimum sodium channel density which is not achieved on the assumptions mentioned above with a ratio of 1:1500 concerning dendrites and ais . Moving the electrode closer to the dendrite reduces the length of the activated region as a consequence of the 70 limit . The rather low maximum sodium conductance gna=8 ms / cm is not enough for nearby stimulation and therefore direct cathodic dendritic spike excitation fails for electrode distances below a limit which is about 56 m for the 5 m dendrite1 . Excitation is however possible when the inward dendritic sodium current shifts the ap initiation site by axial current into the low threshold ais (fig . Maximum membrane voltage increases with stimulus strength in the activated 70 region, still passive excitation is prevented by the hyperpolarized regions on both sides (fig . For an infinite long fiber, reduction of fiber diameter by a factor four causes quite the same numerical values in model evaluation when fiber - electrode distance, compartment length as well as stimulus current is divided by 2 (quadratic relation). This means that thinner dendritic regions are directly excitable for shorter electrode - fiber distances, for example the limit is 28 m for 1.25 m dendrite diameter . In this case, propagating spikes are generated in the small operating window from 25 to 41 a . Higher stimuli cause cathodic block by strong hyperpolarized side lobes that hinder spike propagation (ranck, 1975; rattay, 1986) in a similar way as shown in fig . The geometry of the last example is used to compare dendritic and axonal excitability: moving the electrode from the dendrite (1.25 m diameter) to the distal end of the ais (xelectrode=70 m) with constant axial distance (zelectrode=28 m) reduces cathodic threshold current from 25 to 8.1 a . In this example the ais and the dendrite have quite similar diameters but the ais threshold is less than one - third of the dendrite's value . Furthermore, the electrode with the same axial distance is positioned above the third node of ranvier . In spite of the reduction of diameter (from 1.22 to 1 m) as well as the decrease in the maximum sodium conductance gnav1.6 (from 320 to 160 ms / cm) for compartments of type nr compared to those of type ais, the firing threshold is further reduced by 5% to 7.7 a and cathodic block threshold appears again at 24 a . Above the center of the second internode (xelectrode=518 m, zelectrode=27.6 m) threshold current increases for propagating spikes to 21.8 a and blockade threshold to 45 a . When the electrode is shifted in constant distance along the axis of the neuron, several phenomena (1)the distal end of the axonal initial segment with high nav1.6 channel density is very sensitive to cathodic stimulation and this is the site of spike initiation in many cases. (2)however, the neuron is even more excitable at the third and fourth node of ranvier, in spite of essentially lower ion channel densities . This is a consequence of high activating function values in nodes of ranvier that are favored by the properties of the internodes (coburn, 1989; rattay, 1986, 1989; rattay et al ., 2000). (3)moving the electrode along the thick dendrite in a distance of 100 m from the neuron axis needs about twice the threshold current in comparison to the positions along the axon.moving the electrode with 50 m distance along the neuron shows the following discrepancies (4 and 5) to a quadratic distance threshold current relation as expected from literature (stoney et al ., 1968; tehovnik, 1996; tehovnik et al ., 2006). (4)the thresholds are higher as expected (prediction according to the quadratic rule is shown as dashed blue line in fig . 3a). (5)electrodes above the center of an internode need doubled threshold currents compared to positions above a node of ranvier . This node - internode threshold relation known from peripheral nerve stimulation becomes more and more pronounced when electrode - fiber distance is reduced (rattay, 1987). (6)within a dendritic region (149 m <xelectrode<37 m) anodic threshold is lower than cathodic . This is in agreement with experiments (ranck, 1975) and theory (rattay, 1999). (7)anodic threshold current of the 5 m diameter dendrite example increases in an exponential way with distance from the soma (comp . The rather linear relation of the left part of the green line in the logarithmic current scaling of fig . The thin dendrite has another threshold characteristic as explained below. (8)the extreme node - internode threshold fluctuations seen for cathodic pulses are lost when pulse polarity is reversed. (9)in general thin dendrites have higher thresholds (comp . Thin and thick green and blue lines in fig . The distal end of the axonal initial segment with high nav1.6 channel density is very sensitive to cathodic stimulation and this is the site of spike initiation in many cases . However, the neuron is even more excitable at the third and fourth node of ranvier, in spite of essentially lower ion channel densities . This is a consequence of high activating function values in nodes of ranvier that are favored by the properties of the internodes (coburn, 1989; rattay, 1986, 1989; rattay et al ., 2000). Moving the electrode along the thick dendrite in a distance of 100 m from the neuron axis needs about twice the threshold current in comparison to the positions along the axon . Moving the electrode with 50 m distance along the neuron the thresholds are higher as expected (prediction according to the quadratic rule is shown as dashed blue line in fig . Electrodes above the center of an internode need doubled threshold currents compared to positions above a node of ranvier . This node - internode threshold relation known from peripheral nerve stimulation becomes more and more pronounced when electrode - fiber distance is reduced (rattay, 1987). Within a dendritic region (149 m this is in agreement with experiments (ranck, 1975) and theory (rattay, 1999). Anodic threshold current of the 5 m diameter dendrite example increases in an exponential way with distance from the soma (comp . The rather linear relation of the left part of the green line in the logarithmic current scaling of fig . The extreme node - internode threshold fluctuations seen for cathodic pulses are lost when pulse polarity is reversed . In general thin dendrites have higher thresholds (comp . Thin and thick green and blue lines in fig . Threshold currents are also computed in radial direction for three cell regions: middendritric, soma and ais (fig . Thresholds in table 1 are calculated with charge - balanced triphasic pulses with an interpulse time of 100 s and 100 s per phase . This way they can be compared with our previous data as well as with symmetric biphasic pulse widths of 200 s per phase which are often applied in cortical experiments . As for such pulse durations, the charge per phase is essential for threshold current (nowak and bullier, 1998), half of our reported threshold values are expected when 200 s pulses are applied . For low current stimulation (left part of table 1) deviations between charge - balanced and monophasic pulses note that the spike initiation site or the excitation mechanisms (dendritic spike ais spike anodal break, etc .) Suddenly may change when the electrode is slightly moved . A continuous trend from linear (doubled threshold for doubled distance) to quadratic relationship (fourfold threshold for doubled distance) 3b, table 1 cover also the range above 100 m in order to demonstrate that a quadratic current distance rule ithreshold = kr (with constant k and electrode - neuron distance r) as reported by tehovnik (1996) is a good fit for this area . This behavior changes continuously to a rather linear relation when the electrode enters the region below 50 m where low current stimulation is possible . In contrast to experimental recording, computer simulation allows snapshots of membrane voltages as functions of neuron's length coordinate (fig . 4). We analyze cathodic and anodic stimulation for three electrode positions, always with zel=50 m to be comparable with fig . 3a: (i) above the second node of ranvier (xel=370 m, fig . 4 right traces), (ii) above the distal ais end (xel=70 m, fig . 4 central traces), (iii) above the dendrite, symmetrical to position 1 (xel=370 m, fig . Starting at rest, first compartment responses are proportional to the activating function which is independent of the ion channel composition (rattay, 1986, 1999). They appear because of irregularities of the electric field and due to changes of the neural geometry . With our assumptions irregularities of the electric field in other cases inhomogeneity of extracellular medium and curvature of fibers essentially contribute to high activating function values . This first response is smoothed by intracellular current flow (compare red lines with the activating functions in fig . C) of cathodic stimulation the maxima of the red lines appear at the electrode position which is marked by dashed red lines in fig . 4 and consequently spikes are initiated in the neuron compartment closest to the electrode in cases fig . 4b, but as explained above, the low dendritic sodium channel densities hinder spiking also for higher stimuli, because of the hyperpolarized regions predicted by the negative side lobes of the activating function (comp . The stimulus polarity causes these side lobes of the activating function to become stimulating (comp . 4 g, h. for a fiber with constant diameter the activating function values of the side lobes converge asymptotically to zero when the x distance to the electrode position is increased . In case (fig . 4 g), the rather small activating function values in the ais are, however, still more powerful than the higher values in the dendrite with its low sodium channel density resulting in distant spike initiation, that is more than 400 m away from the electrode . This phenomenon explains the almost exponential increase for anodic threshold current when the electrode is moved distally along the dendrite (left part of green curve in fig . A similar effect, but not as spectacular in its shifting size, is seen for anodic axon stimulation: in case (fig . 4e) causes a mirrored pattern similar to the dendritic excitation of case (fig . Red lines) but the low dendritic sodium channel density together with its higher nav1.2 threshold voltage are the reasons why the spike is initiated within the unmyelinated axon distal to ais . Putting the electrode above the second internode results in an asymmetric activating function with a larger right side lobe value (fig . 4f). Note that this rather small difference in activating function minima favors nodal excitation in spite of half nodal nav1.6 channel densities when compared to the other activating function side lobe in the unmyelinated axon . Increasing the cathodic stimulus of the subthreshold case of fig . 4a from 50 to 57 a causes a delayed spike development for the 5 m diameter dendrite at its distal end (fig ., the primarily excited region is too short for direct spiking but the axial current flow in both directions prefers to stimulate the left side in spite of the rather large hyperpolarization at the end of the pulse (red bottom line in fig . Is assumed to be sealed, the axial current is also restricted to flow backwards which escalates the increase of membrane voltage in that particular area . This effect is also reflected by the asymmetry of the excitation process of the neighboring compartments of the cyan line in fig . 1c with differing response on left side compared to the behavior on the right . When the electrode is slightly moved further away the left hyperpolarized side lobe of the red line in fig . This explains the decrease of threshold as can be seen at the left end of the corresponding thick blue curve in fig . However the effect vanishes when the positive segment of the red curve is reduced, that is before the electrode position reaches the distal end (comp . Blue curve in fig . 3). Reduction of fiber diameter to one - fourth allows direct dendritic spiking for this dendrite changing the polarity reduces the length for direct anodic dendritic spiking (constant threshold values of thin green curve in fig . The aim of the study was to study the effects of external low current stimulation on a cortical model neuron with respect to changing microelectrode position and configuration . In order to enlighten the neural elements which are activated, the electrode has been shifted in the axial direction, showing a varying, non - uniform excitation pattern along the neural axis . A model based on recently reported nav1.2 and nav1.6 channel distribution (hu et al ., 2009) has been used to calculate thresholds for different pulses and varying distances to the electrode by radial offset . During simulations we identified the spike initiation sites with special emphasis on spikes generated in the dendrite by direct stimulation . The performed simulations indicate that the nodes of ranvier and the distal end of the ais are the most excitable structures (fig . 3a) showing the lowest threshold for stimulation which is in accordance with experiments (nowak and bullier, 1998; gustafsson and jonkowska, 1976). In comparison to the sensitive ais and electrode positions close to nodes of ranvier, about 23 times higher currents are needed to excite thick dendrites and even higher thresholds are expected for thin branches (fig . This trend seems to be applicable to various cell types, since gustafsson and jonkowska (1976) revealed similar results with their experiments on spinal motor neurons and spinal border cells . The excitability essentially differs along the neural axis displaying the varying electrical properties of different functional elements of the neuron . Therefore parameter values shall be collected carefully, for example it should be pointed out that different modeling of myelination considering the presence of nav1.2 channels, but with decreased capacitance as formerly described by hu et al . (2009), also leads to a zig - zag behavior of the threshold function as mentioned previously, but not as pronounced as in fig . 3a . Since non - branching long straight excitable fibers with constant diameters show an anodic cathodic threshold ratio in the order of 4:1 (ranck, 1975; rattay, 1986), we focused our simulations on phenomena occurring for the cathodic excitation . Nonetheless, note that only within a small region in the vicinity of the soma dendrites are easier to stimulate with anodic pulses and that the extreme node - internode threshold fluctuations are lost when pulse polarity is reversed . Charge - balanced triphasic pulses with an interpulse time of 100 s and 100 s per phase have also been tested for both polarities, that is anodic first and cathodic first stimuli . When the electrode is placed close to the neuron the thresholds are similar to monophasic cathodic stimulation . Nonetheless we observed an alternative activation of the cell when the electrode is placed in the vicinity of the dendrites . Shortening the electrode - fiber distance allows indirect spike generation in the ais (fig . 2a), but still this type of stimulation demands for electrode positions rather close to the soma . In order to reach their threshold value the neighboring compartments profit from high intracellular resistance . Lower resistances caused by branching or suddenly increased diameters, for example at the border to the soma, not only reduce the possibility of spike development but are also capable of preventing the conduction into the axon . 5b demonstrates a reduction in amplitude to 42% before the thin dendrite spike enters the soma, whereas for the 5 m dendrite the dendrite - soma diameter ratio of 1:4 is secure and the spike enters the soma with full amplitude (fig . Our results indicate that a reasonable hypothesis to explain the sparely distributed population of pyramidal cell bodies when stimulated at low intensities (histed et al ., 2009) is to assume a rather dense concentration of collaterals of many neurons in the close surrounding of the electrode tip . These thin axons and dendrites are capable of generating an ongoing nerve impulse or activate the distant ais via intracellular current flow . Therefore the cell bodies of stimulated pyramidal cells can be situated within a distance of several hundred micrometers to the electrode tip, whereas most of the cell bodies closer to the electrode won't be activated . The performed simulations further approve the ability of dendrites to generate and successfully transmit an ongoing action potential when stimulated extracellularly . (2009) report that the current needed to activate at least one cell was 10 a or less . According to their fig ., 2009), axon and dendrite diameters are estimated with 0.2 m . With our assumptions such tiny dendrites can be directly activated in the range from 10 to 16 a . Our results demonstrate that, by applying a 100 s pulse with an amplitude between 25 and 41 a, a 1.25 m diameter dendrite that passes the electrode tip in 28 m distance is able to evoke a spike, whereas the 5 m thick dendrites needs a minimum distance of 56 m for a local spike and much higher currents . Starting at the resting state, we have demonstrated that regular dendritic spikes need an excited region of minimum size to obtain enough intracellular current for the neighboring segments to reach threshold . In case of external point source stimulation the polarized and depolarized regions are separated by a 70 angle, that is the length of the activated area can be calculated by 1.4 distance to electrode . This area does not depend on ion channel compositions, however the minimum electrode fiber distance for a local spike depends essentially on the sodium and potassium channel densities and the intracellular resistance . The inverse recruitment order for peripheral nerve stimulation which is known since 1933 (blair and erlanger, 1933) states that large diameter fibers have smaller threshold currents . This behavior is also predicted by the activating function both for myelinated and unmyelinated fibers (see methods, rattay, 1989, 1999). Moreover, the strength of dendritic spike propagation is vulnerable to modulation by the extracellular ion concentrations or any other circumstances that change the dendritic voltage - gated channel profile (gasparini et al ., 2004). Although most of these components are beyond the scope of this article, the threshold behavior has also been tested with a more complex model as used by hu et al . Generally, the obtained results were quite similar to the reported ones, demonstrating that dendritic na and k channels play a key role in setting the threshold and determining the shape and forward - propagation of dendritic spikes (gasparini et al ., 2004). (2008) with a simple model of reduced geometrical complexity and an even distribution of only traditional hodgkin huxley style na and k currents which is able to explain properties like rapid spike onset and its high threshold variability . Another obvious reason for the missing effect of the addition of currents ica, ikca and ikm to somatodendritic compartments is their relatively low conductances of gca=0.03, gkca=0.3, gca=0.03 ms / cm (hu et al ., 2009). Since royeck et al . (2008) among other authors report that these ion currents amplify spike after depolarizations and cause the generation of spike bursts, their absence does not influence the initial respond of neurons in the resting start during the onset of microstimulation . Mainen and sejnowski (1996) suggest to include these complex ion channels to expand from single spikes to spike trains, that is repitative firing, which is beyond the purpose of this article . In order to concentrate the analysis on phenomena already observed in a non - branching rectified neuron, we avoided to include the anatomical diversity of pyramidal cells concerning their diverging pattern of dendrites and axons . One should be aware that many more parameters influence the recruitment of extracellular microelectrodes, such as synaptic activities (spruston, 2008; sjstrm et al ., 2008), refractory behavior (miocinovic and grill, 2004), inhomogeneity in ion channel densities (migliore and shepherd, 2002; schaefer et al ., 2007; keren et al ., 2009) as well as branching (manita and ross, 2009), curvature (rattay et al ., 2000; iles, 2005) or irregularities in diameters of cell processes (rattay, 1999). More details on the influence of 3d structures and ion channel distributions will be reported in a forthcoming paper.
Peptide- and protein- hydration is the dominant factor in the stabilization of spatial molecular structure, in the process of protein folding by gating hydrophobic residues, and in the mechanisms of peptide and protein mediated reactions [14]. Water molecules, therefore, can be considered as an integral component of biomolecular systems with dynamic, functional, and structural roles [47]. Investigation of the structural and functional role of water molecules, bound to proteins and peptides, requires a sufficient understanding of the hydration process of their building blocks [1, 2]. The hydration of amino acids and their derivatives at a molecular level, therefore, is of great importance and has been extensively studied with x - ray crystallography [1, 3] and a variety of spectroscopic techniques including multinuclear magnetic resonance spectroscopy [2, 813], ir and raman spectroscopy [1416], icr mass spectrometry, and laser ablation in combination with microwave spectroscopy . We present here, for the first time in the international literature, a comparative investigation of literature d, c, n, and o nmr and crystallographic data in order to provide a coherent hydration model of amino acids and selected derivatives at different ionization states in aqueous solution and in the crystal state . O nmr has received little attention in amino acid and peptide research [2, 12, 13, 19, 20]. This neglect is due to the fact that of the three naturally occurring oxygen isotopes, only o possesses a nuclear spin (i = 5/2). Owing to its electric quadrupole moment (qe = 2.6 10 em) and, thus, broad line widths, and its low absolute sensitivity compared with that of h (~1.1 10), the o- isotope is one of the more difficult to observe by nmr spectroscopy [12, 13, 21, 22]. O nmr studies, therefore, of compounds at natural abundance require high concentrations (> 0.1 m) and extensive signal averaging . Recording of spectra can be greatly facilitated by the use of o enriched samples [2327]. Figure 1(a) illustrates the natural abundance o nmr spectrum of glutamic acid, 0.1 m in o - depleted water at 40c . Despite the extensive signal averaging (number of scans (ns) =3 10) and the total experimental time of 4.2 hours, the achievable signal - to - noise (s / n) ratio is very poor and practically prohibitive for the accurate determination of chemical shifts and line widths . Figure 1(b) illustrates the clear advantages of working with o - labelled glutamic acid (o enrichment 1 at.%). O shieldings of various chemical functional groups are very sensitive for studying hydrogen bonding interactions because of the large chemical shift range of the o nucleus [12, 13]. The effect of solvent - induced hydrogen bonding interactions on (o) of the carboxyl groups is, however, rather small compared with the substantial sensitivity of over 80 ppm to hydrogen bonding interaction of (o) of amide and carbonyl oxygens [12, 13]. Only a single o resonance absorption is observed for the carboxylic group since the shifts of the individual resonance absorptions (c = o) and (oh) are averaged out by rapid intermolecular proton transfer with protic solvents, traces of h2o, and/or through hydrogen bonding aggregates of the cooh groups in organic solvents [12, 13, 23, 24, 26, 28]. Reuben from dilution studies of acetic acid in 1,2-dichloroethane estimated a deshielding effect of ~12 ppm due to breaking of a hydrogen bond involving the carbonyl oxygen of the acid and a shielding effect of 6 ppm due to breaking of a oho hydrogen bond . Therefore, a total shift of only + 6 ppm is expected for the monomeric acetic acid in apolar media (dichloroethane) compared with the dimeric form . Despite the relatively low sensitivity of the o shieldings of the carboxyl group to hydrogen bond interactions, spisni and collaborators attempted to estimate the solvation state of the -carboxyl group of amino acids in the different ionization states . Figures 2(a) and 2(b) show the dependence of (o) of l - alanine and l - proline as a function of molar fraction of dmso in the ph range 7 - 8 and 12 - 13 . Since dmso cannot form a hydrogen bond interaction with the carboxylate group, contrary to the case of h2o, the shielding difference of 1017 ppm between the two solvents was interpreted with the hypothesis that the carboxylate group of these amino acids is hydrated by two water molecules in aqueous solution with one hydrogen bond per carboxylate oxygen . In the acidic ph range (figures 2(c), 2(d)), a nonlinear behaviour of the chemical shift at high dmso molar fractions was observed . For dmso molar fractions up to 0.6, a linear dependence of the chemical shift was observed which, on extrapolation to 100% dmso, results in a shielding of 1517 ppm, the same as in the neutral ph . This was interpreted with the hypothesis that two hydrogen bonds (one to each oxygen) are being ruptured . When the dmso molar fraction is between 0.6 and 0.8, it was suggested that a third molecule of water, which is hydrogen bonded to the hydroxyl hydrogen, is dissociated due to the interaction with dmso . This might explain the deflection from linearity and the plateau - like dependence of the o shielding . The protonated form, therefore, of the carboxyl group of the amino acids is more hydrated with an access of a bound molecule of h2o than the deprotonated form . This conclusion is in qualitative agreement with multinuclear nmr relaxation data (see below). For quadrupolar nuclei, such as d, n, and o, the longitudinal (t1) and transverse (t2) relaxation times are essentially due to quadrupolar interaction (1)1t1=1t2(1+23)2f(,d), where is the nuclear quadrupole coupling constant . The asymmetry parameter varies from 0 to 1 and describes the deviation of the electric field gradient from axial symmetry, and f(, d) is the correlation function, which depends on the rotational diffusion constant d and its relative orientation with respect to the principal axes of the field gradient tensor [12, 13]. When isotropic reorientation is assumed, f(, d) reduces to a single overall correlation time mol which is given by the stokes - debye formula (2a)mol = vmkbt, where vm is the molecular volume, the viscosity of the solution, kb the boltzman constant, and t the absolute temperature . Vm can be estimated as (2b)vm=0.74mwn0, where n0 is the avogadro's number and mw and are the molecular weight and the density of the solute (amino acid), respectively . The removal, therefore, of these impurities is necessary in studies of t2 and t1 relaxation times . Figure 3 illustrates the ph dependence of the o line widths of 0.1 m glycine in h2o . This o line width minimum has been previously explained by a decrease of the molecular tumbling time attributable to a reduction in hydration and, thus, intermolecular association of glycine in the zwitterionic form . In the high ph region, a broad maximum at ph 11 was observed . Addition of 2 mm ethylenediamine - n, n, n,n-tetraacetate (edta) to the original solution resulted in no line width variation in the neutral and high ph region . It can, therefore, be concluded that this broad minimum at ph 11 should be attributed to the effect of paramagnetic impurities and not to a hydration change of glycine in the neutral and high ph region . D t1 relaxation times of cd2 of glycine at acidic ph were shown to be shorter relative to those at neutral ph . This shortening in t1 implies an increase in mol and, thus, in the effective molecular weight mw ((1), (2a), and (2b)), which was interpreted with an increase in the hydration state in the cationic form . Tritt goc and fiat investigated in detail the viscosity and temperature dependence of the o nmr line width of glycine, alanine, proline, leucine, histidine, and phenylalanine at ph 2, 7, and 12.5 . The experimentally observed viscosity / temperature (/t) dependence of the reorientation correlation time was compared with various hydrodynamic models . A model of the hydration state in the primary solvation sphere of the carboxylic group of amino acids in their cationic state was suggested in which two water molecules are hydrogen bonded to the oxygens and one to the hydrogen of the oh group . In the zwitterionic and anionic states, the hydration model of the carboxylate group can be presented by a structure in which one water molecule is hydrogen bonded to each of the oxygens . The o [10, 11] and n nmr line widths of several protein amino acids were measured in aqueous solution to investigate the effect of molecular weight on the line widths (table 1). The n and o line widths, under composite proton decoupling, increase with the bulk of the amino acid, and increase at low ph . Assuming an isotropic molecular reorientation of a rigid sphere and, thus, a single correlation time from overall molecular reorientation (mol), then, the line width 1/2 can be expressed in the following form: (3)1/2=1t2=0+1mw, where mw is the molecular weight, 1 is the contribution to the line width of the quadrupolar coupling constant, density and temperature, (1), (2a), and (2b), and 0 is the solvent viscosity - independent contributions to the line width due to the primary hydration sphere of the amino acids . The linear correlation between 1/2 and mw at ph 6 for both n and o nuclei (figure 4) is in agreement with the hydrodynamic model of (3). Furthermore, the (o) of the amino acid is independent of both the ionization and the degree of hydration of the carboxyl group . The increase in the o line widths at acidic ph (~100 31 hz), relative to those at neutral ph, was interpreted by a change in the rotational correlation time and, thus, effective mw of the amino acids, (3). This implies that the cationic form of the amino acids is more hydrated by an access of 1.3 to 2.5 molecules of water relative to that in the zwitterionic form with lifetimes that are longer than the overall molecular rotational correlation time, presumably 210 ps . In the case of a stochastic diffusion of the amino and carboxyl groups comprising contributions from internal (int) and overall (mol) motions, the correlation time c for n or o is given by (4a)c=mol[a+(b+c)(12/r)intmol+(12/r)int] with (4b)a=34(3cos21)2, b=3sin2cos2, c=34sin4,where is the angle between the rotation axis and the main field gradient (r denotes an r - fold jump mechanism). Since the sum of a, b, and c is equal to 1, (4a) can be rewritten as(5a)c=molamol+imol+i, where (5b)i = (12r)int.equations (5a) and (5b) can be rewritten as (6)c=(1a)i+amol(1a)i2mol+i . Since a and i can be assumed to be constant for all the amino acids, (4a) and (4b) can be written as (7)1/2=0+1mw+2mw+3, where 03 are constants . The minimization of (7) on the basis of the o experimental data gave the mean difference of 35.8 17.3 in mw between ph 0.5 and 6.0 for three different 1/2 values: 250, 350 (figure 4(b)), and 500 hz . The difference in the n line widths at the two ionization states (figure 4(a)) should be attributed to differences in the correlation times and to a decrease in the (n) on deprotonation of the carboxyl group . In the case of the linear model, the influence of variations of values of the (n) to the line width, 1/2, is less for small molecular weights . Therefore, for 1/2 = 70 hz (figure 4(a)), the difference in mw will be a reasonable approximation of the difference in hydration in the two states . The calculated value was found to be 45.2 7.4, which corresponds to an excess of 2 - 3 water molecules in the cationic form compared to that in the zwitterionic form, in reasonable agreement with the o nmr data . More recently, takis et al . Investigated the cc longitudinal relaxation times (t1) and n line widths (1/2) of amino acids and acetyl - amino acids in aqueous solutions at acid and neutral ph . Both c and n values indicate that amino acids and acetyl - amino acids at acid ph interact with an access of one water molecule with respect to their deprotonated form at neutral ph . On the contrary, c and n values of betaines (r3nch(r)coo) crystal structure databases provide a rich source of information to extract details on the architectures and interactions of molecules . This kind of search provides the opportunity to examine the formation of intramolecular and intermolecular hydrogen bond in small molecule crystal structures [33, 34]. Propensities for the hydration of the -carboxylate group of amino acids and their derivatives were derived on the basis of exhaustive searches in the cambridge crystallographic database (csd). Since intermolecular hydrogen bonds are preferred when five- or six - membered conjugated rings are formed, particular attention has been given to the hydrogen bond patterns in the vicinity of the carboxylate group that involves two simultaneous hydrogen acceptors . The concept of five- and six - membered conjugated rings, along with three - center (bifurcated) and 4-center (trifurcated) hydrogen bonds, has been acknowledged and accepted widely as an important factor in determining the structure and function of molecules ranging from inorganic to organic and biological molecules [1, 3539]. Furthermore, port and pullman studied theoretically the formate ion - water interaction as a prototype of the carboxylate group . Three energetically favourable hydration sites were obtained, two equivalent sites on the carboxylate oxygens at the exterior of the ion and one water bridging the two oxygen atoms . The conquest 1.13 program was used for all the statistical analysis described in this paper . Specifically, the csd version 5.32 (november 2010) for small molecules was searched, with the following general search flags: r> 0.5,, and only organic . In order to extract the number of entries present in the current database that form six - membered conjugated rings between the two oxygens of the -carboxylate and the carboxylic group with a molecule of water in the vicinity, the following geometric cut - offs were used: upper limits d = 3 for (ow)ho = c and (ow)ho c, and d = 3.5 for owo = c and owo c 44 hits were obtained for the carboxylate state (figure 5), whereas only one was derived for the protonated form . Figure 5 demonstrates that the oxygen of water, ow, is reasonably close to the carboxylate oxygens and displays a significant preference for the o1c there is a general correlation between hydrogen bond lengths and hydrogen bond angles (figure 6) similar to that observed by jeffrey and maluszynska in the case of water molecules in the hydrates of small biological molecules . Furthermore, crystallographic database searches were performed to identify the propensity for the formation of intramolecular hydrogen bond interaction in the carboxylate (nho) and the carboxylic acid (nho = c) state . Interestingly, 946 and 118 hits were retrieved for the carboxylate and 621 and 6 hits for the carboxylic form in the absence and presence of two molecules of water, respectively . It is evident from figure 7 that in the presence of two bound water molecules there is a significant reduction in the number of structures with intramolecular hydrogen bond interaction for the carboxylate group and, concurrently, a significant increase in the distance (nho). It is important to note that no intramolecular nh3ooc hydrogen bonds were observed for 82 amino acid carboxylates with sp - hybridized c - atoms in agreement with an early survey of amino acid structures determined by neutron diffraction . O shielding changes of amino acids as a function of molar fraction of dmso / h2o, the decrease in the longitudinal relaxation times (t1) of c d and c, and the increase in line widths of n and o at acidic ph relative to those at neutral ph may be interpreted with the hypothesis that the cationic form of amino acids is more hydrated by 1 to 3 molecules of water than the zwitterionic form . Similar behaviour was also observed for acetylated derivatives of amino acids, but not for betaines, between the protonated and deprotonated carboxyl group . Although the precise hydration differences observed for various nuclei deviate somehow, it may be concluded that these hydrated complexes have lifetimes that are shorter than the nmr chemical shift time scale, but presumably longer than the overall molecular rotational correlation time of 210 ps . An exhaustive search in the cambridge crystallographic database (csd) demonstrates a strong tendency of the two oxygens of the deprotonated carboxylate group to form hydrogen bonds with a single molecule of water . Even though statistical analysis of structural parameters in crystals cannot be used in a straightforward way to derive quantitative structural models in solution, it is of interest to note that this mode of six - membered conjugated ring, which is absent in the case of the carboxylic group, might result in a more compact and, thus, less hydrated structure in aqueous solution, in accordance with the nmr data (figure 8). Furthermore, it may be concluded that the bound molecules of water alleviate the nho interaction and very probably this effect is even more pronounced in aqueous solution . From the above, it is evident that the reduced hydration of the carboxylate group, relative to the carboxylic group, should be attributed mainly to the strong tendency of the carboxylate group to form a six - membered conjugated ring with a single molecule of water . Constructively, the tentative models illustrated in figure 8 should be further validated by in silico and experimental approaches . Computational methods complement the experimental results by providing information on the microscope and physicochemical details on the interplay between water and the biomolecule of interest [4447]. For example, introduction of solvent effects into molecular dynamics can provide an atomic description of the folding and unfolding of a protein . Furthermore, there is an array of theoretical approaches that have been utilized for treating nmr shieldings in solution, that can be classified as continuum models [49, 50] and molecular dynamics simulations . Experimental approaches could involve o nmr both in powders and in the crystal state with varying degrees of hydration.
Fabrication of the passive electrode arrays began with solid state phosphorus doping (ph-1000n source, saint gobain, usa, 1000 c for 10 minutes) of p - type device si on a si on insulator (soi, top si ~300 nm, soitec, france) wafer . Removing the buried oxide layer of the soi by wet etching with concentrated hf released the device si as a si nm, retrieved with a slab of the elastomer poly(dimethylsiloxane) (pdms) and transfer - printed to a spin - cast bilayer of poly(methylmethacrylate) (pmma, ~800 nm thick) and polyimide (pi, ~300 nm thick) on a si wafer . Photolithography and reactive ion etching (rie) defined a pattern of electrodes and interconnects in the si nms . Plasma enhanced chemical vapor deposition (pecvd) formed a layer of sio2 (thickness 100 nm) as encapsulation . Patterned etching with buffered oxide etchant removed the sio2 from the electrode regions . Spin casting and patterning a top coating of pi (~300 nm thick) placed the si nm electrodes and interconnects near the neutral mechanical plane . Patterning a mesh structure across the multilayer (i.e. Pi, sio2, pi and pmma) by rie followed by immersion in buffered oxide etchant exposed the base layer of pmma to allow its dissolution in acetone . Retrieval onto a slab of pdms enabled removal of the bottom exposed layer of pi by rie . Transfer onto a film of plga (~30 m thick), facilitated by heating to temperatures close to the glass transition of the plga (55~60 c, lactide / glycolide ratio of 75:25 composition), followed by elimination of the top layer of pi by rie completed the fabrication . Bonding an acf cable to the terminal regions of the si nm interconnects yielded connection points for interfaces to external data acquisition (daq) systems . The fabrication began with growth of 200 nm of thermal oxide on a p - type soi wafer (top si ~320 nm, soitec), photolithography and immersion in buffered oxide etchant to create a mask for solid state phosphorus diffusion (1000 c for 6 minutes) to define the source and drain contacts . Releasing, retrieving and transferring the doped si nms onto a temporary substrate, consisting of si wafer with a bilayer coating of pi / pmma, followed procedures similar to those described for passive electrode arrays . Photolithography and rie etching patterned the si nms into geometries for an 88 array of unit cells, each consisting of two transistors connected in series for purpose of actively multiplexed readout . A thin layer of sio2 (pecvd at 220 c, thickness ~100 nm) served as the gate dielectric . Buffered oxide etching through a photolithographically patterned mask formed openings through the sio2 to expose the source and drain contact regions . Photolithography and lift off in acetone defined a patterned layer of mo (sputter deposited, thickness ~300 nm) for the gate electrodes and metal interconnects . Deposition of a trilayer of sio2 (~300 nm)/si3n4 (~400 nm)/sio2 (~300 nm) by pecvd formed the interlayer dielectric . Photolithography and buffered oxide etching created vertical interconnect access (via) holes for electrical connections between layers . An additional layer of mo (thickness ~300 nm) patterned by photolithography and liftoff defined column select lines . Another trilayer of sio2 (~300 nm)/si3n4 (~400 nm)/sio2 (~300 nm) served as encapsulation, with openings at the locations of the sensing electrodes and peripheral contact pads for interfacing to an external daq system . Selective rie and buffered oxide etching through these multilayer stacks (diluted pi / trilayers of inorganic materials / trilayers of inorganic materials / diluted pi) formed mesh structures that enabled release of active layers from the temporary substrate by dissolving the pmma layer in acetone . Transfer printing steps followed, according to procedures similar to those for the passive electrode fabrication . . 2 is representative of four different acute experiments, each of which lasted 5 - 6 hours . The procedures, which were approved by the institutional care and use committee of the university of pennsylvania, involved an anaesthetized rat with its head fixed in a stereotaxic apparatus . The animal was anesthetized with initially ketamine / xylazine and then isoflurane throughout the craniotomy and neural recordings . A craniotomy exposed a 48 mm region of cortex in either left or tight or in both hemispheres . All recordings were taken in reference to a distant stainless steel bone screw inserted through the skull during the surgery . A commercial stainless steel microwire electrode (~100 um stainless steel wire from california fine wire) placed at 0.5 mm depth from the cortical surface in close proximity to the bioresorbable electrodes served as a control during acute recordings . Neural data was acquired by a fhc multi - channel neural amplifier (fhc inc, bowdoin, me, usa) and an acquisition system (16 bit axon instruments digidata 1322a, axon instruments, foster city, ca). Neural recording data were analyzed offline using clampfit software (axon instruments) and custom matlab software for neural signal analysis . The following procedures were approved by the institutional care and use committee of the university of pennsylvania . One 150-g, sprague - dawley rat was anesthetized with a ketamine (60 mg / kg), dexdomitor (0.25 mg / kg) solution and placed in a stereotaxic frame . A skull screw was placed in the left frontal bone to serve as the reference electrode for the recordings . A pair of needle stimulating electrode were inserted into the left mystacial pad at various locations . Brief electrical currents (~250 - 600 a, 1 ms / phase, biphasic pulse) were passed between the electrodes to activate the intrinsic muscles of the vibrissae, causing a visible protraction of the whiskers . Current amplitude and electrode spacing was adjusted for focal activation, usually 1 - 4 whiskers . An adult long evans rat was anesthetized with isoflurane and placed in a stereotactic frame (david kopf instruments tujunga, ca). Body temperature was maintained with a heating blanket and the eyes were covered with ointment to prevent drying . The skull was exposed and a large craniotomy (4 8 mm) was made between bregma and lambda and laterally to the midline . The electrode was placed on the exposed dura and a slurry of gel foam and saline was layered on top of the electrode . A screw electrode was placed contralaterally to the experimental array, with another such electrode placed posterior to lambda as a ground and reference . The skull and electrodes were then covered with dental cement and the connecting plug was secured on top . The rat was given meloxicam for postoperative pain and allowed to recover on a heating pad . After 1 week the animal was placed in a cage for video / eeg recording . Eeg signals were collected continuously from 3 channels on the array and from the screw . The signals were amplified and low pass filtered at 600 hz (multichannel systems, reutlingen, germany) and sampled at 2000 hz with a 16 bit digitizer (national instruments, austin, tx). Rats (n=14) were anesthetized and transcardiac perfusion was performed using phosphate buffered saline (pbs 10x, cat . #bm-220, boston bioproducts, ashland, ma), followed by 4% paraformaldehyde (pfa, cat . Whole brains were then removed and post - fixed overnight at 4c in the same 4% pfa solution . #57 - 50 - 1, sigma - aldrich, saint louis, mo) at 4c and coronal sections were cut at 20 m using a leica cm3050 s cryostat (leica biosystems inc . ). Serial sections, spanning the entire craniotomy site, were mounted on charged slides and stored at 20c until use . For immunostaining, slides were first immersed in an antigen retrieval solution (0.1 m citrate buffer, ph 6.0, cat #ab64214, abcam, cambridge, ma) and placed in a water bath at 95c for 10 minute . After cooling, sections were rinsed in distilled water, incubated in a blocking solution containing 0.1% triton x-100 (cat . #9002 - 93 - 1, sigma - aldrich) and 5% normal goat serum (cat . Kerrville, tx) for one hour at room temperature (rt) and then incubated overnight at 4 c with the following primary antibodies: anti- glial fibrillary acidic protein (gfap, 1:1000, cat . #smi-22r, covance, princeton, nj), and anti - ionized calcium binding adapter molecule 1 (iba-1, 1:1000, cat . After 320-minute washes in pbs, sections were incubated with the corresponding fluorescent secondary antibodies (alexa fluor 488 goat anti - mouse igg2b, 1:1000, cat . #a-21141, and alexa fluor 568 goat anti - rabbit igg, 1:1000, cat . After the final washes (3 20 min in pbs), the slides were cover - slipped with an anti - fade medium containing the nuclear stain dapi (fluoromount - g+dapi, cat . Control sections were incubated with omission of one or both primary antibodies, adding only the secondary antibodies to exclude false - positive labeling . Slides were examined on an epifluorescence microscope (zeiss axioscope, germany) and images were acquired with a 20 objective and a spot rt3 digital camera, using the spot software 5.1 (diagnostic instruments, sterling heights, mi). Digital images were processed using adobe photoshop 12.0 (adobe systems, san jose, ca). Fabrication of the passive electrode arrays began with solid state phosphorus doping (ph-1000n source, saint gobain, usa, 1000 c for 10 minutes) of p - type device si on a si on insulator (soi, top si ~300 nm, soitec, france) wafer . Removing the buried oxide layer of the soi by wet etching with concentrated hf released the device si as a si nm, retrieved with a slab of the elastomer poly(dimethylsiloxane) (pdms) and transfer - printed to a spin - cast bilayer of poly(methylmethacrylate) (pmma, ~800 nm thick) and polyimide (pi, ~300 nm thick) on a si wafer . Photolithography and reactive ion etching (rie) defined a pattern of electrodes and interconnects in the si nms . Plasma enhanced chemical vapor deposition (pecvd) formed a layer of sio2 (thickness 100 nm) as encapsulation . Patterned etching with buffered oxide etchant removed the sio2 from the electrode regions . Spin casting and patterning a top coating of pi (~300 nm thick) placed the si nm electrodes and interconnects near the neutral mechanical plane . Patterning a mesh structure across the multilayer (i.e. Pi, sio2, pi and pmma) by rie followed by immersion in buffered oxide etchant exposed the base layer of pmma to allow its dissolution in acetone . Retrieval onto a slab of pdms enabled removal of the bottom exposed layer of pi by rie . Transfer onto a film of plga (~30 m thick), facilitated by heating to temperatures close to the glass transition of the plga (55~60 c, lactide / glycolide ratio of 75:25 composition), followed by elimination of the top layer of pi by rie completed the fabrication . Bonding an acf cable to the terminal regions of the si nm interconnects yielded connection points for interfaces to external data acquisition (daq) systems . The fabrication began with growth of 200 nm of thermal oxide on a p - type soi wafer (top si ~320 nm, soitec), photolithography and immersion in buffered oxide etchant to create a mask for solid state phosphorus diffusion (1000 c for 6 minutes) to define the source and drain contacts . Releasing, retrieving and transferring the doped si nms onto a temporary substrate, consisting of si wafer with a bilayer coating of pi / pmma, followed procedures similar to those described for passive electrode arrays . Photolithography and rie etching patterned the si nms into geometries for an 88 array of unit cells, each consisting of two transistors connected in series for purpose of actively multiplexed readout . A thin layer of sio2 (pecvd at 220 c, thickness ~100 nm) served as the gate dielectric . Buffered oxide etching through a photolithographically patterned mask formed openings through the sio2 to expose the source and drain contact regions . Photolithography and lift off in acetone defined a patterned layer of mo (sputter deposited, thickness ~300 nm) for the gate electrodes and metal interconnects . Deposition of a trilayer of sio2 (~300 nm)/si3n4 (~400 nm)/sio2 (~300 nm) by pecvd formed the interlayer dielectric . Photolithography and buffered oxide etching created vertical interconnect access (via) holes for electrical connections between layers . An additional layer of mo (thickness ~300 nm) patterned by photolithography and liftoff defined column select lines . Another trilayer of sio2 (~300 nm)/si3n4 (~400 nm)/sio2 (~300 nm) served as encapsulation, with openings at the locations of the sensing electrodes and peripheral contact pads for interfacing to an external daq system . Selective rie and buffered oxide etching through these multilayer stacks (diluted pi / trilayers of inorganic materials / trilayers of inorganic materials / diluted pi) formed mesh structures that enabled release of active layers from the temporary substrate by dissolving the pmma layer in acetone . Transfer printing steps followed, according to procedures similar to those for the passive electrode fabrication . 2 is representative of four different acute experiments, each of which lasted 5 - 6 hours . The procedures, which were approved by the institutional care and use committee of the university of pennsylvania, involved an anaesthetized rat with its head fixed in a stereotaxic apparatus . The animal was anesthetized with initially ketamine / xylazine and then isoflurane throughout the craniotomy and neural recordings . A craniotomy exposed a 48 mm region of cortex in either left or tight or in both hemispheres . All recordings were taken in reference to a distant stainless steel bone screw inserted through the skull during the surgery . A commercial stainless steel microwire electrode (~100 um stainless steel wire from california fine wire) placed at 0.5 mm depth from the cortical surface in close proximity to the bioresorbable electrodes served as a control during acute recordings . Neural data was acquired by a fhc multi - channel neural amplifier (fhc inc, bowdoin, me, usa) and an acquisition system (16 bit axon instruments digidata 1322a, axon instruments, foster city, ca). Neural recording data were analyzed offline using clampfit software (axon instruments) and custom matlab software for neural signal analysis . The following procedures were approved by the institutional care and use committee of the university of pennsylvania . One 150-g, sprague - dawley rat was anesthetized with a ketamine (60 mg / kg), dexdomitor (0.25 mg / kg) solution and placed in a stereotaxic frame . A skull screw was placed in the left frontal bone to serve as the reference electrode for the recordings . A pair of needle stimulating electrode were inserted into the left mystacial pad at various locations . Brief electrical currents (~250 - 600 a, 1 ms / phase, biphasic pulse) were passed between the electrodes to activate the intrinsic muscles of the vibrissae, causing a visible protraction of the whiskers . Current amplitude and electrode spacing was adjusted for focal activation, usually 1 - 4 whiskers . An adult long evans rat was anesthetized with isoflurane and placed in a stereotactic frame (david kopf instruments tujunga, ca). Body temperature was maintained with a heating blanket and the eyes were covered with ointment to prevent drying . The skull was exposed and a large craniotomy (4 8 mm) was made between bregma and lambda and laterally to the midline . The electrode was placed on the exposed dura and a slurry of gel foam and saline was layered on top of the electrode . A screw electrode was placed contralaterally to the experimental array, with another such electrode placed posterior to lambda as a ground and reference . The skull and electrodes were then covered with dental cement and the connecting plug was secured on top . The rat was given meloxicam for postoperative pain and allowed to recover on a heating pad . After 1 week the animal was placed in a cage for video / eeg recording . Eeg signals were collected continuously from 3 channels on the array and from the screw . The signals were amplified and low pass filtered at 600 hz (multichannel systems, reutlingen, germany) and sampled at 2000 hz with a 16 bit digitizer (national instruments, austin, tx). Rats (n=14) were anesthetized and transcardiac perfusion was performed using phosphate buffered saline (pbs 10x, cat . #bm-220, boston bioproducts, ashland, ma), followed by 4% paraformaldehyde (pfa, cat . Whole brains were then removed and post - fixed overnight at 4c in the same 4% pfa solution . #57 - 50 - 1, sigma - aldrich, saint louis, mo) at 4c and coronal sections were cut at 20 m using a leica cm3050 s cryostat (leica biosystems inc . ). Serial sections, spanning the entire craniotomy site, were mounted on charged slides and stored at 20c until use . For immunostaining, slides were first immersed in an antigen retrieval solution (0.1 m citrate buffer, ph 6.0, cat #ab64214, abcam, cambridge, ma) and placed in a water bath at 95c for 10 minute . After cooling, sections were rinsed in distilled water, incubated in a blocking solution containing 0.1% triton x-100 (cat . #9002 - 93 - 1, sigma - aldrich) and 5% normal goat serum (cat . #gs-0500, equitech - bio inc ., kerrville, tx) for one hour at room temperature (rt) and then incubated overnight at 4 c with the following primary antibodies: anti- glial fibrillary acidic protein (gfap, 1:1000, cat . #smi-22r, covance, princeton, nj), and anti - ionized calcium binding adapter molecule 1 (iba-1, 1:1000, cat . After 320-minute washes in pbs, sections were incubated with the corresponding fluorescent secondary antibodies (alexa fluor 488 goat anti - mouse igg2b, 1:1000, cat . #a-21141, and alexa fluor 568 goat anti - rabbit igg, 1:1000, cat . #a-11011, invitrogen by life technologies, grand island, ny). After the final washes (3 20 min in pbs), the slides were cover - slipped with an anti - fade medium containing the nuclear stain dapi (fluoromount - g+dapi, cat . Control sections were incubated with omission of one or both primary antibodies, adding only the secondary antibodies to exclude false - positive labeling . Slides were examined on an epifluorescence microscope (zeiss axioscope, germany) and images were acquired with a 20 objective and a spot rt3 digital camera, using the spot software 5.1 (diagnostic instruments, sterling heights, mi). Digital images were processed using adobe photoshop 12.0 (adobe systems, san jose, ca).
Integrated care is a common concern for health and social care systems throughout the world . The definition that is used in this study describes integrated care as the .co - ordinated set of services which are planned, managed and delivered to individual service users across a range of organisations and by a range of co - operating professionals and informal carers [2, p. 14]. This definition suggests that there is a need for both inter- and intra - organisational cooperation since multiple organisations and professionals must cooperate and coordinate their services in order to provide care to an individual . When discussing integrated care, elderly care is often in focus [25]. The challenges concerning the complicated elderly care situation involve demographic changes, elderly with multiple care demands and the endeavour to accommodate the care needs of elderly at home for as long as possible [6, 7]. What is more, the trend towards deinstitutionalisation in combination with the move towards advanced palliative care, which previously was provided by the hospital, into the patients home, increases the number of home care interventions [810]. All these factors increase the need for coordinating home care work between all parties involved . Integrated elderly care at home has been studied from various perspectives that have taken different strategies, structures and processes into account, as argued by wijngaarden et al . . Previous studies have focused on policy approaches to integrated care [4, 12], system models [13, 14] and organisational structures . In addition, information technology (it) is often suggested as a way to improve cooperation and coordination and support integrated care [10, 15, 16]. However, research into this field has not explored fully how the daily work is actually carried out in situ . One approach that can be used to explore this perspective is an ethnographic workplace study . In this paper, the aim is to explore how home care workers coordinate their daily work, identify coordination issues in situ and discuss possible actions for supporting seamless and integrated elderly care at home . The concept of cooperation has a long history within the fields of social sciences and sociology . In recent years, cooperation has also been a focus within the field of computer supported cooperative work (cscw). It involves several research disciplines such as computer science, sociology, anthropology, organizational theory and design [17, 18]. The main endeavour with cscw research is to understand the nature and requirements of cooperative work with the objective of designing computer - based technologies for cooperative work arrangement [19, p. 5]. While some of this research focuses on the first part of the cscw acronym, other centres focus mainly on the social aspect in different kinds of cooperative work arrangements [21, 22]. The interest for cscw has grown also in other research areas and in the industry . One of the reasons may be the demands of industry for improved tools that support coordination and help control group activities . Furthermore, a growing interest for cscw can also be found in research areas such as medical and health informatics . At the same time, these medical settings provide a rich domain for studying cooperative work from a cscw perspective . Within cscw, cooperative work is not defined by formal organizational boundaries or structures, but by actual cooperative behaviour . According to schmidt, cooperative work emerges in response to the requirements and constraints of the transformation process and the social environment on one hand and the limitations of the technical and human resources available on the other [18, p. 352]. Bannon argue, people engage in cooperative work when they are mutually dependent in their work and, therefore, are required to cooperate in order to get the work done [19, p. 7, a cooperative work setting furthermore raises the need for coordination of the individual inter - related distributed activities . Coordination in this context implies the need to mesh, allocate, relate, schedule, etc . Activities, actors, and resources with respect to each other [25, p. 689]. Cscw research has highlighted several important aspects of the nature and requirements of cooperative work . In general, it has been shown that supporting cooperative work is a complex issue that requires more than the improvement of information access, communication and coordination [26, 27]. For example, cscw has highlighted the importance of supporting awareness (awareness is here understood as the way in which cooperative actors make sense of and act upon their joint endeavours). In addition, it has been shown that human actions are both situated and flexible according to the social and physical conditions that are in place . As a response to the latter finding, cooperative work is most often explored through workplace studies in situ . These workplace studies often use an ethnographic approach that focus on the work, actions, interactions and technologies typical of complex organizational settings . This methodology was also employed in the present study as described in the next section . Furthermore, in this work, the analysis of the cooperative work focuses on the coordination activities that take place in order to provide integrated care . The present study was carried out in an inter - organisational home care setting in a county in southern sweden during 20022004 . At the studied county, home health care the study was part of a broader project with the general aim to support the cooperative work in home care with mobile it tools . This implies that analysis is conducted through every stage of the research procedure, in the process sharpening the focus of the data collection . The actual methods used to collect data may vary in ethnographic studies . In the present study, data were gathered through observational studies, interviews and group discussions . To gain a general overview of the work domain, some 15 semi - structured interviews were carried out with managers from both the home help service and home health care . This was followed by observational studies that were focused on the cooperation activities taking place in the daily work . During these studies, observation of 30 work shifts taking place during the day, the evening and the night were included in the study . During these studies, field notes were taken and transcribed the day after the observations . In order to obtain a more comprehensive understanding of the views and perceptions of the home care workers, group discussions were conducted . Participants in the group discussions were three district nurses, one assistant nurse, one home help service manager and two home help service workers . The group discussions focused on four themes: problematic issues concerning the inter - organisational division of labour and the division of responsibility between home help service and home health care, problematic issues concerning work activities, problematic issues concerning cooperative activities and, finally, general issues concerning information needs and tools . The aim when analysing data was to identify and categorise common themes, activities and/or issues which can explain how cooperative work and coordination is accomplished in the examined work domain . Therefore, the collected data were indexed according to which theme, activity or issue they illustrated . For this paper, two coordination activities are identified as particularly important for providing integrated care: planning the process and coordination during home care . The work, actions, interactions and relevant technology used in these two activities are described in the result section . Coordination of the home care process can be divided into two main activities; planning the process and coordination during home care . In this paper, these two activities are considered as crucial when providing integrated elderly care at home . The first activity is the starting point for the cooperation between all parties involved while the second describes how the home care workers share information and communicate their efforts during the home care process . During the observations, some interesting situations occurred, which in the group discussions with the care workers were found relevant when improving cooperation and coordination in this setting . The observed situations are described below . In order to deliver integrated elderly care at home, the municipalities and the county councils are required to follow the requirements from the national board of health and welfare . One of these requirements concerns information sharing between care providers while the other deals with cooperative care planning before discharge from hospital . Therefore, when an individual is scheduled to receive care at home after hospital discharge, a care plan meeting with all parties involved is mandatory . During this meeting, the care providers and the care receiver with relatives discuss the home care process . The following care providers attend these meetings: the nurse at the hospital who initiates the meeting, personnel from home health care, the care administrator and physiotherapists from both the hospital and from primary care if needed . The care interventions provided by the home help service and by home health care are different . The home health care provides care interventions defined as health care, such as binding up wounds, giving insulin, taking samples for testing, inserting pharmaceuticals into medical dispenser units and dispensing medicine and eye drops . The home help service provides care interventions defined as care along with social care such as help with food, getting dressed, cleaning, care assistance, practical services and they also respond to alarms . Furthermore, some care interventions are defined by home health care as self - treatment and are, therefore, not provided by the home health care personnel . An elderly patient at the hospital is about to be discharged and is in need of home care . Five persons attend this meeting; the patient himself, a relative, the hospital nurse, the district nurse and the care administrator . The nurse describes the patient s visit to the hospital and what care interventions are needed when the patient returns to his home . The patient needs help with treating a wound with a cream twice a day, a procedure that takes some time . The district nurse responds that this kind of care intervention is classified as self - treatment and should not be conducted by the home health care workers . The hospital nurse informs the home care workers that the patient cannot perform the treatment himself due to his injuries . The care administrator responds that this kind of task is something that certainly should be done by the home health care workers since this task is not something a care receiver could apply for help for and thus cannot be classified as part of the home help service, .besides the home help service unit has no resources for these kinds of care interventions that take so much time . The meeting ends without a final decision about who should take care of the wound . During the group discussion, the home health care workers argued that if the patient is not mentally capable, even simple treatments may be considered as health care interventions . However, some patients are physically incapable to conduct the self - treatment themselves, as in the above observation, and the question then is which organisation is responsible for this person . To determine responsibility in these cases, the question is often asked if the person s health will deteriorate until a health care intervention is needed unless given the treatment in question . Another way of determining responsibility is to decide which organisation would have had responsibility for the patient if he or she had not applied for home help service . In addition to this, it is important to note that for the care receiver, home help service may be more costly than home health care . During the group discussions, the care workers argued for clearer rules or agreements about what organisation should be responsible for which care intervention . The care workers also stated that resources are wasted when both the home help service and home health care are at the care receiver s home at the same time: when one person is making a sandwich while another person gives insulin at the same time, this does not feel as an efficient way to conduct a home visit . However, the care workers also stated that sometimes the unit chiefs or care administrators bend the rules in the best interest of the care receiver and sometimes both the home help service and home health care workers actually conduct care interventions beyond those agreed upon during the care plan meeting . The home help service groups from all shifts store information about the care receiver and the approved care interventions separately in non - digital form . The home health care personnel use a computerised patient record system, which is used within all primary care . However, while working in the patients homes, it is impossible to access this patient record system . To manage cooperation and coordination between the organisations, svop is an acronym for coordinated health care and care planning (in swedish: samordnad vrd- och omsorgs planering). The binder is placed in the care receiver s home and consists of several documents and material which provide the care workers with information for administering home care interventions, for supporting involved individuals in daily work situations and for facilitating communication between the care workers involved . The binder and its use has been described and analysed in more detail in . Particularly important for the present study is the fact that although the current events document facilitates communication, it was rarely used since it was available only in the care receiver s home . This is unfortunate, since some of the information is needed for coordination purposes and important information may be received late, which in turn may affect the care receiver s health condition . To circumvent this problem, workers who add information to the current events document also try to reach the day - shift personnel who need the information by phone . This is further complicated by the fact that the home help service workers during dayshifts (10 persons) share only two mobile phones, making it difficult for district nurses to quickly contact a specific home help service worker . For the night - shift personnel, the notes in the svop binder are the only way to stay informed . To continue, care receivers in need of multiple care interventions can trigger alarms if the need arises . The home help service responds to these alarms and is, therefore, equipped with keys . Not having these keys, the home health care personnel must coordinate their visits according to the home help service s schedule . However, information about the scheduled home help service visits is placed in the svop binder, accessible only during home visits . The following observation shows one consequence of this situation: a district nurse is about to make a home visit . She knows that the home help service usually makes its home visit at a certain time and she, therefore, plans the home visit according to this . When she arrives to the patient, the door is locked and she realises that the home help service worker has not arrived yet . She makes another home visit and attempts to visit the patient later when the home help service has arrived . In addition to this, the information in the svop binder is not always up - to - date for several reasons . In particular, pharmaceutical information, updated by external primary care workers and taken from the patient s medical record, tends to be inaccurate . The following observed situation illustrates an actual consequence of this problem: a district nurse is conducting a home visit in order to insert pharmaceuticals into medical dispenser units . She takes the pharmaceuticals from the packages and in order to insert the right dosage she looks at the prescribed pharmaceuticals document in the svop binder . During this home visit the district nurse consults the prescribed pharmaceuticals document and notices that the dosage of the new pharmaceutical is inconsistent with the prescribed pharmaceuticals document . She, therefore, drives back to the reception to search for the new document . To summarise, the empirical findings clearly show that the tools used for information and communication do not fully support coordination during the home care process, thus forcing care workers to make additional coordination efforts . In order to deliver integrated elderly care at home, the municipalities and the county councils are required to follow the requirements from the national board of health and welfare . One of these requirements concerns information sharing between care providers while the other deals with cooperative care planning before discharge from hospital . Therefore, when an individual is scheduled to receive care at home after hospital discharge, a care plan meeting with all parties involved is mandatory . During this meeting, the care providers and the care receiver with relatives discuss the home care process . The following care providers attend these meetings: the nurse at the hospital who initiates the meeting, personnel from home health care, the care administrator and physiotherapists from both the hospital and from primary care if needed . The care interventions provided by the home help service and by home health care are different . The home health care provides care interventions defined as health care, such as binding up wounds, giving insulin, taking samples for testing, inserting pharmaceuticals into medical dispenser units and dispensing medicine and eye drops . The home help service provides care interventions defined as care along with social care such as help with food, getting dressed, cleaning, care assistance, practical services and they also respond to alarms . Furthermore, some care interventions are defined by home health care as self - treatment and are, therefore, not provided by the home health care personnel . An elderly patient at the hospital is about to be discharged and is in need of home care . Five persons attend this meeting; the patient himself, a relative, the hospital nurse, the district nurse and the care administrator . The nurse describes the patient s visit to the hospital and what care interventions are needed when the patient returns to his home . The patient needs help with treating a wound with a cream twice a day, a procedure that takes some time . The district nurse responds that this kind of care intervention is classified as self - treatment and should not be conducted by the home health care workers . The hospital nurse informs the home care workers that the patient cannot perform the treatment himself due to his injuries . The care administrator responds that this kind of task is something that certainly should be done by the home health care workers since this task is not something a care receiver could apply for help for and thus cannot be classified as part of the home help service, .besides the home help service unit has no resources for these kinds of care interventions that take so much time . The meeting ends without a final decision about who should take care of the wound . During the group discussion if the patient is not mentally capable, even simple treatments may be considered as health care interventions . However, some patients are physically incapable to conduct the self - treatment themselves, as in the above observation, and the question then is which organisation is responsible for this person . To determine responsibility in these cases, the question is often asked if the person s health will deteriorate until a health care intervention is needed unless given the treatment in question . Another way of determining responsibility is to decide which organisation would have had responsibility for the patient if he or she had not applied for home help service . In addition to this, it is important to note that for the care receiver, home help service may be more costly than home health care . During the group discussions, the care workers argued for clearer rules or agreements about what organisation should be responsible for which care intervention . The care workers also stated that resources are wasted when both the home help service and home health care are at the care receiver s home at the same time: when one person is making a sandwich while another person gives insulin at the same time, this does not feel as an efficient way to conduct a home visit . However, the care workers also stated that sometimes the unit chiefs or care administrators bend the rules in the best interest of the care receiver and sometimes both the home help service and home health care workers actually conduct care interventions beyond those agreed upon during the care plan meeting . The home help service groups from all shifts store information about the care receiver and the approved care interventions separately in non - digital form . The home health care personnel use a computerised patient record system, which is used within all primary care . However, while working in the patients homes, it is impossible to access this patient record system . To manage cooperation and coordination between the organisations, svop is an acronym for coordinated health care and care planning (in swedish: samordnad vrd- och omsorgs planering). The binder is placed in the care receiver s home and consists of several documents and material which provide the care workers with information for administering home care interventions, for supporting involved individuals in daily work situations and for facilitating communication between the care workers involved . Particularly important for the present study is the fact that although the current events document facilitates communication, it was rarely used since it was available only in the care receiver s home . This is unfortunate, since some of the information is needed for coordination purposes and important information may be received late, which in turn may affect the care receiver s health condition . To circumvent this problem, workers who add information to the current events document also try to reach the day - shift personnel who need the information by phone . This is further complicated by the fact that the home help service workers during dayshifts (10 persons) share only two mobile phones, making it difficult for district nurses to quickly contact a specific home help service worker . For the night - shift personnel, the notes in the svop binder are the only way to stay informed . To continue, care receivers in need of multiple care interventions can trigger alarms if the need arises . The home help service responds to these alarms and is, therefore, equipped with keys . Not having these keys, the home health care personnel must coordinate their visits according to the home help service s schedule . However, information about the scheduled home help service visits is placed in the svop binder, accessible only during home visits . The following observation shows one consequence of this situation: a district nurse is about to make a home visit . She knows that the home help service usually makes its home visit at a certain time and she, therefore, plans the home visit according to this . When she arrives to the patient, the door is locked and she realises that the home help service worker has not arrived yet . She makes another home visit and attempts to visit the patient later when the home help service has arrived . In addition to this, the information in the svop binder is not always up - to - date for several reasons . In particular, pharmaceutical information, updated by external primary care workers and taken from the patient s medical record, tends to be inaccurate . The following observed situation illustrates an actual consequence of this problem: a district nurse is conducting a home visit in order to insert pharmaceuticals into medical dispenser units . She takes the pharmaceuticals from the packages and in order to insert the right dosage she looks at the prescribed pharmaceuticals document in the svop binder . During this home visit the district nurse consults the prescribed pharmaceuticals document and notices that the dosage of the new pharmaceutical is inconsistent with the prescribed pharmaceuticals document . She, therefore, drives back to the reception to search for the new document . To summarise, the empirical findings clearly show that the tools used for information and communication do not fully support coordination during the home care process, thus forcing care workers to make additional coordination efforts . If seamless and integrated care is to be supplied, the daily work situation needs to be improved . The first issue considers the difficulties that derive from the fact that home help service and home health care are separated between two organisations . The second issue is not related to organisational boundaries, but rather to the fact that the work needs to be coordinated while the care workers are distributed across time and/or space, regardless of organisational belonging . Coordinating home care interventions where home care is divided between two organisations is not a straightforward process . The present study has identified the planning activity, specifically during the care plan meeting, as a particularly problematic subject . The most crucial issue here is, who should assume responsibility for care interventions defined as self - treatment by home health care . This shows that it is difficult to demarcate between treatments considered as health care interventions and treatments that are considered as possible for the patient himself / herself to conduct . There is no established definition of self - treatment and, consequently, each case is judged separately . It is quite clear that this is primarily a policy issue that needs to be resoled at an organisational level . In 2006, the national board of health and welfare initiated an investigation on how to define self - treatment in relation to health care and during 2008/2009 directions will be issued on how to handle this issue . Another possible solution for improving the quality of home care that has been discussed is the merger of the home help service and home health care into one organisation . In 2004, the ministry of health and social affairs investigated how medical and social care including rehabilitation and aids for disabled should be organised to ensure integrated care and service for the elderly [36, p. 14]. The main argument of this bill was that the responsibility for the home help service and home health care should be provided by the municipalities, which would entail a merger of the home help service and home health care . The bill also stated that advanced home health care should remain the responsibility of the county council . Furthermore, it is possible that other services appear that also need to cooperate and coordinate with a merged home help service / home health care team . For example, the county council has recently introduced a trial service called the mobile doctor consisting of a physician from primary care who makes home visits during daytime . Thus, even if the home help service and home health care would merge, there will still be different care providers visiting elderly in their homes . This means that there always will be a need for inter - organisational cooperation and coordination . Furthermore, integrating care by merely merging home health care and the home help service may not be the ultimate solution, as discussed below . The second issue considers the care workers need to coordinate their efforts not only between organisations, but also within their own organisation across space and between work shifts . However, the empirical findings show that the information in the binder is equally important for the intra - organisational coordination taking place between dayshift and nightshift . In this light, the binder supports the core aim of the cooperation and coordination between home care workers, which is the care of the care receiver . Unfortunately, the present study suggests, like, that the svop binder fails to fully serve the need for effective dissemination of information and coordination during the home care process . Additionally, the empirical findings also show that home help service workers lack the mobile phones necessary for communicating effectively with other care providers . Providing home help service workers with mobile phones, thus, coordination during the home care process is clearly not only an organisational matter, but primarily a technical challenge . From this perspective, the most direct way of facilitating information and communication between care workers may be the introduction of it tools that improve information access and asynchronous communication . Certainly, this may improve the care workers possibility to coordinate during the home care process . Following the cscw research, further aspects need to be considered, explored and analysed in order to develop it tools that effectively support coordination . The ethnographic workplace study was conducted in order to understand how coordination is carried out in the daily work, to identify coordination issues in situ and possible actions that may be taken to improve coordination in the studied setting . Therefore, the analysis of the common themes, activities and issues is an important activity during the research process . As in all qualitative research, the data may be influenced by the researcher s point of view a problematic issue that needs to be considered during the research process . This is even more the case when conducting ethnographic studies, since one of the objectives is to capture the members point of view . To evaluate the researcher s interpretation of the empirical findings, common themes, activities and issues have been discussed with the care workers and managers throughout the project, thus confirming the reliability of the data . In cscw, some criticism has been presented in regard to workplace studies intended to inform systems design [38, 39]. It has been argued that the understanding of a particular workplace is a valuable contribution in its own right . It is believed that such an understanding can inform cscw design through raising awareness of important conceptual issues and questioning taken - for - granted assumptions about work activities and how they should be supported [38, p. 321 the assumption in this paper is that this kind of understanding is equally important in the research area of integrated care . The need for coordination in this particular case shows that formal organisational boundaries do not describe the actual behavioural patterns and division of daily work among health care providers . The core aim in the studied setting is to provide the care receiver with good - quality care, and the actors must coordinate their efforts regardless of their organisational belonging to achieve this aim . Bend the boundaries regarding the issue of self - treatment in the best interest of the care receiver . With this in mind, care workers have invented additional tools and routines to support cooperation and coordination in the workplace . Integrated care is clearly much more than organisational boundaries, policies, strategies, structures and processes . In their daily work, people cooperate and coordinate their activities to get the work done, regardless of their organisational belonging . Again, it tools may help to resolve some of the organizational problems faced by home care providers . In care settings, efforts have been made to develop electronic patient records, medical records and health records . These technologies were developed for a particular process or organization, and take the boundaries and structures of this process or organization into consideration . However, as shown in the present study, home care coordination often needs to transcend organizational boundaries . This further emphasises the findings of, which show that the context of elderly care at home involves coordination also with other care providers as well as the inclusion of the care receiver himself / herself and relatives . Furthermore, the present study also stresses the fact that new care providers might visit the care receiver at home in the future . Coordinating home care interventions where home care is divided between two organisations is not a straightforward process . The present study has identified the planning activity, specifically during the care plan meeting, as a particularly problematic subject . The most crucial issue here is, who should assume responsibility for care interventions defined as self - treatment by home health care . This shows that it is difficult to demarcate between treatments considered as health care interventions and treatments that are considered as possible for the patient himself / herself to conduct . There is no established definition of self - treatment and, consequently, each case is judged separately . It is quite clear that this is primarily a policy issue that needs to be resoled at an organisational level . In 2006, the national board of health and welfare initiated an investigation on how to define self - treatment in relation to health care and during 2008/2009 directions will be issued on how to handle this issue . Another possible solution for improving the quality of home care that has been discussed is the merger of the home help service and home health care into one organisation . In 2004, the ministry of health and social affairs investigated how medical and social care including rehabilitation and aids for disabled should be organised to ensure integrated care and service for the elderly [36, p. 14 the main argument of this bill was that the responsibility for the home help service and home health care should be provided by the municipalities, which would entail a merger of the home help service and home health care . The bill also stated that advanced home health care should remain the responsibility of the county council . Furthermore, it is possible that other services appear that also need to cooperate and coordinate with a merged home help service / home health care team . For example, the county council has recently introduced a trial service called the mobile doctor consisting of a physician from primary care who makes home visits during daytime . Thus, even if the home help service and home health care would merge, there will still be different care providers visiting elderly in their homes . This means that there always will be a need for inter - organisational cooperation and coordination . Furthermore, integrating care by merely merging home health care and the home help service may not be the ultimate solution, as discussed below . The second issue considers the care workers need to coordinate their efforts not only between organisations, but also within their own organisation across space and between work shifts . However, the empirical findings show that the information in the binder is equally important for the intra - organisational coordination taking place between dayshift and nightshift . In this light, the binder supports the core aim of the cooperation and coordination between home care workers, which is the care of the care receiver . Unfortunately, the present study suggests, like, that the svop binder fails to fully serve the need for effective dissemination of information and coordination during the home care process . Additionally, the empirical findings also show that home help service workers lack the mobile phones necessary for communicating effectively with other care providers . Providing home help service workers with mobile phones, thus, coordination during the home care process is clearly not only an organisational matter, but primarily a technical challenge . From this perspective, the most direct way of facilitating information and communication between care workers may be the introduction of it tools that improve information access and asynchronous communication . Certainly, this may improve the care workers possibility to coordinate during the home care process . Following the cscw research, further aspects need to be considered, explored and analysed in order to develop it tools that effectively support coordination . The ethnographic workplace study was conducted in order to understand how coordination is carried out in the daily work, to identify coordination issues in situ and possible actions that may be taken to improve coordination in the studied setting . Therefore, the analysis of the common themes, activities and issues is an important activity during the research process . As in all qualitative research, the data may be influenced by the researcher s point of view a problematic issue that needs to be considered during the research process . This is even more the case when conducting ethnographic studies, since one of the objectives is to capture the members point of view . To evaluate the researcher s interpretation of the empirical findings, common themes, activities and issues have been discussed with the care workers and managers throughout the project, thus confirming the reliability of the data . In cscw, some criticism has been presented in regard to workplace studies intended to inform systems design [38, 39]. It has been argued that the understanding of a particular workplace is a valuable contribution in its own right . It is believed that such an understanding can inform cscw design through raising awareness of important conceptual issues and questioning taken - for - granted assumptions about work activities and how they should be supported [38, p. 321 the assumption in this paper is that this kind of understanding is equally important in the research area of integrated care . The need for coordination in this particular case shows that formal organisational boundaries do not describe the actual behavioural patterns and division of daily work among health care providers . The core aim in the studied setting is to provide the care receiver with good - quality care, and the actors must coordinate their efforts regardless of their organisational belonging to achieve this aim . Bend the boundaries regarding the issue of self - treatment in the best interest of the care receiver . With this in mind, care workers have invented additional tools and routines to support cooperation and coordination in the workplace . Integrated care is clearly much more than organisational boundaries, policies, strategies, structures and processes . In their daily work, people cooperate and coordinate their activities to get the work done, regardless of their organisational belonging . Again, it tools may help to resolve some of the organizational problems faced by home care providers . In care settings, efforts have been made to develop electronic patient records, medical records and health records . These technologies were developed for a particular process or organization, and take the boundaries and structures of this process or organization into consideration . However, as shown in the present study, home care coordination often needs to transcend organizational boundaries . This further emphasises the findings of, which show that the context of elderly care at home involves coordination also with other care providers as well as the inclusion of the care receiver himself / herself and relatives . Furthermore, the present study also stresses the fact that new care providers might visit the care receiver at home in the future . Two important issues of the studied home care setting must be addressed to support seamless and integrated care: the division of care interventions that are defined as self - treatment and the coordination difficulties that arise from the fact that workers are distributed in time and/or space . The first issue concerns the fact that care is provided by two organisations and can be resolved through policymaking or through the merger of the home help service and home health care into one organisation . However, since informing and communicating between time shifts within an organisation was shown to be more difficult than between organisations, this may not help resolve the second issue . A more effective solution to this issue may be the introduction of it tools that support distributed access to information and communication between all care workers involved, regardless of organisational belonging . The findings of this paper thus suggest that integrated care needs to be developed not only between organisations but also within each organisation . In the daily work, cooperation and coordination cooperative work goes beyond organisational boundaries and integrated care must be developed with this situation in mind, beyond organisational boundaries . In view of this, a core subject for future practice and research is to develop it tools that reach beyond formal organisational boundaries and processes while remaining adaptable in view of future structure changes . Yves couturier, mss, phd, researcher at the geriatric institute of the sherbrooke university and at the research centre for research in youth, science teaching and learning, department of social services, university of sherbrooke, sherbrooke, canada alison petch, dr . Director research in practice for adults, blacklers, dartington hall trust, totnes, uk isabella scandurra, phd, medical informatics, chief science officer, apri \| ehealth, sjlevad, sweden and maria hgglund, phd, medical informatics, karolinska institutet, health informatics centre, stockholm, sweden
Aspergillus species are ascomycetes that are classified in the form subdivision deuteromycotina, as many of them do not show a sexual reproductive phase . Generally, they are common ubiquitous saprophytes in soil and on dead organic substrates . Being classic opportunistic pathogens, invasive infections by aspergillus species almost exclusively develop in immunocompromised patients, while localized infections and allergic bronchopulmonary aspergillosis occur in individuals without immunosuppression . Generally, the species aspergillus fumigatus represents the most common inducer of invasive and allergic manifestations, followed by a. terreus, a. flavus, and a. niger [1, 2]. Invasive aspergillosis (ia) considerably contributes to the morbidity and mortality among immunocompromised individuals, including patients with haematological malignancies, recipients of haematological stem cell and solid organ transplants, aids patients, and patients treated with immunosuppressive regimens due to autoimmune diseases . The most important single risk factor is prolonged and profound neutropenia (<500 neutrophils/l for more than 10 days) [1, 46]. Over the last decades, invasive fungal infections, particularly aspergillosis, have become more frequent due to a higher number of immunocompromised patients (new chemotherapy regimens, increasing number of solid organ transplant recipients, and immunosuppressive regimens) and extended survival time in hiv patients (haart therapy) [1, 79]. On the side of the pathogen, several characteristics and various putative virulence factors that may facilitate the infection have been described for a. fumigatus . It differs from nonpathogenic species by its growth at 37c; furthermore, it is rapidly growing and has very small conidiospores (35 m). These include melanin and a hydrophobic protein - coat layer on the surface of conidia that may help to protect them against recognition, ingestion and/or elimination by complement and phagocytes [1014]. Various proteases that may help to pass tissue barriers and to degrade proteins of the immune response are secreted by the fungus [1517], and mycotoxins like gliotoxin might also contribute to undermine the host defence [1820]. The most important path of aspergillus infections is via inhalation of the conidia into the respiratory tract . As conidia of pathogenic aspergillus species are very small, they can be inhaled deeply into the lung and even into the pulmonary alveoli . In immunocompetent individuals, conidia are effectively phagocytosed and eliminated by alveolar macrophages and infiltrating neutrophils [12, 21, 22], but in the case of immunologic deficits, they are able to germinate and to penetrate the lung tissue, thus causing an invasive pulmonary aspergillosis . Infections of the lung are the by far most frequent type of ia . By penetration of blood vessels, aspergillus can disseminate and invade other organs, including the heart, the liver, and the central nervous system (cns). Cerebral aspergillosis occurs in 10%20% of all cases of ia and thus is the most common extrapulmonary form . Neuropathologic features include hemorrhagic infarcts and/or necrosis, vascular thrombosis, meningitis, granuloma, and formation of solitary as well as multiple abscesses [6, 2325]. According to the division of bacterial and mycotic diseases (dbmd), the incidence of aspergillosis is 1 - 2 per 100,000 per year . Incidence rates of ia in high - risk populations depend on the respective group and rise up to 24% in patients with prolonged and profound neutropenia . Furthermore, ia is the most expensive opportunistic infection in immunosuppressed patients, with annual treating costs in europe of approximately 200 million . In - hospital stays complicated by ia cause additional costs of 75,000 per patient . Despite antimycotic therapy and surgical interventions, the fatality of ia is high and depends on the degree of immunosuppression and on the affected organs . Without treatment, the mortality is nearly 100%, while under treatment the overall case - fatality rate is nearby 60% and rises to more than 90% in cases of cns aspergillosis [1, 6, 26]. Complement consists of approximately 30 fluid - phase and membrane - bound proteins that cooperate to form the cascade . Regulatory factors control and modulate its activity, and cellular receptors mediate the interaction between complement factors and immune cells . Representing a potent component of the innate host defence and an interface to adaptive immunity the most outstanding roles are the direct and indirect defence against infections, the stimulation and regulation of b- and t - cell response, and the disposal of debris [2731]. Hepatocytes are the main producers of complement factors; however, several other cell types participate in the synthesis . Activation of the complement system is triggered by a multiplicity of danger signals, such as pathogen - associated molecular patterns (pamps), antigen / antibody complexes, and the presence of transformed cells, apoptotic cells, or cell debris . Three different activation pathways start the complement cascade, all of them resulting in the cleavage of the central complement factor c3 by proteolytic enzyme complexes (c3 convertases), and subsequently leading to the common terminal pathway (figure 1). In the classical pathway, binding of complement factor c1q to immunoglobulin class g or m (igg, igm) of antigen - antibody complexes represents the initial step . Alternatively, the globular heads of c1q can interact with microbial surfaces that had been covered by pentraxins, a class of soluble pattern recognition molecules . The thereby induced conformational changes of c1q subsequently activate the associated proteases c1r and c1s, which cleave the factors c4 and c2 . The resulting fragments form the c3 convertase c4b2a . In the lectin pathway, foreign carbohydrate molecules on the surface of pathogens are recognized by mannose - binding lectin (mbl) or the related ficolins . Mbl - associated serine proteases (masps) cleave c4 and c2, and the fragments build up the c3 convertase c4b2a, which is identically equal to the one of the classical pathway . Ficolin-2 can also interact with pentraxin - covered microbes, thus starting the lectin pathway in an alternative manner . Interestingly, mbl was recently described to support c3 cleavage by a c2 bypass mechanism, which results in activation of the alternative pathway . The alternative pathway is triggered via activating foreign surfaces and creates an amplification loop by spontaneous reaction of c3 with h2o (c3(h2o)); alternatively, c3b generated by the other pathways represents the starting trigger . Surface - bound c3b associates with factor b, which is then cleaved by the plasma serine protease factor d. these steps result in the formation of the c3 convertase c3bbb [27, 36]. Proteolytic cleavage of c3 by one of the c3 convertases is the common and central step of all three activation pathways . This split generates the fragments c3a and c3b, which are two important components that mediate a multitude of complement functions (see below). The product c3b associates with the c3 convertases, thus forming the c5 convertases, which cleave factor c5 into c5a and c5b . This step initiates a chain of assembly processes of the proteins c6, c7, c8, and c9 . The bound and polymerized c9 units create the terminal complement complex (tcc) that can form a pore in the target lipid bilayer, called membrane attack complex (mac). Targeted cells, bacteria and viruses die or are inactivated by efficient disruption of the membrane integrity [31, 37]. Beneath the mac formation and direct pathogen destruction, complement displays several additional antimicrobial mechanisms aiming to neutralize invading microbes and to restore body homeostasis . Surface - bound c3b undergoes internal cleavage steps; the derived products ic3b, c3d, and further, coat and label the pathogens for phagocytosis (opsonization). Effector cells with specific membrane - bound complement receptors (crs) recognize the opsonizing complement fragments, ingest the labeled pathogens, and eliminate them . Furthermore, the interaction of the opsonized particles with cr - bearing immune cells results in their activation and their increased proliferation . The receptor cr3, a heterodimer of cd11b and cd18, is regarded to be the most important mediator for complement - driven phagocytosis . Being expressed on phagocytes like dendritic cells, neutrophils, macrophages, and microglia, it interacts with ic3b on the pathogen [27, 38]. The complement receptors cr3 and cr4 allow adhesion of cells to cells of the same and other cell types, respectively, (homotypic and heterotypic adhesion). Immune cells can bind via these receptors to their ligands on endothelium of the blood vessels, a prerequisite for penetration through the vessel wall into the tissue and migration to the site of infection and inflammation . Surface - bound ic3b can be further cleaved proteolytically to generate the opsonizing fragment c3d . Binding of c3d - opsonised pathogens to the corresponding complement receptor cr2 (cd35) on b cells induces cross - linkage with the b cell receptor complex, a process that lowers the threshold for b cell activation by the specific antigen by several orders of magnitude . Cleavage of c3 and c5 generate the potent anaphylatoxins c3a and c5a, respectively, which exert several biological functions by binding to their corresponding cellular receptors c3ar, c5ar (cd88), and c5l2 . They provoke chemotactic attraction of immune cells to the site of infection and an increase of vascular permeability [40, 41]. Furthermore, c3a and c5a trigger an efficient proinflammatory response by stimulating cytokine synthesis and secretion [40, 41]. Various cell types harboring the corresponding anaphylatoxin receptors on their surface react on ligand binding with cell activation, stimulation of cell specific signaling pathways, or of oxidative burst [27, 42]. The complement cascade needs a tight control to prevent host damage by cell / tissue lysis and excessive inflammation . A variety of both soluble and membrane - bound regulators can influence all steps of the complement cascade, with the c3/c5 convertases as main control targets [27, 37, 4345]. Under normal conditions, these regulators should protect all body cells against auto - attack by the complement system . The serine protease factor i cleaves both c4b and c3b and is thereby supported by various cofactor molecules . C4 binding protein (c4 bp) and factor h (fh) are fluid - phase proteins that enable the cleavage of c4b and c3b, respectively, moreover, the membrane - anchored molecules complement receptor 1 (cr1, cd35) and membrane cofactor protein (mcp, cd46) support the degradation of both c3b and c4b . In addition, fh and c4 bp accelerate the decay of assembled c3 convertase; cr1 affects both c3 and c5 convertases . Decay accelerating factor (daf, cd55) is another notable membrane - bound regulator that efficiently prevents the assembly and promotes the disintegration of both c3 and c5 convertases [27, 4345]. In the terminal pathway, the membrane - anchored cd59 (protectin) binds to c8 in the c5b-8 complex and thus inhibits further incorporation and polymerization of c9 units to form the mac [27, 4345]. The potency of complement represents a valuable tool to attack invading pathogens and to defend the host against penetration and dissemination . One particular advantage of complement lies in the fact that activation can start within seconds after contact with the microbe and ends with a multifaceted spectrum of antimicrobial reactions . However, the fact that microbial infections occur in a considerable proportion, already implicates that the pathogens have developed appropriate counterstrategies to avoid elimination, thus starting a vicious circle of reaction and counterreaction . Furthermore, the antimicrobial effector mechanisms of the complement system might also harbour harmful consequences for the affected host . As known from several infectious and noninfectious diseases, chronic or exceeding complement - mediated inflammation putative mechanisms for such complement - induced tissue damage may include a fulminant inflammatory reaction and opsonization of surrounding bystander cells with subsequent lysis . Aspergillus conidia and hyphae activate the complement system via all three pathways [4648] (figure 1). Initiation of the complement cascade by resting conidia is mediated predominantly by the alternative pathway . However, when the conidia begin to swell and transform into hyphae, there is a progressive involvement of the classical pathway . These differences in the activation pathways are reflected by different kinetics; the slowest initiation is seen with resting conidia . Furthermore, mbl as a pattern recognition molecule of the lectin pathway is able to bind to carbohydrate structures on the surface of aspergillus and promotes complement activation via the lectin pathway, which results in the deposition of c4 . As mentioned above, mbl can support c3 cleavage by a c2 bypass mechanism after contact with a. fumigatus conidia, resulting in activation of the alternative pathway and avoiding formation of the classical pathway c3 convertase . This mechanism is not restricted to a. fumigatus, but can also take place in the presence of a. terreus, a. niger, and a. flavus . Mbl generally seems to be a molecule of high significance for innate defence against a range of pathogens . In several studies, it was shown to bind to various sugars on the surfaces of viruses, bacteria, yeasts, fungi, and protozoa [4851]. Further evidence for the crucial role of mbl arises from findings in patients with chronic necrotizing pulmonary aspergillosis and mouse models of pulmonary aspergillosis [52, 53]; these facts suggest mbl as a promising molecule for prophylaxis and therapeutical treatment (see below). A further mechanism for complement activation driven by aspergillus involves the interaction with the pattern recognition molecule pentraxin-3 (ptx-3). When a. fumigatus is opsonized with ptx-3, the complement cascade can be activated either by interaction between ptx-3 and c1q via the classical pathway, or by interaction between ptx-3 and ficolin-2 via the lectin pathway . After seroconversion, anti - aspergillus antibodies in the serum can trigger the start of the classical complement pathway . The thesis that complement represents a central tool in antifungal host defence is supported by several findings . Furthermore, recognition by the complement system and activation of the cascade seems to interfere with efficient dissemination in the host . This conclusion is strongly indicated by the fact that the level of complement deposition on different aspergillus species correlates inversely with their pathogenicity: highly virulent species like a. fumigatus and a. flavus bind less c3 on their surface than nonpathogenic species like a. glaucus or a. nidulans . The antimicrobial potency of the complement cascade appears to be independent from direct killing via formation of a mac; presumably, the thick fungal cell wall block the formation of a pore by the c9 polymers and the subsequent lysis of the cells . Opsonization of the fungal surface with c3-derived fragments are presumably the most relevant complement - associated weapon, stimulating efficient phagocytosis or release of damaging compounds, oxidative burst and killing by monocytes, bronchoalveolar macrophages, and polymorphonuclear cells [46, 47, 57]. The capacity to opsonize pathogens and to exert antifungal effects strictly depends on the available complement levels in the respective compartment of the body . The complement concentrations in the central nervous system (cns) are low and thus only allow a rather weak deposition . Consequently, the complement amounts in the cerebrospinal fluid are unable to induce a significant oxidative burst in immune cells and to result in reduced fungal viability, thus making the cns a highly vulnerable organ . However, the brain cells react to the fungal presence with an upregulation of complement synthesis to enable better opsonization and therefore a more efficient clearance of the fungus . Mice deficient in complement factor c5 can exert the early opsonization processes with c3 fragments, but are unable to fulfil the complete cascade . When infected with a. fumigatus, these mice show decreased resistance and lower 50% lethal conidia dosage for a disseminated infection [59, 60]. Since this enhanced susceptibility is unlikely to be due to absent mac formation in the fungal cell membrane, it might be supposed that the inability to form the anaphylatoxin c5a could be the relevant deficit in these mice [59, 60]. C5a exerts a wide range of proinflammatory effects; by binding to its receptor c5ar, c5a recruits inflammatory cells to the site of infection, enhances cellular adhesion, and stimulates oxidative metabolism . In addition, c5a can trigger the release of lysosomal enzymes and of inflammatory mediators such as tumor necrosis factor - alpha (tnf-) and interleukin-6 (il-6) [61, 62]. Furthermore, a higher susceptibility to aspergillosis in c5-deficient mice might be attributed to missing tcc . Low doses of this soluble complex were shown to bind to the membrane of a range of cell types, thereby triggering various effects like activation, rescue from apoptosis, and secretion of prostaglandins, which are important regulators of the immune response [6367]. The potency of aspergillus to cope with the complement system and to undermine its mechanisms for elimination determines how successful the fungus can establish an infection . Aspergillus has developed a complex repertoire of effector mechanisms for this purpose (table 1). Single or multiple abscess formation is a characteristic feature of aspergillosis, particularly in the central nervous system (cns). The fungal hyphae are found in brain blood, vessels with invasion through vascular walls into adjacent parenchymal tissue . Fungal brain abscesses may arise from these sites of localized parenchymal infection . In this case, white blood cells collect in the affected part of the brain, and fibrous tissue forms around this area, creating a mass . Cns abscesses typically present with headache, focal neurological abnormalities, and/or seizure, which is the consequence of local destruction or compression of adjacent brain tissue . Mature fungal abscesses exhibit a central necrotic area with fungal hyphae, surrounded by a capsule of newly formed fibrous tissue . The formation of abscesses represents a host mechanism to inhibit further spreading of invading pathogens . However, this sealing off not only inhibits fungal dissemination, but also forms some kind of protection shields against the complement attack . Immunohistochemical staining revealed that effect: whereas the fibrous surrounding tissue was intensely stained for complement proteins, the central necrotic area contained only minor complement concentrations . No deposition of complement factors on the fungal surface in the abscess was visible, implying that the encapsulation protects the fungus within the abscess from any efficient complement attack . Putative complement recognition sites on the conidial surface of a. fumigatus are optimally masked to minimize the stimulus for complement activation . Experiments aiming to identify the relevant fungal structure indicated that melanin could play a substantial role for masking; for this purpose, knock out mutants lacking enzymes of the melanin biosynthesis pathway were used [10, 11, 76]. Disruption of the gene alb1, which encodes a polyketide synthase in the synthesis of melanin, results in increased opsonization of the conidia with c3 and in a better ingestion by human neutrophils . Deposition of pigments on the conidial surface might mask the c3 binding sites, and disruption of the alb1 gene might expose these sites and thus allow enhanced c3 binding . A mouse model confirms this function of alb1 in fungal pathogenesis, since the alb1-deficient mutant of a. fumigatus turned out to be less virulent than the wild - type fungus [11, 76]. Inactivation of the gene for the pigmentation protein arp1 similarly increased the deposition of c3 on conidia . This pigment seems to be a central element of aspergillus against the host defence, as it is also involved in scavenging reactive oxygen species (ros) and inhibits the acidification of phagolysosomes of alveolar macrophages, monocyte - derived macrophages, and human neutrophil granulocytes after ingestion of conidia [12, 13, 77]. As mentioned above, the activity of the complement cascade is strictly limited by several fluid - phase inhibitors . Immunofluorescence analysis, adsorption assays and flow cytometry studies showed that aspergillus acquires fh, factor h - like protein 1 (fhl-1), factor h - related protein 1 (fhr-1), and c4 bp from the host [70, 71]. Fhl-1 is a splicing product of the fh gene, and fhr-1 is a related protein belonging to the fh family . Bound to the conidial surface, fh maintained its regulatory activity and could act as a cofactor for the factor i - mediated cleavage of c3b . As a consequence of covering the fungal surface with these complement inhibitors, all three pathways might be downmodulated . The attachment molecules on aspergillus are not yet known, whereas the corresponding binding regions within fh were recently described . One of them was identified within n - terminal short consensus repeats (scrs) 1 to 7 and a second one within c - terminal scr 20 . A. fumigatus not only acquires complement inhibitors from the host, but also produces and releases its own soluble factor that inhibits complement activation and opsonization of the fungus [72, 73]. This complement inhibitor (ci), which is also synthesized by a. flavus, selectively abolishes activation of the alternative pathway and interferes with c3b - dependent phagocytosis and killing . The exact chemical composition of ci is as yet unknown; it contains 15% protein and 5% polysaccharide . Recent own results raise the possibility that this described ci or a closely related activity also contributes to the pathogenesis in cerebral aspergillosis, since immunohistochemical studies show deposition of c1q and c4, but not of c3, on the fungal hyphae in the cns . Studies by sturtevant revealed the synthesis of a proteolytic enzyme that is able to degrade c3 (, reviewed in:). This is confirmed by own experiments showing complement - degrading proteolysis in the supernatant of aspergillus when grown in cerebrospinal fluid (csf). The fungus - induced degradation of complement in csf evoked a drastic reduction of the opsonization of the fungal hyphae . In parallel, the fungal serine protease alp1 might participate in complement degradation and thus be partly responsible for the complement evasion [15, 17]. To date, degradation of c1q, c3, c4, c5, mbl, and factor d were shown [15, 17]. The high morbidity and lethality of invasive aspergillosis strongly demands for an expansion of the current treatment options . The antimycotic therapy might be completed by new approaches aiming to strengthen the host immune response against the fungus . Putative approaches could be to increase the available complement concentrations or to improve the efficiency of complement attack by undermining the fungal evasion strategies . Appropriate strategies that target the complement system may aim to the following . Our own studies about cerebral aspergillosis showed a clear correlation between the complement levels in the csf and the capacity of csf to opsonize fungal hyphae and designate them for phagocytic killing . A therapeutic increase of mbl concentrations in invasive aspergillosis might be an appropriate approach, since patients with chronic necrotizing pulmonary aspergillosis show more frequently mbl haplotypes that encode for low levels of the protein than healthy control persons . Further support comes from a murine model of invasive pulmonary aspergillosis: those mice with externally administered recombinant mbl reveal better survival, compared to untreated animals . Detailed studies confirmed that mbl - treated mice show a significant increase in the levels of the proinflammatory cytokines tnf- and il-1, together with a marked decrease of anti - inflammatory il-10 and of fungal hyphae in the lung . Blocking of the fungal surface pigments by specific antibodies or peptides might be a hypothetical approach that could help to expose the c3 binding sites and thus improve complement deposition and ingestion of conidia by phagocytes . Another approach might target the acquisition of the negative complement regulators fh, fhl-1, and c4 bp to the fungal surface . For candida albicans, some molecules that bind c4b and fh have recently been identified [78, 79], while the attachment sites on aspergillus are still unknown but might include related molecules . Blocking antibodies, designed peptides or other inhibitors against these complement regulator binding molecules might help to make the fungus more vulnerable towards complement attack . A similar approach might be developed for the aspergillus - derived complement inhibitor described by washburn [72, 73]. Our own results open the possibility to neutralize the fungal protease(s) that is / are secreted by aspergillus to degrade complement proteins . Two different strategies were tested by first in vitro experiments: the neutralization of the protease by specific inhibitors or interference with the production of this proteolytic enzyme . In our studies, we could prevent the complement degrading activity by serine protease inhibitors . However, a therapeutically used protease inhibitor must be highly specific, since a general block of serine proteases might be fatal for the host . Alternatively, our experiments exhibited that the secretion of complement - degrading enzymes strictly depends on the availability of nitrogen sources . Thus, the supply of amino acids in the infected host might downmodulate the secretion of the relevant fungal protease that cleaves the complement factors of the host . Despite new antifungal drugs and improved medical treatment, invasive aspergillosis remains a dangerous threat for immunocompromised patients, as the innate immune defence is the most crucial weapon against this infection . The complement system is of particular importance, as it harbours multiple effects against infectious diseases, bridges the elements of the human defence network by a multitude of factors, and helps to preserve the homeostasis of the body . The presence of fungal pathogens is detected by different pattern recognition molecules; three pathways guarantee the activation of the complement cascade by resting, swollen, and germinating conidia as well as by hyphae of aspergillus . Direct lysis of fungal cells by the membrane attach complex (mac) appears to be of minor importance for the antifungal defence . Presumably, attraction and activation of immune cells (monocytes, pulmonary macrophages, and polymorphonuclear neutrophils) are the most essential mechanisms . Anaphylatoxins (c3a, c5a) chemotactically recruit immune cells to the site of the infection and induce further inflammatory reactions . Opsonization of conidia and hyphae with complement fragments like c3b and ic3b mediate phagocytosis, oxidative burst, and release of damaging compounds by binding to corresponding receptors on immune cells . However, highly virulent aspergillus species have evolved mechanisms to evade the attack by complement . They hide from recognition, acquire complement regulatory molecules from the host, and secrete proteases to degrade complement factors . The multifaceted interactions between complement and aspergillus represent promising approaches for future therapeutic strategies that may help to improve the outcome of invasive aspergillosis.
Isolation of rat brain mitochondria all procedures involving animals were approved by the children's hospital of pittsburgh (animal protocol number chp upmc arcc 1805) and were in compliance with principles of laboratory animal care and current laws of the united states . Rat brain mitochondria were isolated from the cortex of adult sprague - dawley rats . After removal, tissue was minced and homogenized in ice - cold isolation buffer i, which contained 225 mm mannitol, 75 mm sucrose, 5 mm hepes buffer (ph adjusted to 7.3 with koh), 0.1 mg / ml fatty acid - free bovine serum albumin, 1 mm tetrapotassium edta, and 12% percoll . The homogenate thus obtained was carefully layered on the top of a discontinuous gradient of percoll (24 and 42%) prepared using the same buffer . The preparation was then centrifuged at 16,000 g for 10 min . The fraction containing the mitochondria located between 42 and 24% percoll was carefully withdrawn by a syringe and washed from percoll twice by pelleting in isolation buffer i. the resulting mitochondrial suspension was diluted in isolation medium ii, which was similar to isolation buffer i, except for the concentration of edta (0.1 mm) and lack of albumin, and spun down at 12,000 g for 10 min . The deposit of mitochondria was homogenized in isolation buffer ii at a final protein concentration of 20 mg / ml and stored on ice until use . The protein concentration in the mitochondrial samples was determined using a protein assay kit . Mitochondria prepared in this way were active for at least 56 h, as determined by their ability to maintain a stable transmembrane potential in the presence of oxidizable substrates . Fluorescence measurements measurements were performed in a stirred cuvette mounted in a shimatzu rf-5301 spectrofluorimeter maintained at 37 c . Mitochondria (0.2 mg / ml protein) were added to 1.5 ml of the basic incubation medium, which contained 125 mm kcl, 5 mm mgcl2, 10 mm tris, 10 mm hepes (ph adjusted to a particular value between 6 and 8 with koh or hcl), 10 m egta, 5 mm succinate or 5 mm glutamate, and 5 mm malate as oxidizable substrates . Due to a tendency of precipitation at alkaline ph in the presence of inorganic phosphate, concentration of mgcl2 was reduced to 0.5 mm . Hydrogen peroxide hydrogen peroxide was measured using a2 m concentration of the fluorescent amplex red dye in the presence of 1 unit / ml horseradish peroxidase as previously described (26). Measurements were carried out at excitation / emission wavelengths of 560 nm (slit 1.5 nm)/590 nm (slit 3 nm), respectively . H2o2 generation was calibrated by constructing calibration curves using known h2o2 concentrations in the standard incubation buffer together with amplex red and horseradish peroxidase but without mitochondria at all ph values 68 . At acidic ph, the correction coefficients calculated from the titration curves for different medium ph were as follows: ph 6.0, 0.68; ph 6.5, 0.90; ph 7.0, 1.00; ph 7.5, 1.08; ph 8.0, 1.10 . These correction coefficients were applied to compensate for a difference in h2o2 reporting system response when determining rates of ros release at a given ph . Mitochondrial transmembrane potential (m) was estimated using fluorescence quenching of the cationic dye safranin o that is accumulated and quenched inside energized mitochondria . The excitation wavelength was 495 nm (slit 3 nm) and emission 586 nm (slit 5 nm), and the dye concentration used was 2.5 m (11). Respiration of mitochondria respiration of mitochondria was measured by a clark type oxygen electrode (hansatech co.; oxygen electrode unit dw1). The water - jacketed chamber was maintained at 37 c and constantly stirred with a magnetic stirring bar . Statistical analysis was performed using student's t test for comparison of two groups or one - way analysis of variance with bonferroni's correction for multiple comparison implemented in prism 4.0 software (graphpad software, san diego, ca). To study the ph - dependent component in the mechanism of reactive oxygen species generation in mitochondria, we used three types of incubation media with different impacts on the ph and electrical components of proton motive force through the inner membrane: condition 1, the medium without permeant anions, such as pi; condition 2, the medium of condition 1 supplemented with pi; condition 3, the medium of condition 2 supplemented with nigericin . Rat brain mitochondria respiratory characteristics measured in condition 2 and ph variations from 6 to 8 with succinate or glutamate plus malate as substrates are presented in fig . 1 . These data show that our mitochondrial preparations are good in coupling between respiration and phosphorylation in physiological range of ph . At ph 7, the addition of oligomycin inhibits respiration, whereas the addition of adp (state 3 (st 3)) essentially stimulates it, so that the ratio of respiration rates (state 3/state oligomycin (st 3/st oligo)) was 5.6 0.6 (n = 3) for succinate and 6.5 0.3 (n = 6) for glutamate plus malate . At alkaline ph, adp stimulated respiration to a smaller degree, possibly because the lack of protons in the medium limits the process . The first condition provides the maximal value of ph (maximal ph in mitochondrial matrix (phin) at a given ph of incubation medium (phout) and minimal value of . Pi co - transported with h (or in exchange to oh) decreases ph (and respectively increases). Therefore, the second condition stabilizes phin at a value less alkaline compared with the first condition; it provides the h distribution between electrical and chemical components similar to that in mitochondria of living cells . Figure 1.ph dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (a) and glutamate and malate (b) as substrates . Basic incubation medium (see experimental procedures) supplemented with 1 mm pi was used . Statistical analysis was as follows . A, rates of respiration in state 3 taken at ph 8 were significantly different from that at ph 67 (n = 34;, p <0.05, t test). B, rates of respiration in state 3 taken at ph 8 were significantly different from that at ph 67.5; rates at ph 7 and 7.5 were significantly different from each other (n = 6;, p <0.05;, p <0.001, t test). Ph dependence of mitochondrial respiration in state 3 (triangles), state 4 (squares), and state 4 with added oligomycin (inverse triangles), with succinate (a) and glutamate and malate (b) as substrates . Basic incubation medium (see experimental procedures) supplemented with 1 mm pi was used . A, rates of respiration in state 3 taken at ph 8 were significantly different from that at ph 67 (n = 34;, p <0.05, t test). B, rates of respiration in state 3 taken at ph 8 were significantly different from that at ph 67.5; rates at ph 7 and 7.5 were significantly different from each other (n = 6;, p <0.05;, p <0.001, t test). Nigericin as an h / k exchanger adjusts ph according to the k gradient across the inner membrane . Since k is the major osmogenic component, mitochondrial volume normally is stabilized when k concentrations inside and outside are the same and therefore under the third condition phin = phout . Since the chemical component of h is close to zero, could reach a higher value than under the other conditions (fig . 3a (squares) shows that the rate of ros production under condition 1 is 3 times increased when the ph of media changed from 6 to 7, whereas the levels of membrane potential decreased (fig . Usually, high is considered to be a factor promoting ros production (factor iii in the introduction). Here the situation is controversial; ros production is increased when is decreased . Obviously, substrate and oxygen concentration (factors ii and i) remain the same for all conditions; therefore, medium ph is the only factor correlating with increase of ros . At more alkaline ph (7.5 and 8), 4 demonstrates, the loss of membrane potential can be delayed by the presence in the medium of the agents known to inhibit permeability transition pore opening; cyclosporine a together with adp and oligomycin . This fact indicates that the loss of membrane potential in this condition is due to permeability transition pore opening . Additions at the time points indicated by the arrows were as follows: nigericin, 100 nm; fccp, 200 nm . Nigericin increases membrane potential of mitochondria oxidizing succinate at ph 68 . Additions at the time points indicated by the arrows were as follows: nigericin, 100 nm; fccp, 200 nm . In the presence of 1 mm of pi (condition 2), which makes the conditions more physiological, the behavior of the analyzed system is similar to that under condition 1 . As fig . 3a (triangles) shows, the rate of ros production steeply increased upon increase of medium ph, reaching a maximum at ph 7.5, while membrane potential (fig . The decrease of ros production at ph 8 correlates with a slight decrease of respiration rate (fig . 3c, triangles). When pi and nigericin were present in the medium (condition 3), the rates of ros production also increased along with the medium ph increase from 6 to 8 (fig . 3c, circles) repeated the pattern when only pi was present (condition 2), reaching maximum at ph 7.5 . If ph = 0, the whole pattern remains qualitatively similar to that observed in the presence of pi (condition 2). The ph dependences presented in fig . 3 clearly show that ph itself, and not ph, is an important determinant of ros production . As can be seen, at ph ranging from 6 to 7, when mitochondria do not undergo permeability transition, ros production under condition 1 is higher than under conditions 2 or 3, which could indicate an important role of matrix ph . The difference in ros production can be seen when the conditions change in the course of the experiment, producing qualitatively the same outcome (i.e. The addition of pi or nigericin always resulted in decrease of the ros generation rate despite the increase in (fig . 5, a and b) because of trading the ph component of proton motive force to the electric one). The difference in ros production between the three conditions presented in fig . In addition to the use of pi and nigericin, application of the ionophores fccp and valinomycin can be instrumental in manipulating matrix ph to study its role in mitochondrial ros generation . Both of these ionophores can decrease membrane potential; however, protonophore fccp acidifies the matrix by increasing proton leak, whereas valinomycin causes increase of matrix ph by carrying k into the matrix, decreasing and, thus, facilitating the proton ejection by the electron transport chain . To stress the qualitatively different effects of these ionophores, we applied a small concentration of fccp that did not substantially change and a concentration of valinomycin that essentially depolarized mitochondria (fig . 6b shows, fccp induced a dramatic decrease of ros production, wheras valinomycin temporarily increased it . Figure 3.effect of medium ph on rate of ros release (a), level of membrane potential (b), and rates of respiration of mitochondria oxidizing succinate (c). Basic medium (see experimental procedures) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm pi (triangles); condition 3, basic medium with 1 mm pi and 100 nm nigericin (circles). Statistical analysis was as follows . A, ros rates taken at all conditions and all ph values were significantly different from each other (n = 310, p <0.05), with an exception for conditions 2 and 3 at ph 8 (one - way analysis of variance test). Within the same condition, ros rates taken at each ph were significantly different from that at the adjacent ph value (n = 310, p <0.05), with the exceptions of the rates taken at ph 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at ph 7.08.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 38;, p <0.05, analysis of variance test) and also from the membrane potential at ph 6.06.5 for the same condition (n = 3; +, p <0.05, t test). C, at ph 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 38; +, p <0.05, analysis of variance test). In condition 1, rates at ph 8.0 were significantly different from that at all other ph values (n = 78;, p <0.0001, t test); in condition 3, rates at ph 7.5 were significantly different from that at ph 6.0 and 6.5 (n = 34; #, p <0.05, t test). Effect of medium ph on rate of ros release (a), level of membrane potential (b), and rates of respiration of mitochondria oxidizing succinate (c). Basic medium (see experimental procedures) was used in all experiments with different supplementations: condition 1, basic medium alone (squares); condition 2, basic medium with 1 mm pi (triangles); condition 3, basic medium with 1 mm pi and 100 nm nigericin (circles). Statistical analysis was as follows . A, ros rates taken at all conditions and all ph values were significantly different from each other (n = 310, p <0.05), with an exception for conditions 2 and 3 at ph 8 (one - way analysis of variance test). Within the same condition, ros rates taken at each ph were significantly different from that at the adjacent ph value (n = 310, p <0.05), with the exceptions of the rates taken at ph 7.5 and 8 in condition 3 (t test). B, the levels of membrane potential measured at ph 7.08.0 for condition 1 were significantly different from that in condition 2 or 3 (n = 38;, p <0.05, analysis of variance test) and also from the membrane potential at ph 6.06.5 for the same condition (n = 3; +, p <0.05, t test). C, at ph 8.0, rates of respiration measured in conditions 2 and 3 were significantly different from that in condition 1 (n = 38; +, p <0.05, analysis of variance test). In condition 1, rates at ph 8.0 were significantly different from that at all other ph values (n = 78;, p <0.0001, t test); in condition 3, rates at ph 7.5 were significantly different from that at ph 6.0 and 6.5 (n = 34; #, p <0.05, t test). In the experiments described above, succinate was applied as a substrate . Used in much higher concentrations than normally produced in krebs cycle, it stimulates high ros production rate, and this was convenient to study the process, although the ros production rates were much higher than could be observed under physiological conditions . This high concentration of succinate could be observed, however, at pathologies like ischemia (27, 28). More physiological substrates, glutamate and malate, produce much smaller amounts of ros (11, 29). However, as fig . 7a illustrates, the rates of ros release in this case also significantly increased with the increase of medium ph . These experiments were performed in the presence of both pi and nigericin to ensure the conversion of the ph component of proton motive force into the electric one (condition 3). Fig . 7, b and c, shows that, indeed, the addition of inorganic phosphate and nigericin did increase membrane potential the same way as it was observed with succinate as substrate . Figure 4.spontaneous depolarization of mitochondria takes place in the medium without pi (condition 1) at ph 8 . A, membrane potential 2 m csa, 200 m adp, and 2 m oligomycin were present in the medium before the addition of mitochondria (curve 2). Spontaneous depolarization of mitochondria takes place in the medium without pi (condition 1) at ph 8 . A, membrane potential; b, ros . 2 m csa, 200 m adp, and 2 m oligomycin were present in the medium before the addition of mitochondria (curve 2). 7b) and dramatically stimulated ros generation (11, 29). 8 shows that upon application of rotenone, regardless of depolarization, the rate of ros generation increased with the increase of medium ph . Thus, all presented data show the same pattern of ros production increase in conjunction with the alkalization of medium . In a study performed recently by lambert and brand (30), the authors came to the conclusion that ph gradient through the inner mitochondrial membrane determines ros production . Our results clearly show that even in the absence of ph gradient (figs . 3a (circles), 7a, and 8) medium ph is an essential factor defining ros production by the respiratory chain, and ph increase itself induces the increase in ros generation these data are in agreement with an early study presented by turrens and boveris (31), where a simpler model of submitochondrial particles was used . The authors have shown that the rate of superoxide generation increased in conjunction with an increase of medium ph from 7 to 9.2 in the presence of nadh or succinate as substrates and inhibitors rotenone or antimycin a, respectively . Figure 5.inorganic phosphate (a) and nigericin (b) increase the value of membrane potential but decrease the rate of ros release . The same relative scale of fluorescence was used for both dyes . A, membrane potential (traces 13) and ros (traces 46). Basic incubation medium, ph 7, was used (see experimental procedures); 1 mm pi was present (traces 1 and 4), or no pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (mt) (all traces), 1 mm pi (traces 3 and 6) and 200 nm fccp (traces 13) were made at the time points indicated by arrows . B, basic incubation medium without pi was used; ph was adjusted to 6.5 . Trace 1, ros; trace 2, membrane potential . At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm fccp (trace 2). Inorganic phosphate (a) and nigericin (b) increase the value of membrane potential but decrease the rate of ros release . A, membrane potential (traces 13) and ros (traces 46). Basic incubation medium, ph 7, was used (see experimental procedures); 1 mm pi was present (traces 1 and 4), or no pi was present (traces 2, 3, 5, and 6). The additions of mitochondria (mt) (all traces), 1 mm pi (traces 3 and 6) and 200 nm fccp (traces 13) were made at the time points indicated by arrows . B, basic incubation medium without pi was used; ph was adjusted to 6.5 . Trace 1, ros; trace 2, membrane potential . At the time points indicated by arrows, the following additions were made: mitochondria (all traces), 100 nm nigericin (all traces), and 200 nm fccp (trace 2). The ph dependence of ros production could be explained mechanistically, as outlined in the introduction . Proton binding could be a limiting step in semiquinone radical - ubiquinol transformation; in this case, the decrease of proton concentration must stabilize semiquinone radical . Ohnishi and trumpower (32) directly measured that semiquinone radical concentration in respiratory chain steeply increased with the increase of ph . This supports the proposed role of medium ph in mitochondrial ros generation by respiratory chain . Figure 6.ionophores valinomycin and fccp have opposite effect on the rate of ros release by brain mitochondria . A, recordings of membrane potential basic medium without pi (ph 6) was used . At the time points indicated by arrows, the following additions were made: mitochondria (mt) (all traces), 20 pm valinomycin (traces 1a and 1b), and 2.5 nm fccp (traces 2a and 2b). Concentration of k ions in the medium was 125 mm . To achieve a complete depolarization, ionophores valinomycin and fccp have opposite effect on the rate of ros release by brain mitochondria . A, recordings of membrane potential basic medium without pi (ph 6) was used . At the time points indicated by arrows, the following additions were made: mitochondria (mt) (all traces), 20 pm valinomycin (traces 1a and 1b), and 2.5 nm fccp (traces 2a and 2b). Concentration of k ions in the medium was 125 mm . To achieve a complete depolarization, the experimental results of (30) are in line with the ones presented here, and we believe that they could be better interpreted in terms of absolute value of matrix ph . Indeed, ph = phin - phout could be increased by two ways (either by increasing phin or by decreasing phout) with a different outcome . Higher phin would stabilize semiquinone radical on the matrix side of the membrane, thus stimulating ros production, and this was observed in the work (30) as well as in the present study . However, lowering outside ph (increase of outside proton concentration) would counteract the dissociation of protons from ubiquinol and formation of semiquinone radical (reaction 2). In the latter case 3a indicates that in the same medium ph, ros production is different, apparently because the matrix ph is different as defined by the respective conditions of incubation . The decrease of matrix ph induced by the addition of pi and nigericin (switch from condition 1 to condition 2 or 3) is accompanied by an increase of (fig . 3b), which is expected to stimulate ros production . However, the ros generation rate decreased (figs . 3a (triangles and circles) and 5), thus indicating that in these circumstances the effect of ph dominates over the opposite effect of . Figure 7.effect of medium ph on rate of ros release (a) and level of membrane potential of mitochondria oxidizing complex i substrates glutamate and malate (b and c). Basic incubation medium was supplemented with 1 mm pi (a and c) and 100 nm nigericin (a). For traces of membrane potential (b and c), ph was adjusted to the values indicated by each trace . The additions of mitochondria (mt), 1 mm pi, 1 m rotenone, 100 nm nigericin, and 200 nm fccp were made at the time points indicated by the arrows . All data sets for ros rates were statistically different from each other (n = 4;, p <0.05, t test). Effect of medium ph on rate of ros release (a) and level of membrane potential of mitochondria oxidizing complex i substrates glutamate and malate (b and c). Basic incubation medium was supplemented with 1 mm pi (a and c) and 100 nm nigericin (a). For traces of membrane potential (b and c), the additions of mitochondria (mt), 1 mm pi, 1 m rotenone, 100 nm nigericin, and 200 nm fccp were made at the time points indicated by the arrows . All data sets for ros rates were statistically different from each other (n = 4;, p <0.05, t test). The mentioned above comparison of the three conditions could give an approximate quantitative estimation of ph gradient and the role of matrix ph in ros production . In the presence of nigericin, matrix ph is the same as external ph; therefore, subtracting the ros production rate under condition 3 from that under two other conditions (as indicated in fig . 9), we can deduce the role of matrix ph difference in ros production . As fig . 9 shows, at ph 6, the difference between ros production rate under the condition when pi and nigericin are absent (condition 1) and when they are present (condition 3) is 650 pmol / min / mg protein . The reason for this difference is different matrix ph, which is higher in the absence of pi and nigericin (condition 1). The same difference in ros production (650 pmol / min / mg prot) could be induced by a 0.5-unit ph shift from ph 6 to 6.5 in the presence of both pi and nigericin (under condition 3) (fig . Therefore, if under condition 1 matrix ph defines this difference in ros production, it must be not less than 6.5 when ph of the medium is 6 . Figure 8.acidic ph inhibits rotenone - induced ros generation by rbm in the presence of glutamate and malate . Basic incubation medium supplemented with 1 mm pi was used; concentration of rotenone was 1 m . Data sets taken at ph 6.0, 6.5, and 7.0 were significantly different from that at ph 7.5 and 8.0 (n = 58;, p <0.01;, p <0.001, t test). Acidic ph inhibits rotenone - induced ros generation by rbm in the presence of glutamate and malate . Basic incubation medium supplemented with 1 mm pi was used; concentration of rotenone was 1 m . Data sets taken at ph 6.0, 6.5, and 7.0 were significantly different from that at ph 7.5 and 8.0 (n = 58;, p <0.01;, p <0.001, t test). Figure 9.difference between the rates of ros release calculated from the data presented in fig . 3 . Results of subtraction of ros release rates are plotted versus corresponding ph of the medium . Circles, condition 1 to condition 3; squares, condition 2 to condition 3 . Values of difference between condition 1 and condition 3 (circles) at ph 6.5 and 7 were significantly different from that at ph 6 (n = 310;, p <0.01, t test); values of difference between condition 2 and condition 3 (squares) at ph 7 were significantly different from that at ph 8 (n = 310;, p <0.05, t test). Difference between the rates of ros release calculated from the data presented in fig . Results of subtraction of ros release rates are plotted versus corresponding ph of the medium . Circles, condition 1 to condition 3; squares, condition 2 to condition 3 . Values of difference between condition 1 and condition 3 (circles) at ph 6.5 and 7 were significantly different from that at ph 6 (n = 310;, p <0.01, t test); values of difference between condition 2 and condition 3 (squares) at ph 7 were significantly different from that at ph 8 (n = 310;, p <0.05, t test). At ph 7, the difference between ros production under conditions 1 and 3 is even higher (1300 pmol / min / mg protein (fig . 9, circles)). Using the same reasoning as above, we can deduce that under condition 1 at ph 7, an even higher difference between external and internal ph should be expected . Thus, high ph must result in decrease, and it was measured as fig . Moreover, so high a ph difference assumes extremely low intramitochondrial proton concentrations; this could be a limiting factor restricting respiration rate when mitochondria are in state 3, as demonstrated by fig . This indirect estimation of ph gradient by ros production is in agreement with measurements made by conventional methods, 0.51.4 ph units (30, 33, 34). Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the krebs cycle, which provide nadh, complex i substrate, and succinate, complex ii substrate . Respiratory complex i accepts electrons from nadh, oxidizing it to nad, and delivers these electrons to ubiquinone (q). Proton transport in complex iii is coupled with electron transport in accordance with the generally accepted ubiquinone / ubiquinol (q / qh2) cycle mechanism, which is shown in more detail . The overall reaction performed by complex iii is as follows, qh2 + 2cyt cox + 2hn q + 2cyt cred + 4hp(i.e . It oxidizes ubiquinol, reducing cytochrome c and releasing two h to the cytosolic side (positive or p - side; this is reflected in the index of released h)). In addition, it translocates two protons from matrix (negative or n - side) to cytosol . Ubiquinol (qh2) delivers its first electron to fe, releasing two protons at the p - side of the inner mitochondrial membrane and producing semiquinone radical (sq). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (q) dissociates from the complex . Free q binds at the n - side and receives two electrons from cytochrome bh, resulting from oxidation of two qh2 molecules, thus producing subsequently sq and qh2, taking protons from the n - side . Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the krebs cycle, which provide nadh, complex i substrate, and succinate, complex ii substrate . Respiratory complex i accepts electrons from nadh, oxidizing it to nad, and delivers these electrons to ubiquinone (q). Proton transport in complex iii is coupled with electron transport in accordance with the generally accepted ubiquinone / ubiquinol (q / qh2) cycle mechanism, which is shown in more detail . The overall reaction performed by complex iii is as follows, qh2 + 2cyt cox + 2hn q + 2cyt cred + 4hp(i.e . It oxidizes ubiquinol, reducing cytochrome c and releasing two h to the cytosolic side (positive or p - side; this is reflected in the index of released h)). In addition, it translocates two protons from matrix (negative or n - side) to cytosol . Ubiquinol (qh2) delivers its first electron to fe, releasing two protons at the p - side of the inner mitochondrial membrane and producing semiquinone radical (sq). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (q) dissociates from the complex . Free q binds at the n - side and receives two electrons from cytochrome bh, resulting from oxidation of two qh2 molecules, thus producing subsequently sq and qh2, taking protons from the n - side . Ros levels are physiologically important as a metabolic signal (35, 36). Ros overproduction is one of the main factors inducing apoptosis (37). Here we show that mitochondrial matrix ph is one of the factors controlling ros signaling . Higher matrix ph in the absence of pi and nigericin (condition 1) at medium ph more than 7 resulted in spontaneous depolarization, probably due to permeability transition as figs . 3b (squares) and 4a illustrate . In these conditions, mitochondria experience dramatic perturbations with the loss of all gradients across the inner membrane . This resulted in a substantial decrease of ros release, which requires functional integrity of the mitochondrial membrane (figs . 10 that sq is not produced if the electron efflux from the fes center is blocked . On the other hand, matrix ph was shown to be an essential factor in induction of permeability transition (38). Evidence from the literature indicates that the induction of permeability transition may occur in the presence of inorganic phosphate and/or ca (39). Since these compounds were not present in the medium of condition 1, our results demonstrate that ph itself could regulate permeability transition; this proves the conclusion of our previous theoretical study (40). The permeability transition was not observed under conditions 2 and 3; however, the alkalization - induced increase of respiration rate when remained constant (figs . 3c, triangles and circles) indicates that ros affects membrane permeability, increasing leaks . In the presence of pi and nigericin (under condition 3), respiration increased more steeply with ph increase despite slower ros production (fig . This could be due to the fact that in the presence of both pi and nigericin (condition 3) is higher and that the leak strongly depends on . At ph 8, respiration decreases (remains unchanged), apparently due to the decrease in proton leak, since external h concentration decreases . Steep inhibition of respiration observed in condition 1 at ph 7.5 and 8, in all likelihood, is the consequence of the loss of cytochrome c in the course of permeability transition . The data discussed above leave no doubts that change in matrix ph is vital; it controls such physiologically important processes as ros production and permeability transition . Proton translocation coupled with electron transport itself cannot change ph extensively, because a small amount of translocated protons results in high increase of electric potential, which stops the process . However, if proton translocation is coupled with an influx of some cations, such as ca or k, it could result in a considerable alkalization of matrix . Mitochondria are also permeable for ca, and it is possible that ca overload of cells and, consequently, mitochondria could take place under some stress conditions . Although proton leak normally is compensated by respiratory proton translocation, it could set a dynamic steady state with matrix less alkaline . Application of uncoupler fccp shown in fig . 6 modulates this situation . In cells, the activation of ucps can down - regulate the level of ros by controlling the current of protons into the matrix without considerable compromise of mitochondrial atp production . To demonstrate the phenomenon of ph regulation of ros production by the respiratory chain, mitochondria were studied in the conditions of artificially high succinate levels . In this case, they produce ros with a higher rate than when respiring on physiological substrates glutamate and malate (although with similar ph dependence, as figs . Ph, evidently, is not the only factor defining ros production rate . If the levels of ubiquinol are high and, according to mass conservation, the levels of ubiquinone are low, the lack of electron acceptor ubiquinone results in a block of electron efflux from cytochrome bh and, subsequently, bl (see fig . The levels of cytosolic side semiquinone radical and, consequently, the rate of ros production must be high . Oxidation of succinate, available in excess, produces much higher levels of ubiquinol than would be produced by complex i, since ubiquinone reduction by respiratory complex ii is not related with proton translocation (as in complex i) and is not restricted by the proton electrochemical gradient . We suggest that this is the main reason why mitochondria, when respiring on succinate, produce much more ros than when they respire on complex i substrates . It should be noted that the increase of matrix ph stabilizes not only matrix side but also cytosolic side semiquinone . The stabilization of matrix side semiquinone slows down the electron transport in the chain upstream, thus increasing the life - time of semiquinone radical bound to cytosolic side . This rationale is in accordance with the finding that complex iii of mitochondria respiring on succinate produces substantial amounts of ros on the cytosolic side of the membrane (42, 43). The whole set of experiments presented here supports the proposed ph - dependent mechanism of ros generation in mitochondrial respiratory chain and its link with mitochondrial permeability transition . It allows us to understand the mechanism by which ucps down - regulate the level of ros in cells . The specific properties and quantitative data featuring mitochondrial ros generation could be a basis for a more detailed study aimed at exploration of the mechanism of ros production.
Myositis is an acute, sub - acute, or chronic inflammation of skeletal muscles . A previously healthy 12-year - old boy was admitted to driscoll children s hospital in corpus christi, tx, usa, with a 9 day history of fever, and 7 day history of pain and weakness in the lower extremities . Three days prior to admission, he was seen by a nurse practitioner and diagnosed with otitis media and pharyngitis . His temperature was 36.8c and the physical examination was significant for antalgic, broad - based gait and tender gastrocnemius soleus muscles . Sensation and deep tendon reflexes were normal with absent romberg s and babinski s sign . The white blood cell count was 8300 cells per cubic mm with 60% neutrophils and 40% lymphocytes, hemoglobin was 13.5 g / dl, and the platelet count was 172,000 cells per cubic mm . Creatinine phosphokinase (cpk) was elevated at 2394 /l, aspartate aminotransferase (ast) was 222 /l and, alanine aminotransferase (alt) was 143 /l . A diagnosis of myositis was made and the patient was given analgesics and oral prednisone as dermatomyositis and polymyositis were amongst the differential diagnosis . Rhabdomyolysis was considered in the differential diagnosis and the patient was started on one and a half maintenance intravenous fluids . Our patient s clinical course was not severe and did not develop the complications usually associated with rhabdomyolysis . On day 2 of his hospital stay, he was able to stand up with support and cpk levels came down to 1713 cerebrospinal fluid analysis showed a protein concentration of 62 mg / dl and white blood cell count of 4 . A multiplex respiratory viral pcr was positive for rhinovirus and negative for influenza a and b, parainfluenza, metapneumovirus and adenovirus . Arboviral titers for west nile virus was negative and so was the rocky mountain spotted fever titers . On the day 3 of his hospitalization, serum was evaluated by use of an indirect immunofluorescence antibody (ifa) test kit for immunoglobulin g (igg) to rickettsia typhi and rickettsia rickettsii (focus diagnostics, cypress, ca, usa). The complete recovery of the patient took about 48 hours which included 100 mg of doxycycline given twice daily by mouth . He was discharged home on doxycycline to complete a 10 day course and prednisone was discontinued . Myositis can be caused by strenuous activity, muscle trauma, illicit drug injections, genetic diseases, connective tissue disorders, diabetes and infections . Infectious myositis can involve single or multiple muscle groups in the limbs with the proximal muscles being predominantly affected the exception being trichinosis which commonly involves orbital muscles . Signs and symptoms of myositis include fever, malaise, muscle swelling, weakness and pain that is worse with movement . With this presentation, other diseases that should be considered are polymyositis, dermatomyositis, rhabdomyolysis, guillain barre syndrome, cellulitis, osteomyelitis, deep vein thrombosis, metabolic and other inflammatory myopathies (table 1). Rhabdomyolysis was considered as one of the top differential diagnosis for our patient and given intravenous fluids . It presents 1 - 2 weeks after the bite of an infected flea although a history of such exposure is rarely elicited . . The rash may not be present in 50% of the patients with murine typhus . Doxycycline is the treatment of choice, which our patient was prescribed at the time of discharge . It has been known that murine typhus resolves even without treatment . To our knowledge, this is the first pediatric case reported of murine typhus associated with myositis and paraparesis . Physicians practicing in or near rickettsia typhi - endemic areas need to consider this in the differential diagnosis of children with evidence of myositis . Making this diagnosis may have a significant impact on the course of the disease because of the availability of antimicrobial therapy against rickettsial organism.
Microarrays, once a selectively used expensive tool, have become increasingly common due to their falling costs and increased credibility over the past 10 years . In contrast to the bulk of dna sequencing, which has been taken over by large centers that automatically submit sequencing reads to centralized databases (e.g. Genbank), the majority of microarray expression data is still generated by smaller laboratories addressing particular biological questions . Given the diversity of expression possibilities in the cell and the stochastic nature of transcription and of microarrays themselves, previous studies have found computational analysis of large sets of microarrays (compendia) to be a powerful means of identifying strong biological signals between genes and across conditions (14). Historically these compendia have been generated as large internally controlled projects from a single laboratory, often excluding smaller datasets from independent laboratories . Yet, the many small microarray datasets generated worldwide represent a large and underutilized resource for genome - scale analyses such as compound mode of action identification (5) and network inference (6,7). The creators of the geo database at ncbi (8) and the arrayexpress database at ebi (9) have sought to address this opportunity by providing a central repository of expression data for large and small laboratories . While valuable first initiatives, the geo and arrayexpress databases are not yet structured in a way that facilitates efficient exploration or analysis of the data . Four main obstacles exist: first, submitting microarray datasets to repositories is more difficult than submitting sequences to genbank the data itself is more complicated, requiring submission formats that are beyond the means of many non - computational researchers . This problem has been addressed to some degree as more journals require submission of microarray data to geo or arrayexpress . The second obstacle is the presence of platform - specific biases in expression data due to the use of many different microarray platforms in a compendium . These biases obfuscate the interpretation of the integrated dataset . For dual - channel arrays, the situation is often further complicated by the lack of a single physiological reference condition used across all arrays in the platform . Hence, the first step in the analysis of an array compendium is often to segregate data into sets with a uniform reference condition and a consistent array type . This time - consuming step often reduces the compendium to a far less expansive dataset . The third obstacle is the lack of uniformity in the format of expression data, even within a single expression platform . The data deposited in geo and arrayexpress does not necessarily employ a uniform preprocessing approach, nor is the raw intensity data always provided with the deposits . The fourth obstacle is the incompleteness and inconsistency in the curation of metadata describing the details of each experimental condition . Each expression profile run for a given species can have a different genetic background, media, growth conditions and any number of chemicals, which might have an effect on the cell's expression . Such data is fundamental to the meaningful interpretation of expression data . Even when provided, this metadata is found as unstructured prose in the database deposit or in the methods sections of each publication . Ideally, this metadata would be collected in a computable format with uniform units across all laboratories . Although standards like miame (10) promote the human interpretation of experimental conditions, the standard is unevenly applied and it does not facilitate computational analysis . To address the latter three of these problems, we have constructed the many microbe microarrays database (m). M currently contains over 1000 microarrays for escherichia coli (507), saccharomyces cerevisiae (530) and shewanella oneidensis (14), all of which were collected and combined from individual investigators, geo (8), arrayexpress (9) and asap (11). To avoid problems with platform - specific biases, the expression data is uniformly normalized to enable web - based or offline (via a database dump) analysis without further user - dependent normalization . This facilitates analysis of the data across all laboratories and conditions, even by non - expert users . A set of web - based browsing and analysis tools is provided to facilitate efficient interrogation of the dataset without extensive computational skills . Importantly, experimental metadata in m is human curated from each microarray publication converting each chemical and growth attribute into a structured and computable set of experimental features with consistent naming conventions and units . Finally, all versions of the database builds are maintained and accessible in perpetuity on the website to facilitate the future interpretation and comparison of results published on m data . The various attributes of m comprehensive data and metadata, uniform normalization, access to raw data dumps, a computable structure, versioning of the database and web - based analysis tools facilitate both efficient human interrogation of the dataset and machine - based computational analysis . Moreover, the consistency and uniformity of the dataset facilitates downstream comparison of results and findings based on the dataset . Large microarray depositories like geo and arrayexpress focus on the archiving of expression data as used in specific publications . These archives play an essential role in biological science by allowing transparent replication of microarray analyses by other researchers . Experimenters using the same array platform often use different normalization methods for their analyses, so that data downloaded from different projects on geo or arrayexpress are unlikely to be directly comparable . A geo dataset contains a collection of biologically and statistically comparable microarray samples processed using the same platform . Unfortunately, there is a significant delay between when a sample is submitted to geo and when it is available as a geo dataset . Only one - fifth of the number of samples in m were available from geo datasets (figure 1a and b). Figure 1.all of the available e. coli affymetrix antisense2 expression data for the transcription factor lexa and its known target reca were downloaded from ncbi geo profiles (a) and from m compendium e_coli_v3_build_1 (b and c). Ncbi geo profile data is derived from ncbi geo datasets that contain only a subset of the data in geo, therefore many more samples were available for plotting from m (445) than from geo (85). The correlation between lexa and its known target was higher when the raw data was uniformly normalized with rma (c) rather than normalizing each microarray individually with mas5 (a and b). All of the available e. coli affymetrix antisense2 expression data for the transcription factor lexa and its known target reca were downloaded from ncbi geo profiles (a) and from m compendium e_coli_v3_build_1 (b and c). Ncbi geo profile data is derived from ncbi geo datasets that contain only a subset of the data in geo, therefore many more samples were available for plotting from m (445) than from geo (85). The correlation between lexa and its known target was higher when the raw data was uniformly normalized with rma (c) rather than normalizing each microarray individually with mas5 (a and b). We have initially chosen to include only single - channel affymetrix microarrays in m. the photolithography process used by affymetrix allows all laboratories to start with a very consistent substrate for hybridization . In addition, the single - channel design eliminates the need for a common reference condition for all arrays . Thus, in contrast to two - color array designs, data from different laboratories and projects can be integrated without artifacts due to an inconsistent reference condition . The remaining systematic biases in the affymetrix platform are due to researcher - specific differences in the rna preparation and hybridization protocols . However, when the raw probe - level microarray data (cel files) are normalized as a group with rma (12), we find that these systematic researcher biases are small relative to the biological changes that occur across experimental conditions (7). In addition, the rma normalized data tends to have higher correlation between the expression of transcription factors and their known targets (figure 1b and c). To employ the rma normalization approach in m, all expression profiles for a particular array design (e.g. The e. coli antisense 2 array) are collected, uniformly normalized and deposited as a build. Periodically, we add new expression profiles for a particular array design, renormalize all data, and release a new build. This ensures that all experiments in any build are uniformly normalized and comparable across conditions . The renormalization process may result in small changes in the expression values of all profiles . Thus, all builds are labeled with a version number that references the underlying mysql schema of the database and a build number that denotes the particular set of microarray data (e.g. E_coli_v3_build_2 uses mysql schema version 3 and is the second compendium built for e. coli). This system, like the build system used by the human genome assembly, allows computational researchers to specify the exact dataset used for a particular analysis . The experimental condition information underlying each microarray sample is the most under - utilized aspect of compendia collected from multiple disparate sources . In scientific language there are typically multiple units that can be used to describe a particular aspect of an experiment . For example, the amount of glucose added to a media can be described in weight / volume, as a percent solution, or using molarity . To promote large - scale analyses of the relationship between experimental conditions and the expression values of each gene, we provide the quantitative and qualitative features of each experimental condition cataloged in a consistent framework suitable for computation . We use human curation to convert the condition metadata in each publication into consistent units and naming conventions, and we use computer validation to provide data integrity . To facilitate large - scale computational analyses of compendium data, we provide bulk downloads of the normalized expression data in m. for each build, we provide separate files containing normalized data for all genes, all genes + intergenic regions, and all genes + intergenic regions + control probes . We also provide flat files containing the gene names, probe set names and curated experimental condition information . In addition, we provide the raw cel files as a tar archive for researchers interested in using or developing other normalization methods . For more targeted analysis and data exploration, m allows the flexible construction, visualization and download of custom datasets . Users can select any subset of the experiments in m using checkboxes or by selecting projects that represent larger groups of experiments (typically a project is the set of microarrays available in a single publication). Similarly, users can choose a subset of genes by typing or uploading a list of gene and/or probe names . Genes can also be selected by differential expression as measured by t - test, z - test or fold change (e.g. Choose all genes with a significant expression change between experiments a, c, e versus b, d, f, g as measured by a t - test with a user - chosen significance threshold). Once a user selects a set of genes and experiments, the data can be downloaded or visualized . Although, there are many existing general plotting tools and a few software visualization products dedicated to microarrays, it is often convenient to be able to choose a few conditions of interest, type in a few genes and see a quick plot of the data . M currently provides heat plots (with and without clustering), expression histograms (for individual genes and groups of genes), scatter plots and a genome browser for visualization of expression in a genome context (figure 2) (13). Figure 2.custom datasets constructed on m can be visualized with scatterplots (a), histograms of individual genes (b), heatplots (c), histograms of collections of genes (d) and in their genome context using a genome browser (e). Custom datasets constructed on m can be visualized with scatterplots (a), histograms of individual genes (b), heatplots (c), histograms of collections of genes (d) and in their genome context using a genome browser (e). All browsing, analytical and download features of m3d are accessed from the same page, the analysis page, on the website . This page guides the user step - by - step through the process of database selection, experiment selection, gene selection, visualization, analysis and download . At each step, a user's selections are saved in a cookie, enabling the user to return to and modify any prior selection without losing any other selections . Context - specific help is provided on each page by mousing over the [?]' Symbol . The power of the internet resides in its interconnectivity . Biological databases like ncbi (14), ecocyc (15) and regulondb (16) provide easy linking mechanisms so that other databases can automatically generate hyperlinks to their content . M provides a simple mechanism for generating links to m genepages, which contain a basic set of plots for each gene . In addition all of the plots on m are generated using a simple url syntax so that websites can easily include plots generated by m on their own sites . For example, strong correlation is often found between the expression of a transcription factor and its targets, a website cataloging known or predicted regulatory interactions might find it useful to provide a scatter plot of the transcription factor's expression versus its target gene's expression to allow users to see if expression data currently supports the regulatory interaction . The url syntax for this and other plots can be found by clicking the help tab on the main menu of m. raw cel files are periodically collected from the arrayexpress and geo microarray databases . Upon accumulation of approximately 50 new chips for a particular species, all of the old and new microarrays are normalized together into a new compendium build . For researchers preferring to submit cel files directly to m, we can generate a template submission to geo, which the researcher can then edit as desired.
Pharmacists embedded in primary care medical practices, such as those modeled on the patient - centered medical home, are playing increasing roles in interdisciplinary primary healthcare teams.13 however, with limited pharmacist resources and high patient to pharmacist ratios, it is important to identify which patients would benefit most from consultation with a primary care pharmacist . We have previously completed a similar assessment of patient requirements for dietician services in primary care.4 while commonly used approaches to identify patients for pharmacist care include physician referrals, drug- or disease - specific programs (eg, chronic disease management), and patients recently discharged from hospital, an alternative strategy is to focus on patients at increased risk of medication - related problems (mrps).2,57 mrps are events or circumstances involving drug therapy that actually, or potentially, interfere with an optimum outcome for the patient.8,9 several studies have identified factors associated with an increased risk of mrps,1014 and short, self - administered surveys have been developed to help identify patients at risk of mrps.12,15 our objective was to determine the feasibility of using a locally adapted, self - administered, paper - based, patient survey, to identify patients at risk of mrps, in a convenience sample of patients at a single academic family medicine clinic . We conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . At the time of the survey, seven family physicians, two family medicine resident doctors, two full time clinic nurses (one dedicated to chronic disease management), and two academic clinical pharmacists (each working ~0.2 full - time equivalent) provided service to approximately 7,000 patients . All patients registered to be seen by one of the clinic physicians were eligible to participate in this survey, unless they were less than 18 years old, could not read english, or were not responsible for self - management of their medications . After registration at the reception desk, a medical office assistant asked eligible patients if they would be willing to participate . Those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room, and asked to complete the survey while waiting to be seen by their physician . The university of alberta health research ethics board (reb) approved the study protocol . We used the previously validated, ten - item mrp questionnaire, developed by barenholtz levy, and the modified five - item questionnaire, developed by langford et al, to create an adapted ten - item questionnaire (figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population, several changes were made . The two most significant changes made were: 1) the focus of question 3 (present in both surveys) was changed from high risk medications to a chronic diseases focus, and 2) question 9, does someone else bring any of your medications to your home for you? Was replaced (as all patients attending the family medicine clinic were ambulatory) with do you sometimes worry about the long - term effects of your medications? To determine feasibility, we recorded patient - reported time to complete the questionnaire, and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . To estimate the number of patients at high risk for an mrp, we calculated a summary risk score . Responses, after collapsing question 3 into a single yes or no response . As done by others in the literature, we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons, we did a sensitivity analysis, in which we examined the number of patients who were at risk, while restricting analysis to the five questions developed by langford et al.15 we conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . At the time of the survey, seven family physicians, two family medicine resident doctors, two full time clinic nurses (one dedicated to chronic disease management), and two academic clinical pharmacists (each working ~0.2 full - time equivalent) provided service to approximately 7,000 patients . All patients registered to be seen by one of the clinic physicians were eligible to participate in this survey, unless they were less than 18 years old, could not read english, or were not responsible for self - management of their medications . After registration at the reception desk, a medical office assistant asked eligible patients if they would be willing to participate . Those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room, and asked to complete the survey while waiting to be seen by their physician . The university of alberta health research ethics board (reb) approved the study protocol . We used the previously validated, ten - item mrp questionnaire, developed by barenholtz levy, and the modified five - item questionnaire, developed by langford et al, to create an adapted ten - item questionnaire (figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population, several changes were made . The two most significant changes made were: 1) the focus of question 3 (present in both surveys) was changed from high risk medications to a chronic diseases focus, and 2) question 9, does someone else bring any of your medications to your home for you? Was replaced (as all patients attending the family medicine clinic were ambulatory) with do you sometimes worry about the long - term effects of your medications? To determine feasibility, we recorded patient - reported time to complete the questionnaire, and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . To estimate the number of patients at high risk for an mrp, we calculated a summary risk score . Responses, after collapsing question 3 into a single yes or no response . As done by others in the literature, we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons, we did a sensitivity analysis, in which we examined the number of patients who were at risk, while restricting analysis to the five questions developed by langford et al.15 demographic characteristics, summary risk scores, and time to complete the questionnaire are presented in table 1 . From patient reports in answer to the questionnaire, the median time to complete the questionnaire was 2 minutes (interquartile range: 1.53 minutes). There were no comments from the medical office assistants, or physicians approached during regular staff meetings, to suggest that the questionnaire was intrusive on the clinic s workflow . The questionnaire was not distributed by medical office assistants to consecutive patients, because research was not considered to be a priority in their responsibilities, and clinical tasks sometimes intervened . Forty - three respondents had three or more risk factors identified by the full ten - point questionnaire, and 26 respondents met this criterion in the sensitivity analysis . In view of the limited data, and bearing in mind that this was a pilot study, no further mining of the data was undertaken . The findings of this pilot study suggest that distribution of a mrp questionnaire at the point of care is feasible in a busy family medicine clinic, and that there was a strong signal for potential mrps in the sample studied . However, there may have been a selection bias introduced by the office assistants, in terms of who they approached to participate, especially when they were busy . We have identified issues relating to the efficiency of this approach (consecutive patients were not all approached), and we suggest that asynchronous methods to systematically screen larger numbers of patients, such as the use of electronic medical record data, should also be explored to supplement the direct approach . This anonymous pilot study, as approved by the reb, did not permit the pharmacists to act upon the mrps identified, whereas a chart survey would allow this . This study adds to existing literature that demonstrates in practice the clinical utility of a mrp screening tool, and demonstrates its potential to generate pharmacist referrals . While our overall estimate of patients at high risk for an mrp was double that obtained by langford et al15 (43% versus 18%, respectively), our sensitivity analysis, using only langford s questions, demonstrated more similar proportions (26% versus 18%). The difference is likely related to the change in focus of one question, from high risk medications to the presence of common chronic diseases . In view of the differences between the two studies, unfortunately, comparisons with the sample studied by barenholtz levy are not possible, as they did not report summary risk scores, nor use a cut - off to define patients as being at high risk.12 perhaps one of our study s biggest strengths was its use of medical office assistant staff to screen patients, rather than using research personnel or clinic pharmacists . From a practical standpoint, part - time clinic pharmacists are not available to screen all patients attending clinics . Despite this strength, since our study was an uncontrolled, single site, cross sectional survey based on a convenience sample, the time taken to achieve 100 completed surveys, as well as our estimates of the proportions of patients at high risk of mrps, may be unreliable . Framing the survey as research to the office assistants (rather than quality improvement) may have contributed to the prolonged recruitment period; this was also contributed to by the deliberate arms length policy of the investigators, to test the feasibility of the intervention in the practice context . Volunteer bias or preselection by medical office assistants could have led to overestimation of those at high risk . Finally, as demonstrated by our sensitivity analysis, the reliability of this estimate is influenced by the number of questions included in the survey, and the arbitrary nature of the cut - off points to define high risk . Future study is required to compare different methods of patient identification for primary care pharmacists, determine what threshold accurately defines high risk of an mrp, and, ultimately, the effect of pharmacist intervention on risk of mrps . In conclusion, our results suggest that screening patients at the point of care, using a self - administered mrp questionnaire, was feasible, and that a large proportion of those who complete the questionnaire will have multiple risk factors for mrps . This is similar to the opportunity found in our dietician study.4 however, other strategies, including electronic medical record screening or focusing on patients attending the chronic disease management nurse, may represent complementary methods to increase screening efficiency, and allow for more systematic screening of clinic patients for risk of mrps . Our findings support the integration of a pharmacist into family medicine practices, as a member of the patient - centered medical home . Future work will include further content and construct validation of the revised tool and its predictive ability for measures of health care utilization . Additionally, we plan to compare the predictive accuracy and feasibility of an automated electronic medical record medication list screening approach for identifying patients at high risk of mrps.
Obesity has become a heavy public health problem in the united states, with a prevalence among adults increasing to 32% from 13% between the 1960s and 2004 . Currently, 66% of adults and 16% of children and adolescents are overweight or obese . Although obesity has long been recognized as an independent risk factor for cardiovascular diseases and diabetes mellitus, newer research points to obesity as an important risk factor for chronic kidney diseases (ckds) [24]. In 1974, weisinger et al . Subsequent studies confirmed that obesity could induce renal injury, namely, obesity - related glomerulopathy (org) [68]. A large - scale clinicopathologic study including 6818 renal biopsies from 1986 to 2000 revealed a progressive increase in biopsy incidence of org from 0.2% in 19861990 to 2.0% in 19962000 . The tenfold increase in incidence of org over 15 years suggests a newly emerging epidemic . The clinical characteristics of subjects with org typically manifest with nephrotic or subnephrotic proteinuria, accompanied by renal insufficiency [810]. Histologically, org presents as focal segmental glomerulosclerosis (fsgs) and glomerular hypertrophy or glomerular hypertrophy alone and relatively decreased podocyte density and number and mild foot process fusion [8, 11, 12]. Clinically, it is distinguished from idiopathic fsgs (i - fsgs) by its lower incidence of nephrotic syndrome, more benign course, and slower progression of proteinuria and renal failure [8, 11]. Potential mechanisms by which obesity affects renal physiology include altered renal hemodynamics, insulin resistance, hyperlipidemia, activation of renin - angiotensin - aldosterone system (raas), inflammation, and oxidative stress . Increases in both glomerular filtration rate (gfr) and renal plasma flow (rpf) were observed in obese subjects and animals [13, 14]. This likely occurs because of afferent arteriolar dilation as a result of proximal salt reabsorption, coupled with efferent renal arteriolar vasoconstriction as a result of elevated angiotensin ii (angii). These effects may contribute to hyperfiltration, glomerulomegaly, and later focal glomerulosclerosis [8, 9]. Insulin resistance can raise the transcapillary pressure gradient and cause hydrostatic pressure and hyperfiltration by reducing norepinephrine - induced efferent arteriolar constriction, leading to glomerular hypertrophy and sclerosis . Hyperinsulinemia also has been shown to stimulate the synthesis of growth factors such as insulin - like growth factor- (igf-) 1 and igf-2 and transforming growth factor-1 (tgf-1), which accelerate production of extracellular matrix and promote glomerular hypertrophy and sclerosis [17, 18]. Hyperlipidemia may promote glomerulosclerosis through mechanisms that involve engagement of low - density lipoprotein receptors on mesangial cells, direct podocyte toxicity, oxidative cellular injury, macrophage chemotaxis, and increase renal expression of sterol regulatory element - binding proteins (srebp-1 and srebp-2), resulting in the renal accumulation of cholesterol and triglycerides and together with significant renal increase of fibrogenic cytokines [19, 20]. Obese subjects usually have increases in plasma renin activity, angiotensinogen, angiotensin - converting enzyme activity, and circulating angii, which trigger or promote renal damage by renal hemodynamic changes and nonhemodynamic pathways such as hyperinsulinemia, oxidative stress, and inflammation [2123]. Inflammatory abnormalities and oxidative stress are characteristic findings of obesity and play important roles in the renal damage associated with obesity, which will be discussed in detail in the following . Recent studies have demonstrated that obesity causes chronic low - grade systemic inflammation and thus contributes to the development of systemic metabolic dysfunction that is associated with obesity - related disorders and renal disease [2427]. Levels of some inflammatory markers and cytokines such as c - reactive protein (crp), tumor necrosis factor- (tnf-), interleukin-6 (il-6), and macrophage migration inhibitory factor (mif) are elevated, whereas concentrations of adiponectin, a protein hormone that exerts anti - inflammatory activities, are reduced in obesity [2834]. Leptin is a 16-kda - peptide hormone encoded by obese (ob) gene that is mainly produced by adipose tissue . Leptin serves as a regulator of energy balance by binding to the full - length leptin receptors obese receptor b (ob - rb) in the hypothalamus, leading to reduction in food intake and elevation in temperature and energy expenditure . Leptin receptors can be classified as secreted - forms (ob - re), short - forms (ob - ra, c, d, and f) mainly expressed in peripheral tissue, and long - forms (ob - rb) predominantly expressed in hypothalamus . The kidney expresses abundant concentrations of the truncated isoform of the leptin receptor ob - ra, but only a small amount of the full - length receptor ob - rb . Leptin production is associated with increased size of adipocytes and is positively correlated with the body mass index (bmi). Increased circulating leptin, a marker of leptin resistance, is common in obesity . Obesity - induced leptin resistance injures numerous peripheral tissues including kidney, liver, myocardium, and vasculature [36, 38]. Leptin results in the development of renal disease by binding to its specific receptors in renal endothelial cells and mesangial cells . In glomerular endothelial cells, leptin stimulates cellular proliferation, tgf-1 synthesis, and type iv collagen production [36, 39]. In mesangial cells, leptin upregulates synthesis of the tgf- type ii receptor, but not tgf-1, and stimulates glucose transport and type i collagen production through signal transduction pathways involving phosphatidylinositol-3-kinase . However, both those cell types increase their expression of extracellular matrix in response to leptin . Transgenic mice with leptin overexpression demonstrated an increase in collagen type iv and fibronectin mrna in the kidney . Leptin is involved in the development of glomerulosclerosis through a paracrine tgf- pathway (between glomerular endothelial and mesangial cells) that promotes the deposition of extracellular matrix, proteinuria, and, eventually, glomerulosclerosis . Infusion of leptin into normal rats for 3 weeks fosters the development of focal glomerulosclerosis and proteinuria . Leptin also has proinflammatory actions through its interaction with mediators of innate and adaptive immunity and crp . Leptin regulates components of innate and adaptive immunity, including t lymphocytes and monocytes / macrophages [42, 43]. Central leptin administration in ob / ob mice accelerates renal macrophage infiltration through the melanocortin system . Leptin stimulates central t - cell production and a peripheral shift in favor of t helper (th) 1 adaptive immune responses (proinflammatory) as opposed to th2 responses (anti - inflammatory). Leptin has been shown to modulate adaptive immunity by enhancing t - cell survival and stimulating production of proinflammatory cytokines such as ifn- and il-2 . Leptin also has structural and functional resemblance to proinflammatory cytokines, such as il-6, and may modulate crp, a leptin - interacting protein . Therefore, these direct and indirect effects of leptin on the kidney, including stimulating cellular proliferation and hypertrophy, increasing extracellular matrix expression, and exhibiting proinflammatory activities, may partially explain obesity - related kidney disease . Adiponectin is a 30 kda adipocyte - derived protein hormone encoded by the adipose most abundant gene transcript 1 (apm1), which plays a role in the suppression of inflammation - associated metabolic disorders . Adiponectin is highly abundant in human serum, but its level is decreased in most obese animal and human subjects, particularly in those with visceral obesity [4951]. Recent clinical studies show a negative association of adiponectin in obese patients, [52, 53] suggesting that adiponectin may play a key role in the development of obesity - related albuminuria and alteration of renal function . Studies with the adiponectin knockout mouse provide evidence that adiponectin can regulate podocyte function and thus contribute to the initial development of albuminuria [37, 53]. Sharma et al . Showed that knockout of adiponectin in mice increased albuminuria and caused fusion of podocyte foot processes . In cell culture studies with podocytes, (zo-1) to the plasma membrane, and reduced the renal predominant nadph oxidase nox 4, largely via a 5-amp - activated - protein kinase- (ampk-) dependent pathway . Treatment of the adiponectin knockout mice with exogenous adiponectin was able to decrease albuminuria and improve podocyte morphology . Chronic hyperadiponectinemia significantly alleviated the progression of proteinuria in early - stage diabetic nephropathy by several mechanisms . It led to an increase in nephrin expression, improvement of the endothelial dysfunction due to decreases in endothelin 1 (et-1) and plasminogen activator inhibitor 1 (pai-1), and an increase in endothelial nitric oxide synthase (enos) expression in the renal cortex . Recent studies suggest that adiponectin exerts anti - inflammatory effects by suppressing tnf--induced activation of nuclear factor-b (nf-b) in human aortic endothelial cells and aortic smooth muscle cells through inhibition of ib phosphorylation [55, 56] and inhibition of vascular cell adhesion molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1) expression, thereby reducing monocyte adhesion and macrophage - induced cytokine production and crp expression in human adipose tissue . Human adipose tissue expressed crp, which was negatively correlated with adiponectin expression in adipose tissue . Low levels of adiponectin are associated with higher levels of highly - sensitive c - reactive protein (hs - crp) and il-6, two inflammatory mediators that are involved in the initiation and progression of atherosclerosis and renal disease . Therefore, hypoadiponectinemia contributes to development of a low - grade systemic chronic inflammation state, suggesting that hypoadiponectinemia may play a causative role in the systemic and vascular inflammation commonly found in obesity and obesity - related disorders, including renal injury, through its proinflammatory effects . Resistin, also known as adipocyte - specific secretory factor (adsf) or as found in inflammatory zone (fizz), is a cysteine - rich 12.5-kda polypeptide that belongs to a small family called resistin - like molecules (relms) [60, 61]. In rodents, resistin is secreted from white adipocytes [62, 63]. In human, it is produced largely by macrophages and expressed in adipose tissue predominantly by nonadipocyte resident inflammatory cells [6466]. Current evidence suggests that resistin has been variably associated with obesity, insulin resistance, inflammation, and renal dysfunction . Resistin levels are elevated in both genetic and diet - induced animal models of obesity [62, 67]. Studies of obese subjects have frequently noted higher serum levels of resistin as well as direct correlations between resistin level and adiposity as measured by bmi [68, 69]. There has been a link between circulating resistin and low - grade inflammation that accompany obesity . Resistin is associated with elevated crp and white blood cells, suggesting that the role of resistin may be a component of obesity - related inflammation . It has recently been found that resistin involves in the regulation of proinflammatory cytokine expression . Resistin strongly upregulates il-6 and tnf- in human peripheral blood mononuclear cells (pbmcs) via nf-b pathway . Human resistin enhanced secretion of proinflammatory cytokines, tnf- and il-12 in macrophages by nf-b - dependent pathway . Studies also show that increased levels of resistin in patients with ckd are associated with declined renal function and inflammation [73, 74]. This suggests that resistin may play an important role in obesity and obesity - associated disease by triggering the release of other proinflammatory cytokines . A growing body of evidence indicates that obesity - related glomerulopathy is associated with upregulation of inflammatory mediators . Obesity leads to adipose tissue macrophage infiltration in white adipose tissue and increased levels in proinflammatory cytokines . Several inflammatory mediators released from adipocytes and macrophages, such as tnf-, il-6, il-1, crp, monocyte chemoattractant protein 1 (mcp-1), pai-1, and mif, contribute to a low level of chronic inflammatory state in obesity and may be responsible for renal injury in obesity - associated glomerulopathy . An emerging pattern of gene expression was observed in adipose tissue in mice fed high fat - fed diets, indicating a shift toward global upregulation of inflammatory genes, including tnf-, il-6, and mcp-1 . Tnf-, a proinflammatory cytokine, is predominantly produced by macrophages infiltrating adipose tissue [77, 78] and can also be produced by the kidney . This cytokine is involved in the genesis of inflammation and contributes to obesity - associated insulin resistance [8082]. Within the kidney, angii, advanced glycation end - products (ages), and oxidized low - density lipoprotein (ldl) a recent study demonstrates that tnf- reduces the expression of klotho, a protein expressed by renal cells, through an nf-b - dependent mechanism, which contribute to renal injury . Tnf- enhances the expression of pai-1 in human adipose tissue and plasma pai-1 levels in obesity subjects and is responsible for reduced fibrinolysis and also a component of extracellular matrix, leading to renal fibrosis and terminal renal failure [8890]. Tnf- also has been shown to induce the expression of mcp-1 via p38 mitogen - activated protein kinase (mapk) signaling pathway in renal mesangial cells . Mcp-1, a key regulator in recruiting monocytes to the glomeruli, may also contribute to renal damage at a later stage of kidney disease in obesity . Il-6 is another important proinflammatory mediator systemically secreted from adipose tissue and locally produced in the kidney . Studies have demonstrated the positive relationship between bmi and plasma il-6 concentrations [9294]. Studies also suggest that il-6 plays a key role in the development of renal disease . Endogenous il-6 enhances the degree of renal injury, dysfunction, and inflammation caused by ischemia / reperfusion by promoting the expression of adhesion molecules and subsequent oxidative stress . Transgenic knockout of il-6 ameliorates renal injury as measured by serum creatinine and histology . Blocking the il-6 receptor prevents progression of proteinuria and renal lipid deposit as well as the mesangial cell proliferation associated with severe hyperlipoproteinemia . Il-6 also stimulates the synthesis of crp, which is well known as both a marker and important risk factor of atherosclerosis in the general population and ckd patients . Mif was initially described as an immunomodulatory factor isolated from the supernatants of t lymphocytes and was found to inhibit the random migration of macrophages . Subsequent studies have indicated that mif acts as a proinflammatory cytokine and pituitary - derived hormone that potentiates endotoxemia . Immunologically induced crescentic antiglomerular basement membrane glomerulonephritis, treatment with anti - mif antibody reduced proteinuria, prevented the loss of renal function, attenuated histological damage including glomerular crescent formation, inhibited renal leukocytic infiltration and activation, and reduced il-1 expression by both intrinsic kidney cells and macrophages . Thus, mif is a key mediator of the inflammatory and immune response and plays a pathological role in immune - mediated renal injury . Oxidative stress is caused by an imbalance between increased production of reactive oxygen species (ros) and/or reduced antioxidant activity, leading to oxidative damage to cells or tissue including lipids, proteins and dna . It is known that oxidative stress is involved in pathological processes of various diseases, such as cancer, diabetes mellitus, hypertension, and cardiovascular disease . Studies have suggested that obesity is associated with increased oxidative stress [103, 104]. Analysis of oxidative markers in obesity subjects indicates that oxidative damage is associated with increased bmi and percentage of body fat [105, 106]. Conversely, parameters of antioxidant capacity are inversely related to the amount of body fat and central obesity [107, 108]. The possible mechanisms of obesity - related oxidative stress include increased oxygen consumption and subsequent production of free radicals derived from the increase in mitochondrial respiration, diminished antioxidant capacity, fatty acid oxidation, lipid oxidizability, and cell injury causing increased rates of free radical formation [104, 109, 110]. It is also reported that the increase in obesity - associated oxidative stress is due to the presence of excessive adipose tissue accumulation . Accumulated adipose tissue generates an immune response leading to the secretion of proinflammatory cytokines, including tnf-, il-1, and il-6, which lead to increased generation of ros . Excessive fat accumulation also stimulates nicotinamide adenine dinucleotide phosphate (nadph) oxidase activity, which contributes to ros production . Ros, in return, augmented the expressions of nadph oxidase (nox) subunits, including nox4 and pu.1 in adipocytes, establishing a vicious cycle that augments oxidative stress in white adipose tissue and blood . Oxidative stress in adipocyte seems to be responsible for the low - grade proinflammatory state commonly observed in obesity [113, 114]. Oxidative stress is increasingly viewed as a major upstream component in cell - signaling cascades involved in inflammatory responses, stimulating the expression of proinflammatory cytokines . Ros activate redox - sensitive transcription factors, particularly nf-b, inducing the release of proinflammatory cytokines and the expression of adhesion molecules and growth factors, including tnf-, il-6, il-1, tgf-1, connective tissue growth factor, igf-1, platelet - derived growth factor, and vcam-1 [115, 116]. H2o2 stimulates il-4 and il-6 gene expression and cytokine secretion by an apurinic / apyrimidinic - endonuclease / redox - factor-1- (ape / ref-1-) dependent pathway . Ros increased the expression levels of pai-1, il-6, and mcp-1 through nadph oxidase pathway . Oxidized high - density lipoprotein (hdl) enhances proinflammatory properties such as tnf- and mcp-1 in renal mesangial cells partly via cd36 and ldl receptor-1 and via mapk and nf-b pathways . Oxidized ldl can stimulate tnf- synthesis from renal cells and initiate local effects of renal damage . Increased ros production and mcp-1 secretion from accumulated fat may cause infiltration of macrophages and inflammation in adipose tissue of obesity . Moreover, enhanced macrophage migration induces the release of proinflammatory cytokines, which further stimulates the generation of ros [119, 120]. Therefore, oxidative stress - induced cytokine production is likely to further increase oxidative stress levels, setting a vicious cycle that may promote the progression of kidney damage in obesity . Oxidative stress has been commonly identified in obesity - related renal diseases and may be the mechanism underlying the initiation or progression of renal injury in obesity [122, 123]. Previous studies suggest that oxidative stress triggers, at an early age, the onset of kidney lesions and functional impairment in zucker obese (zo) fa / fa rats, a good model of obesity - related renal disease, in absence of hyperglycaemia, hypertension, and inflammation . Ros are highly reactive molecules that oxidize lipids and proteins, cause cellular injury, and promote glomerular and renal tubule injury and associated proteinuria . Ros are produced by various cells, such as vascular cells, inflammatory cells, and renal cells, and have distinct function on different types of cells, such as endothelial dysfunction, inflammatory gene expression, and renal tubule ion transport . A major source for vascular and renal ros is a nox family of nonphagocytic nad(p)h oxidases, including the prototypic nox2 homolog - based nad(p)h oxidase, as well as other nad(p)h oxidases, such as nox1 and nox4 . Numerous reports indicate that within the kidney, nad(p)h oxidase, an enzyme that produces superoxide (o2) by transferring electrons from nadh / nadph to molecular oxygen and thereby forming o2, h, and nad / nadp, is capable of modulating renal epithelial ion transport [130, 131]. Nap(d)h oxidase - derived ros can alter renal pressure natriuresis and blood pressure regulation through its effects on renal hemodynamics and renal tubular sodium transport . Recent data suggest that nadph oxidase - mediated oxidative injury to the proximal tubule, like that seen in the glomerulus, contributes to proteinuria in insulin - resistant states . Oxidative stress also plays an important role in the pathogenesis of renal damage through its effects on vascular biology . Ros are generated by all types of vascular cells, including endothelial, smooth muscle, and adventitial cells . Ros influence vascular cell growth, migration, proliferation, and activation [132, 133]. Physiologically, ros can mediate cellular function, receptor signals, and immune responses on vascular cells . In pathophysiological condition, ros contribute to progressive vascular dysfunction and remodeling through oxidative damage caused by decreased nitric oxide (no) bioavailability, impaired endothelium - dependent vasodilatation and endothelial cell growth, apoptosis or anoikis, endothelial cell migration, and activation of adhesion molecules and inflammatory reactions [134, 135]. Obesity accelerates the progression of renal injury, associated with augmented inflammation in adipose and kidney tissues . Studies in il-6 transgenic mice suggested that high concentrations of il-6 contribute to development of renal injury . Treatment with anti - il-6 receptor antibody mr16 - 1 prevented progression of proteinuria, renal lipid deposit, and the mesangial cell proliferation in hypercholesterolemia - induced renal injury . Inhibition of tnf- by etanercept, a tnf- antagonist, also decreased blood pressure and protected the kidney through reduction of renal nf-b, oxidative stress, and inflammation . Tnf- blockade increases renal cyp2c23 expression and slows the progression of renal damage in salt - sensitive hypertension . Tnf- inhibition also reduces renal injury in deoxycorticosterone - acetate- (doca-) salt hypertensive rats via suppression of renal cortical nf-b activity . Treatment of adiponectin - knockout mice with adenovirus - mediated adiponectin results in amelioration of albuminuria, glomerular hypertrophy, and tubulointerstitial fibrosis and reduces the elevated levels of vcam-1, mcp-1, tnf-, tgf-1, collagen type i / iii, and nadph oxidase components . Excessive fat accumulation contributes to macrophage infiltration in adipose tissue and increased production of proinflammatory cytokines, such as tnf-, il-8, and il-6 [142145]. Consequently, it is possible that weight loss may be a potential method to reduce inflammation . Evidence indicates that weight loss induced by nutritional intervention or gastric surgery markedly improves the systemic and adipose tissue inflammatory states linked to obesity [143, 146, 147]. Studies by gene profiling analysis have shown that caloric restriction - induced weight reduction leads to the regulation of a wide variety of inflammation - related molecules in human adipose tissue . Weight loss globally improves the inflammatory profile of obese subjects through a decrease of proinflammatory factors and an increase of anti - inflammatory molecules in white adipose tissue . Roux - en - y - gastric - bypass- (rygb-) induced weight loss has been shown to reduce mcp-1, il-18, il-6, and tnf- concentrations [149, 150]. A longer - term weight reduction induced by rygb in corpulence also prevails in regulating circulating cytokine concentrations . Weight loss also ameliorates the low - grade inflammation state that leads to glomerular dysfunction in obesity . In morbidly obese individuals with glomerular hyperfiltration, weight loss by surgical interventions normalizes glomerular filtration rate (gfr) and reduces blood pressure and microalbuminuria . Weight loss improves renal function as shown by reduced levels of serum creatinine and improved creatinine clearance . Since ros play a key role in the pathogenesis of renal injury such as glomerulosclerosis and tubulointerstitial fibrosis, approaches to reduce oxidative stress by antioxidants supplementation, nutritional and surgical interventions may have renoprotective effects . Garcinia protects against obesity - induced nephropathy by attenuating oxidative stress through reduced lipid peroxidation and levels of oxidized ldl . The obese zucker rat is a good model for studying obesity - related kidney disease because it develops proteinuria, glomerular hypertrophy, and focal segmental glomerulosclerosis [155157]. Using these rats, it has been demonstrated that nephropathy is associated with oxidative stress, and supplementation with an antioxidant ebselen improved kidney damage by ameliorating proteinuria and renal focal and segmental sclerosis . Chronic ebselen therapy also improved vasculopathy with lipid deposits, tubulointerstitial scarring, and inflammation . For example, administration of grape seed proanthocyanidin extract (gspe), an efficient phytochemical antioxidant, can protect against the nephrotoxicity effects induced by cisplatin and gentamicin [159, 160] and reverse experimental myoglobinuric acute renal failure . Quercetin, a flavonoid that exhibits antioxidant properties in many diseases, could also protect the rat kidney against lead - induced injury and improve renal function . Thus, antioxidants may be a potential therapeutic to prevent the renal damage in org . Nutritional and surgical interventions are additional approaches to reduce oxidative stress and prevent kidney injury in obesity . Caloric restriction and protein restriction reduce free radicals and ros formation and inhibit accumulation of oxidative biomarkers in animal models . In genetically obese animals, diet restriction can prevent or greatly delay the onset of specific degenerative lesions, in particular glomerulonephritis associated with obesity . Since adipose tissue mass in obesity contributes to oxidative stress, bariatric surgery - induced weight loss also results in decreasing systemic oxidative stress in adiposity . Weight loss induced by diet restriction or bariatric surgery not only improves inflammation state but also reduces oxidative stress state in obesity, which may protect renal function in obesity - related glomerulopathy . Obesity causes chronic low - grade inflammation and systemic and local oxidative stress, which may play a pivotal role in the initiation or progression of obesity - associated glomerulopathy . Elevated inflammation in obesity is the result of the production of adipokines and increased inflammatory cytokines and decreased anti - inflammatory factors . Oxidative stress is triggered by an imbalance between increased production of ros and/or reduced antioxidant activity . Both inflammation and oxidative stress induce damage to renal tubule and glomerulus and result in endothelial dysfunction in the kidney . Therefore, anti - inflammation and antioxidant interventions may be the potential therapies to prevent and treat obesity - related renal diseases.
All chemicals and reagents were of high performance liquid chromatography (hplc) or analytical grade . Pyrrole (99%), thiophene (99%), and tetrabutylammonium perchlorate (98%) were purchased from sigma - aldrich (schnelldorf, germany). Medical steel wires (ni cr, = 750 m) were purchased from b. braun surgical s.a . Polypyrrole (ppy) and polythiophene (pth) coatings were synthesized by electropolymerization with a linear sweep voltammetry technique . The three - electrode cell was filled with electrolyte solution consisting of 100 mm pyrrole / thiophene (fig . 4) and 250 mm tetrabutylammonium perchlorate in acetonitrile.fig . 4chemical structures of ppy (a) and pth (b) spme fiber coatings chemical structures of ppy (a) and pth (b) spme fiber coatings polymerization was performed using medical steel wires as working electrodes . An ag / ag electrode was applied as a reference electrode, with platinum net bent into a cylinder as a counterelectrode . For ppy, a potential range from 0.2 to + 2.5 v and seven scans second, an additional 14 scans in the higher potential range from 0.2 to 2.7 v were used (fig . 5electropolymerization of pyrrole (a) and thiophene (b) electropolymerization of pyrrole (a) and thiophene (b) the morphologies of the polypyrrole and polythiophene fibers were investigated using a scanning electron microscope (leo 1430vp; carl zeiss smt, oberkochen, germany) coupled with a backscattered electron (bse) detector . Small angle x - ray scattering (saxs) data were collected using a nanostar system (bruker axs gmbh, karlsruhe, germany) with pinhole collimation and a two - dimensional hi star detector with resolution of 1,024 1,024 pixels, mounted on an x - ray tube with a copper anode and equipped with crossed gbel focusing mirrors . Samples were mounted between two mica windows in sample holders with thickness of 1 or 2 mm . The sample - to - detector distance was 650 mm, and the exposure time for a single frame was 10,000 s. each sample was measured three times, and the obtained data were averaged . The saxs data were recorded within the scattering vector range of 0.15 nm <s <3.5 nm (where s = 4sin/, 2 is the scattering angle, and is the x - ray wavelength). The saxs data were corrected for the detector response and normalized to the intensity of the incident beam, and the background scattering (empty holder) was subtracted using the saxs_nt v4.1 program package (bruker axs gmbh, karlsruhe, germany). Electropolymerization was performed using a homemade electrochemical cell coupled with a high performance potentiostat galvanostat (pgstat128n series; metrohm - autolab b.v ., all chemicals and reagents were of high performance liquid chromatography (hplc) or analytical grade . Pyrrole (99%), thiophene (99%), and tetrabutylammonium perchlorate (98%) were purchased from sigma - aldrich (schnelldorf, germany). Medical steel wires (ni cr, = 750 m) were purchased from b. braun surgical s.a . Polypyrrole (ppy) and polythiophene (pth) coatings were synthesized by electropolymerization with a linear sweep voltammetry technique . The three - electrode cell was filled with electrolyte solution consisting of 100 mm pyrrole / thiophene (fig . 4) and 250 mm tetrabutylammonium perchlorate in acetonitrile.fig . 4chemical structures of ppy (a) and pth (b) spme fiber coatings chemical structures of ppy (a) and pth (b) spme fiber coatings polymerization was performed using medical steel wires as working electrodes . An ag / ag electrode was applied as a reference electrode, with platinum net bent into a cylinder as a counterelectrode . For ppy, a potential range from 0.2 to + 2.5 v and seven scans second, an additional 14 scans in the higher potential range from 0.2 to 2.7 v were used (fig . 5electropolymerization of pyrrole (a) and thiophene (b) electropolymerization of pyrrole (a) and thiophene (b) the morphologies of the polypyrrole and polythiophene fibers were investigated using a scanning electron microscope (leo 1430vp; carl zeiss smt, oberkochen, germany) coupled with a backscattered electron (bse) detector . Small angle x - ray scattering (saxs) data were collected using a nanostar system (bruker axs gmbh, karlsruhe, germany) with pinhole collimation and a two - dimensional hi star detector with resolution of 1,024 1,024 pixels, mounted on an x - ray tube with a copper anode and equipped with crossed gbel focusing mirrors . Samples were mounted between two mica windows in sample holders with thickness of 1 or 2 mm . The sample - to - detector distance was 650 mm, and the exposure time for a single frame was 10,000 s. each sample was measured three times, and the obtained data were averaged . The saxs data were recorded within the scattering vector range of 0.15 nm <s <3.5 nm (where s = 4sin/, 2 is the scattering angle, and is the x - ray wavelength). The saxs data were corrected for the detector response and normalized to the intensity of the incident beam, and the background scattering (empty holder) was subtracted using the saxs_nt v4.1 program package (bruker axs gmbh, karlsruhe, germany).
The maxillary anterior teeth should be positioned as close as possible to the positions originally occupied by natural teeth to achieve a natural appearance in making complete dentures.1 it is necessary to refer to anatomic landmarks to achieve this goal.2 the incisive papilla is a small, pear shaped eminence composed of a pad of fibrous connective tissue overlying bony exit of nasopalatine blood vessels - nerves and one of the significant anatomical landmark in locating maxillary anterior central incisors position in complete denture fabrication procedure . Harper3 stated that the incisive papilla was stable that was obtained by caliper measurements on pre - extraction and post - resorption models of the same cases over seven years . Although the shape and the localization of the papilla shows a wide range of variation,58 the centre of the papilla is commonly used as a reference point in denture construction and studies.24,915 when artificial teeth is set in proper positions, which were determined from the incisive papilla, the foundation is correctly laid for natural speech, pleasing appearances and normal function.3 the horizontal relationship between incisive papilla and maxillary central incisors has been already investigated by several authors . Harper3 suggested that the incisal edges of the maxillary central incisors should be 5 to 8 mm at the horizontal direction in front of the centre of the papilla . Ortman and tsao6 stated that the most anterior part of the maxillary central incisors and the posterior of the incisive papilla was 12.45 mm . Many studies showed that incisal edges of the maxillary central incisors were positioned 8 to 10 mm in front of the centre of the incisive papilla.2,4,68,1020 another anatomical landmark that have been suggested in complete denture construction is pterygomaxillary notch - incisive papilla plane.21 fu et al22 found that the pterygomaxillary notch - incisive papilla plane tends to be more parallel to the occlusal plane that uses mesio - labial incisal edge of maxillary right central incisor as anterior reference point and mesio - buccal cusp tips of maxillary first or second molars as posterior reference point . Although, several studies investigated the horizontal relationship between incisive papilla and the maxillary central incisors,24,10,11,16,17,20,21,2325 the authors did not find specific information in the literature relative to vertical distance between the incisive papilla and the maxillary central incisors . Thus, the aim of the present study was to determine the vertical distance between the maxillary central incisors and centre of the incisive papilla . Dental student volunteers from suleyman demirel university faculty of dentistry were solicited by a written announcement to participate in the study . The inclusion criteria were: no restoration and tooth loss on the upper jaw, well - arranged maxillary anterior teeth, no orthodontic treatment, angle class i maxillomandibular relationship, no pathology that affects the dentition or the surface texture and shape of teeth, no incisal wear on the dentition and no gummy smile in the subjects . The volunteers were examined by one of the investigators of the study and one hundred volunteers (58 women, 42 men) were selected from the students that have the inclusion criteria with a draw . The ages of the subjects were ranged from 19 to 22 years, with a mean age of 20.371.1 years . Upper jaw impressions were taken on the subjects by using stock impression trays (teknik dental rostfrei, istanbul, turkey) and irreversible hydrocolloid impression material (tulip, cavex holland, haarlem, holland). In order to correctly register the incisive papilla and reduce soft tissue distortion the stone casts were obtained using ada type iii dental stone (gilidur, fachbereich dental, ludwigshafen, germany). Each stone cast was trimmed following the same procedures to produce a flat base that was parallel to the occlusal plane (anterior reference point: mesio - labial incisal edge of upper right central incisors; posterior reference points: mesio - buccal cusp tips of upper first molars). Two mm standard transparent resin plate and a spirit level (veto, istanbul, turkey) were used to achieve the parallelism of the described occlusal plane to the base of the stone casts . The centre of the incisive papilla was found measuring the planar distance between the most anterior and posterior borders of the incisive papilla that was on the median line of the palate using a digital caliper which had a precision level at 0.01 mm (mitutoyo corporation, tokyo, japan) (figure 1) and halving this distance . The midpoints of the incisive papilla were marked on each stone cast (figure 2). A digital caliper that was fixed perpendicular to the horizontal bar of a surveyor (rotaks dent a.s ., istanbul, turkey) was used as a measurement instrument (figure 3). Stone casts were mounted to the surveyor by the help of a spirit level to obtain a certain level in the horizontal plane . The midpoint of the flat edge panel of the depth caliper was marked with graphite . The flat edge plane of the depth caliper had contacted to the mesial incisal edges of the maxillary central incisors and the midpoint of the flat edge plane of the depth caliper was matched with the midpoint of the maxillary central incisors (figure 4). The pin of the depth caliper was touched to the midpoint mark of the incisive papilla . The mounted casts were moved by sliding and the first measurement accepted as a starting point . The measurements were done by two investigators using the same instrument at two separate times . The average values of two investigators measurements were calculated and analyzed with spss 13.0 for windows (spss inc ., the kappa scores for the assessment of intra and inter observer agreement were higher than 0.75 which implies substantial agreement between the observers (tables 1 and 2). The mean vertical distance between maxillary central incisors and midpoint of the incisive papilla on the stone casts was 6.700.81 mm . A common average vertical position of the maxillary anterior teeth to the constant landmarks has been found by measuring lots of casts of natural healthy teeth . It is important to orient the stone casts in a standardized manner during measurements so that the results achieved can be applied when artificial teeth are being set in complete denture bases . The occlusal plane is a common reference for mounting stone casts and in general, occlusal plane done parallel to the bases of stone cast in standardize manner . Fu et al22 found the pterygomaxillary notch - incisive - papilla plane (hip) tends to be parallel to the natural occlusal plane that defined as anterior reference point was mesio - labial incisal edge of upper right central incisor and posterior reference points were mesio - buccal cusp tips of upper first or second molars . In the present study, a standard transparent resin sheet was positioned in contact with the maxillary right central incisors and the mesio - buccal cusp tips of first molars to orient the stone cast to the standardized occlusal plane . This occlusal plane orientation was used, for it is more comparable to the pterygomaxillary notch - incisive - papilla occlusal plane.22 harper3 stated that the incisive papilla is a dependable basis for reproducing the horizontal and vertical position of the maxillary central incisors . Watt9 stated that the anterior portion of the maxilla undergoes extensive resorptive changes following tooth removal and the anatomic location of the incisive papilla can be used only as a guide in the setting of denture teeth . Grave16 suggested that using the border of the incisive papilla as the part likely to be least affected by the changes in anatomy of the region . In the previous studies, the midpoint of the incisive papilla has been used as a reference point for tooth arrangement . Most of the previous studies used the midpoint of the incisive papilla as a reference point.2,4,1015,25,26 the midpoint of the incisive papilla has also been used as a reference point for tooth arrangement and occlusion rims in contemporary dental practice . Pterygomaxillary notch - incisive - papilla occlusal plane also uses the centre of the incisive papilla as anterior reference point.22 for these reasons; the centre of the incisive papilla was used as a reference point for measurements in the present study . Ortman and tsao6 stated that the most anterior part of the maxillary central incisors and the posterior of the incisive papilla was 12.45 mm . The measurement methods of all these studies were in 2-dimensional, however park et al28 found similar results (11.961.37) in their 3-dimensional measurement on a virtual model between the maxillary anterior teeth and incisive papilla . The results of these studies that used 2 and 3 dimensional measuring methods show that a sensitive measuring method is reliable and can be repeatable . Chalsuthipan and boonsiri29 investigated the relationship between the incisive papilla, maxillary central incisors and canines in thai population . The authors found that the vertical distance from the most distal point of the incisive papilla to incisal edge of the central incisors was ranged from 6.94 to 7.23 mm with the mean of 7.08 mm.29 the present study supports the findings of chalsuthipan and boonsiri.29 the mean value difference between chalsuthipan and boonsiri s study29 and the present study was 0.38 mm . The mean value variation may be caused by the reference point differences on the incisive papilla because the authors used the most posterior border of the incisive papilla . The most posterior part of incisive papilla is the most far from the occlusal plane and maxillary central incisors . On the anterior region of the maxilla, the vertical relationship between incisive papilla and central incisors can be used in determination of occlusal plane with the reference of other anatomical landmarks together; however the clinical applicability of this relationship in complete denture construction needs further investigation . This study investigated the vertical distance between maxillary central incisors and centre of incisive papilla . The dentist and laboratory technician can be use the results of this study as a guideline in fabrication of maxillary occlusal rims and in determination of the position of the occlusal plane with the reference of other anatomical landmarks together . However, the wax rim should be modified intraorally to incorporate individual characteristics, and the anterior tooth should be arranged on modified wax rim . Also, further investigations are needed in different age, gender, race, dental and skeletal morphology groups.
In recent years, nanoparticles have been used increasingly for biomedical applications, including drug or gene delivery, imaging, sensing, or photothermal therapy . In particular, gold nanoparticles (nps) have been suggested as highly useful sensitizing agents in phototherapy due to their unique size and shape - dependent optical properties, high absorption coefficients, ease of synthesis, biocompatibility, and their ability to hold a variety of functional ligands . It is well - known that citrate - stabilized gold nps are endocytosed by cells and remain in intracellular vesicles . Moreover, targeting of specific sites inside the cell by functionalization of the surface with cell penetrating peptides or peptides containing nuclear localization sequences has been reported . Alternatively, to selectively target specific types of cells, gold nps can be modified with suitable antibodies . This approach has been studied for potential use in photothermal cancer therapy, where cancer cells overexpressing human epidermal growth factor receptor 2 (her2) or epithelial growth factor receptor (egfr) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies, respectively . The nps were then irradiated with light within their plasmon resonance absorbance band to heat the cells to temperatures leading to cell death . Most interestingly, it has also been demonstrated that endocytosis of gold nps by cancer cells and subsequent irradiation of such intracellular nps can lead to cell death even at irradiation levels that are not high enough to cause significant heating . This nonthermal route to laser - induced cell death has been ascribed to an as yet not fully characterized photochemical reaction, although irradiation of endocytosed nps was reported to be accompanied by increased levels of reactive oxygen species . The use of such a photochemical mechanism could be of great advantage in situations where different types of cells coexist in close vicinity, since it would allow for more selective targeting of particular cells, whereas due to the fast diffusion of heat over the relevant length scales photothermally induced cell death will affect all cells within the irradiated volume more or less indiscriminately, as long as some of them contain nps . Irradiation of gold nps with continuous wave (cw) lamp or laser light has been shown to lead to photogeneration of singlet oxygen (o2) in vitro, suggesting that this highly reactive species, which is widely used in photodynamic therapy, may be involved in the photochemical pathway of cell killing by gold nps . In vitroo2 photogeneration by irradiation of spherical gold nps with short laser pulses or cw laser light at comparable powers and intensities has also been reported . In this study, it was suggested that the mechanism of singlet oxygen photogeneration may involve hot electrons, i.e., the highly excited conduction band electrons which upon absorption of a short laser pulse by a np can reach quasi - equilibrated energy distributions corresponding to temperatures of several thousand degrees . This seems somewhat surprising, since cw light at the intensities used does not yield hot here we present new experimental results on the photogeneration of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light, as well as theoretical work pertaining to the underlying mechanism(s), which so far had not been addressed . We show that electron temperatures in excess of 2000 c are easily achieved in pulsed laser irradiation experiments, whereas cw light under similar conditions yields electronic temperatures of at most 10 c above room temperature . Thus, the photogeneration of o2 by gold nps proceeds by different mechanisms under different irradiation conditions; the implications for the further development of medical applications of the effect are discussed in detail . Furthermore, we also found that even a moderately thick, but dense, ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface, which also has important consequences for practical applications . Citrate - stabilized spherical gold nps with 15 and 46 nm diameter were prepared according to the turkevich frens and a seeded growth method, respectively . Gold nanorods (nrods) were synthesized using the seeded - growth method reported by dickerson et al . With slight modifications . More details of the np preparation are provided in the supporting information . Nps were characterized using uv vis spectroscopy (genesys 10 uv), differential centrifugal sedimentation (cps instruments dc24000), and transmission electron microscopy (tem, fei tecnai spirit microscope at 120 kv) (see figures s1 and s2 in the supporting information). The uv vis absorbance spectrum of nrods showed a transverse plasmon resonance band at 522 nm, and a longitudinal plasmon resonance band at 798 nm and tem revealed nrods to have a length of 40 nm and a diameter of 12 nm . Functionalization with thiolated peg ligands or peptides was achieved by overnight incubation with excess ligand, followed by repeated centrifugation for excess ligand removal . The capping ligands used here were peg - oh (hs-(ch2)11-(eg)4-oh), mpeg5000 (hs-(ch2)2-(eg)n - o - ch3, average mw 5000 g mol) and peptide c - tat (primary sequence calnnagrkkrrqrrr); see figure s3 for the structures of the peg ligands . O2 was detected via the bleaching of 1,3-diphenylisobenzofuran (dpbf), which is widely used for this purpose . All experiments involving dpbf were carried out in the dark . Because dpbf is not soluble in neat water, all experiments were conducted in 50/50 (v / v) mixtures of water and ethanol . A fresh solution of dpbf (3.1 mg, 0.115 mm, a412 nm = 2) in etoh (100 ml) was kept stirring in the dark . A 10 mm quartz cuvette and a 3 mm stirrer bar were left in aqua regia (1:3 hno3:hcl) for 15 min and thoroughly rinsed multiple times with milli - q water (mq h2o) and etoh . In the clean cuvette with stirrer bar, either mq h2o (600 l) or np solution (600 l) was mixed with the ethanolic dye solution (600 l). Where appropriate, the np concentration was adjusted prior to mixing to yield an absorbance of 0.4 at 532 nm in the final solution . The cuvette was sealed with an airtight lid and parafilm, and the uv vis absorbance spectrum was recorded (genesys 10 uv). The sample was then placed on a stirring plate (in the dark), and its absorbance spectrum measured every 10 min for 30 min to ensure the solution was stable . For the irradiation experiments, the cuvette containing 1200 l of sample solution was placed on a stirring plate in front of the laser . For experiments with nps, the cuvette was fitted with heat fins using non - silicone heat transfer paste and cooled using a fan . No significant increase of the cuvette temperature beyond a slight warming was observed . For most experiments, the sample was irradiated at 1000 mw (unless stated otherwise) using a 532 nm continuous - wave diode pumped solid state laser (laser quantum opus 532) with a 1/e beam diameter of 1.85 mm; for some experiments the beam was expanded to 8 mm diameter using a lens, as stated explicitly . The sample was irradiated for 10 min and then removed from the laser setup to record the absorbance spectrum; this was repeated until the sample had been irradiated for 60 min in total . Irradiation of gold nrods in the longitudinal plasmon resonance band at 800 nm was performed in the same setup, but using a titanium: sapphire laser (coherent mira 900), aligned on the auxiliary cavity, which prevents mode - locking and thus provides continuous - wave laser operation at 800 nm with 1000 mw power; the beam was expanded to 3.4 mm using two lenses . Pulsed laser irradiation was performed in the same setup, but using the second harmonic of a q - switched nd: yag laser (quantel brilliant) (532 nm, 5 ns pulse length, 10 hz repetition rate, 3.5 mm beam size, 15 mj pulse energy). For experiments that included nps, the absorbance of dpbf at 412 nm was calculated by subtracting the np absorbance at 412 nm (obtained from neat np samples) from the measured sample absorbance . The concentration of dpbf in the samples always yielded an initial absorbance very close to 1 at 412 nm; for comparative data analysis, the irradiation - time - dependent dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation time . 1,3-diphenylisobenzofuran (dpbf) readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . Dpbf strongly absorbs light at 412 nm (figure 1a), but due to the loss of the -system of isobenzofuran, the product does not absorb light at this wavelength; it is this loss of absorbance that is used to detect the presence of singlet oxygen . Here, dpbf was chosen as singlet oxygen sensor as it has no absorbance at 532 or 800 nm, the wavelengths used for laser irradiation of gold nanoparticles in their plasmon resonance bands, and no photobleaching of dpbf was expected to occur upon irradiation in the absence of nps . Contrary to this expectation, some bleaching was observed under our cw irradiation conditions, with the dpbf absorbance decreasing by ca . 10% upon irradiation at 1 w (37 w cm) for 60 min (figure 1a). As shown in figure 2a, and described in more detail in the supporting information, this photobleaching of dpbf has two phases: (i) a rapid phase, extending over the first 1020 min of irradiation under the conditions used here, and (ii) a slower phase, which on the time scale investigated appears linear with time . We found that phase i depends on laser power, beam size, and the presence of oxygen, whereas the slope of the time dependent bleaching after 20 min, phase ii, is essentially independent of the presence of oxygen and of the beam power / intensity in the range used here (see figure s4 for details). It should be noted that similar effects have been reported previously, with cw irradiation at 514 nm for 60 min at significantly lower powers (40 mw) than employed here, leading to a ca . 5% decrease of the absorbance of dpbf in benzene, although no explanation was suggested in that report . The oxygen dependence of phase i is in agreement with direct photogeneration of singlet oxygen by visible light, which has been suggested to be the reason for dpbf photobleaching upon irradiation with light at wavelengths above 470 nm . However, the slower phase ii is not affected by removing oxygen from the solution (see figure s4b and figure 3) and hence cannot be ascribed to singlet oxygen formation . At the present moment, the mechanism of both phases of dpbf photobleaching remains unclear, although both effects are highly reproducible and bleaching does not occur when the sample is not exposed to light (see figure s4a). Photobleaching of the dpbf absorbance upon cw irradiation at 532 nm, 1 w (37 w cm), in a 50/50 (v / v) mixture of water and ethanol: (a) in the absence of nps and (b) in the presence of 15 nm citrate - stabilized spherical gold nps . Shown are absorbance spectra taken at intervals of 10 min from before the irradiation up to a maximum irradiation time of 60 min; the arrows indicate the direction of change . (a) time dependence of the photobleaching of the dpbf absorbance at 412 nm, a412(dpbf), upon cw irradiation at 532 nm, 1 w (37 w cm), in the absence and presence of citrate - stabilized spherical nps with 15 and 46 nm diameter; shown here are the results from several individual experiments (dashed lines) and the average (solid lines), after subtraction of the np absorbance and normalization to 1 at time zero; see the experimental section for details of data treatment and analysis . (b) effect of the nps alone, calculated by subtracting the photobleaching effect of dpbf in the absence of nps from the results obtained in the presence of nps; the solid lines in (b) are linear fits of the data in the range 2060 min . Gradient of the time - dependent dpbf absorbance photobleaching in the irradiation time window 2060 min for different samples under cw irradiation at 532 nm, 1 w (37 w cm). For experiments in the presence of nps, the np concentration was adjusted to yield an absorbance of 0.4 at 532 nm . Experiments for dpbf in the absence of nps and in the presence of 15 nm spherical nps were also undertaken after bubbling the sample with nitrogen for 10 min, as indicated (+ n2). The error bars correspond to the standard deviation of several repeat experiments, and indicates statistically significant differences with respect to the experiment on dpbf only, as determined by the anova f - test at p <0.001; it should be noted that the results for 15 or 46 nm spherical nps without nitrogen bubbling or a peg capping layer were found to be different to all other results at this statistical significance level . No repeat experiment was undertaken for irradiation of nanorods at 532 nm, but the same result (no additional bleaching in the presence of nanorods) was obtained for irradiation at 800 nm (figure s5). In the presence of citrate - stabilized spherical gold nps, the photobleaching of dpbf upon irradiation at 532 nm, i.e., within the nanoparticle plasmon resonance band, is significantly increased (figure 1b). The time dependence of the dpbf photobleaching in the absence of nps and in the presence of nps with 15 and 46 nm diameter is shown in figure 2a . It can be clearly seen that the presence of nps leads to a significant increase of the dpbf photobleaching effect and that larger nps yield a larger effect although the np absorbance was adjusted to be the same for all samples, so that the same amount of light was absorbed . The additional photobleaching of dpbf caused by the presence of nps can be clearly ascribed to the generation of reactive oxygen species (ros), since purging of the samples with nitrogen removes this effect (see below). Moreover, previous experiments had shown that light irradiation of citrate - stabilized spherical gold nps leads to the characteristic luminescence of o2 at 1280 nm and does not result in the generation of superoxide, o2, or hydroxyl, oh, radicals, which leads us to conclude that the predominant ros species produced here is singlet oxygen, o2 . The fact that significant dpbf photobleaching occurs even in the absence of nps requires careful consideration for the analysis of these data . Like dpbf photobleaching in the absence of nps, the effect in the presence of nps shows two phases . However, subtraction of the effect observed when only the dye is present yields an essentially linear time dependence for the additional effect ascribed to the nps on the time scale of the experiment (figure 2b), indicating that the np - induced photogeneration of o2 is essentially constant over our experimental time interval . Moreover, the second phase of direct dpbf photobleaching is largely independent of the laser power, at least down to 0.1 w, i.e., significantly lower powers than those used for most of the experiments reported here, as shown in detail in the supporting information (figure s4a), and therefore should be independent of the presence of nps whose absorbance leads to a decrease of the laser power along the beam path, allowing a direct comparison of the results obtained in the time frame of 2060 min . Moreover, this slower phase of direct dpbf photobleaching is also independent of the presence of oxygen . For these reasons, only data starting at 20 min irradiation will be used for quantitative comparisons . Figure 3 summarizes the main results obtained here after np photoexcitation at 532 nm . It is very obvious that citrate - stabilized spherical gold nps induce significantly faster dpbf photobleaching than is observed in their absence (black); this is found for 15 nm gold nanoparticles (red) but is even more pronounced for larger nps with 46 nm diameter (blue). Increasing the size of the spherical nps from 15 to 46 nm increases the np - induced dpbf photobleaching (i.e., the additional effect, after subtracting the direct dpbf photobleaching effect) by ca . It is interesting to note that unlike the direct dpbf photobleaching, which is not affected by nitrogen purging, the np - induced additional bleaching is significantly reduced by nitrogen purging, almost down to the level of the direct dpbf photobleaching effect . This strongly supports the conclusion that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching . Furthermore, capping the 15 nm gold nps with peg - oh, a moderately large ligand (figure s3), essentially removes the np - induced bleaching and reduces the observed effect to the level of the direct dpbf photobleaching . Gold nanorods (nrods) with a length of 40 nm and a diameter of 12 nm, which have two plasmon resonance bands, namely the transverse band at 522 nm and the longitudinal one at 798 nm (figure s1a), stabilized with a capping layer consisting of a mixture of a peg polymer (mpeg5000) and a peptide (c - tat) (see the experimental section), were also investigated . Irradiation at 532 nm, i.e., in the transverse plasmon resonance band, showed no additional dpbf bleaching above the direct dpbf effect (figure 3). Irradiation with 1 w (11 w cm) cw laser power at 800 nm, i.e., in the longitudinal plasmon resonance band (figure s5), showed reduced direct dpbf photobleaching compared to irradiation at 532 nm, but again no additional photobleaching was observed in the presence of nrods . These results show that the nrods used here, which have a ligand layer consisting of a mixture of a peg polymer and a peptide, do not induce the formation of o2 upon laser irradiation, independent of the plasmon resonance which is photoexcited . Irradiation at 532 nm in the presence and absence of citrate - stabilized spherical gold nps with 15 nm diameter was also investigated using laser pulses with a pulse duration of 5 ns (figure 4). In this case, the uv vis spectra show a slight broadening of the np plasmon resonance band at 520 nm during the first 5 min of irradiation, suggesting that some aggregation occurs; after the initial 5 min, however, the nps remain stable . Analysis of the spectra shows that photobleaching of dpbf in the absence of gold nps is similar to the results obtained using cw laser irradiation at comparable average powers (figure s4a). In the presence of gold nps with 15 nm diameter, on the other hand, the np - induced bleaching effect (i.e., the effect remaining after subtracting the direct dpbf photobleaching effect) which is induced by pulsed laser irradiation is larger than that caused by cw irradiation by almost 1 order of magnitude, in spite of the significantly lower laser power employed during the pulsed irradiation experiments (0.15 w vs 1 w), which leads to a correspondingly lower number of absorbed photons; compare figure 4b (pulsed irradiation) with figure 3 (cw irradiation). Photobleaching of the dpbf absorbance upon laser irradiation with 5 ns laser pulses at 532 nm, 0.15 w, 10 hz repetition rate (corresponding to a power density of 1.5 w cm and a pulse energy density of 0.15 j cm). (a) absorbance spectra in the presence of citrate - stabilized spherical gold nps with 15 nm diameter, taken at intervals of 5 min from before the irradiation (gray) up to a maximum irradiation time of 30 min; the arrow indicates the direction of change . (b) gradient of the time - dependent dpbf photobleaching (measured at 412 nm) in the irradiation time window of 2030 min in the absence and presence of nps . It is straightforward to estimate the quantum yield of np - induced dpbf photobleaching from the absorbed laser power and the observed rate of absorbance bleach . For the citrate - stabilized spherical nps with 15 and 46 nm diameter under cw irradiation at 532 nm, this yields values of 5 10 and 8 10, respectively . Thus, less than one of each 1 million photons absorbed by a np leads to the photobleaching of a dpbf molecule under cw irradiation . For irradiation of 15 nm nps with 5 ns laser pulses, on the other hand, the quantum yield of np - induced dpbf photobleaching is 3.5 10, i.e., almost 2 orders of magnitude larger than for cw irradiation, but still very small . Diffusion of oxygen over the lifetime of o2 (approximately 6 s in 50/50 water / ethanol) covers a distance larger than the average distance between dpbf molecules at the concentrations used here, and hence it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be detected . 10 m, which is close to the concentration used here, is sufficient to detect 50% of photogenerated o2 . Thus, the observed low quantum yield of dpbf photobleaching indicates a very low quantum yield of o2 photogeneration by gold nps (i.e., number of o2 generated for each photon absorbed), having values of the order of ca . 10 for cw irradiation and ca . 10 for irradiation with nanosecond laser pulses . Our results show that irradiation of citrate - stabilized spherical gold nps at 532 nm, i.e., in their surface plasmon band, with pulsed or cw laser light leads to the production of o2, detected here by monitoring the bleaching of dpbf absorbance . However, the rate of o2 production is much larger when using short laser pulses than when using cw light of comparable intensity . Whereas ca . 24% of the dye is bleached after only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at an average power of 150 mw (figure 4), only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power (1 w) (figure 2). This difference allows one to draw important conclusions on the mechanism of o2 production by irradiation of gold nanoparticles . Irradiation of spherical gold nps at 532 nm leads to the excitation of their plasmon resonance, which can be described as a coherent oscillatory motion of the conduction band electrons; this oscillation dephases and decays on the sub-100 fs time scale, with only a very small radiative contribution, so that most of the excitation energy is retained as electronic excitation in the form of electron hole pairs . Since the photon energy is close to the minimum energy required for direct excitation of d - band electrons into the conduction band of gold, a minor contribution of this excitation mechanism cannot be ruled out; however, d - band holes are filled by conduction band electrons on the 10 fs time scale, yielding essentially the same outcome as excitation of the plasmon resonance band . The excited electrons initially have a nonthermal energy distribution, and often are referred to as primary hot electrons, although the concept of temperature does not strictly apply to such a distribution . They rapidly (within less than 500 fs) equilibrate by electron electron scattering to yield a fermi distribution corresponding to an elevated temperature and can then be referred to as hot electrons . Thiol bond dissociation at the surface of gold nps and have been suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses . They lose their energy on the time scale of a few picoseconds by interaction with the lattice (electron phonon scattering) with coupling times that are essentially size - independent for nps above 10 nm in diameter, although they strongly depend on the amount of energy deposited due to the temperature - dependent electronic heat capacity . Because the lattice heat capacity is much larger than the electronic heat capacity, this leaves the electrons and the lattice in equilibrium at a temperature which is significantly lower than the initial electronic temperature; finally, cooling occurs by heat transfer to the solvent and heat diffusion on the time scale of 10 to a few 100 ps, strongly depending on np size . Since the dissipation of the absorbed energy proceeds on the picosecond time scale, excitation with a nanosecond laser pulse yields a highly nonequilibrium situation during the duration of the laser pulse where energy is continuously deposited into the electronic system and at the same time flows through the lattice into the surrounding solvent . It is therefore not straightforward to predict the temperature of the hot electrons achieved in such experiments . We used the two - temperature model for the electron and phonon heat baths coupled to finite - element heat transfer and diffusion simulations in the surrounding solvent to estimate this temperature; details of these simulations are given in the supporting information . As shown in figure 5, under our conditions the electrons are expected to reach a temperature of 2100 c, whereas the lattice reaches temperatures of the order of 1400 c and the solvent in the immediate vicinity of the np a temperature of about 900 c . Time - dependent temperatures of the conduction band electrons (red), lattice (black), and first solvent layer (blue), calculated for our experiments using nanosecond - laser pulse excitation (15 nm spherical nps in 50/50 etoh / water, 5 ns laser pulses with 0.15 j cm intensity, solid lines) and for the experiments described in ref (24) (40 nm spherical nps in 80/20 etoh / water, 7 ns laser pulses with 0.03 j cm intensity, dashed lines) using the two - temperature model for the electron and phonon heat baths, coupled to finite - element heat transfer and diffusion simulations in the surrounding solvent (see supporting information for details); time zero corresponds to the center of the laser pulse . A lattice temperature of 1400 c, which is above the melting temperature for solid gold, albeit present for only a few nanoseconds, might be sufficient to cause temporary melting of the nanoparticle, although the pulse intensities used here are still below the reported threshold for size reduction of gold nps of 15 nm diameter by nanosecond laser pulses . Nevertheless, a minor effect on the shape and/or size of the nps, especially at the upper end of the size distribution, cannot be ruled out and may be related to the minor np spectral changes observed upon irradiation which suggest some aggregation to occur (see above). Because of the slower heat dissipation around larger nps, these are heated to higher temperatures and hence are more likely to fragment, in agreement with experimental results on nanosecond - laser pulse induced fragmentation, which also show that fragmentation is finished after 5 min under conditions similar to the ones used here . Since our nps were prepared without excess citrate, the resulting increase in the ratio of surface area to volume may indeed cause some aggregation . It also cannot be ruled out that the solvent near the np surface temporarily forms bubbles, although the pulse intensities used here are still below the reported threshold for bubble formation by nanosecond pulses for 15 nm nps . Such bubbles could lead to better thermal insulation, thus potentially increasing the maximum temperatures, but on the other hand, they might prevent oxygen from reaching the np surface, thus reducing the chance of o2 formation . The formation of o2 from the triplet ground state by interaction with a photoexcited sensitizer requires a change of the electron spin and hence cannot result from dipole dipole (frster) interaction, but only from dexter - type electron exchange coupling; the latter can be described as simultaneous transfers of an electron from one of the 2 * molecular orbitals on oxygen to a photogenerated hole on the sensitizer and of an electron with opposite spin from a high - energy excited sensitizer level to the same or the other 2 * orbital, resulting in the formation of the or singlet oxygen state, respectively . This mechanism requires significant overlap of the relevant electronic wave functions and hence only occurs at short distances of at most 10 . Since the photoexcited hot electrons have such a short lifetime, this reaction can only occur if an oxygen molecule happens to be in the vicinity of the np or is temporarily adsorbed to its surface at the moment of excitation . A similar reaction occurs on photoexcited si nanocrystals, although in this case the reaction is more efficient than for au nps due to the much longer lifetime of the photoexcited excitons in si . Thus, the short lifetime of hot electron excitation in gold nanoparticles easily explains the low quantum yield of o2 photogeneration which is observed here . The excitation of electrons to temperatures exceeding 2000 c means that a significant number have sufficient energy to excite an oxygen molecule to the state, which has an energy of 0.98 ev above the ground state (see figure 6). The number of hot electrons available at the higher energy and the number of holes available at the lower energy involved in this two - electron exchange reaction can be estimated from the density of states of gold and the fermi distribution, as described in more detail in the supporting information . For example, for a spherical np with 15 nm diameter at an electron temperature of 2100 c, there are 260 electrons within an energy interval of 0.1 ev around the state . (it should be noted that the relevant energy interval is the width of this state for an oxygen in the vicinity or temporarily adsorbed onto a np, which is not known, so only relative numbers will be used here .) Thus, the electron temperatures achieved in our experiment are sufficient for a significant population of hot electrons and holes at the relevant levels . Schematic diagram showing the population probability f(e) for a np electron state at energy e near the fermi level, ef, under different conditions: (a) in equilibrium at room temperature, (b) at an electron temperature of te = 2100 c after electron electron equilibration (hot electrons), and (c) immediately after the absorption of photons by single electrons (primary hot electrons, with population changes highly exaggerated to make them visible). Also shown are the energies of the ground - state triplet () and lowest - excited singlet state () of oxygen as well as the next singlet state () under the assumption that ef is equidistant from the and energies . Excitation of an oxygen molecule to o2 requires the simultaneous transfer of an electron from the oxygen to a hole at the energy of the state and of a hot electron with the opposite spin and an energy at the (or) level to the oxygen molecule . The large number of equilibrated hot electrons available during a nanosecond - laser pulse also rules out the primary hot electrons as the main source of o2 photogeneration . The number of primary hot electrons available during the pulse duration can be estimated from the number of photons which are absorbed per nanosecond, multiplied by their lifetime, which is less than 500 fs . However, these primary hot electrons populate np states at energies from the fermi level ef to ef + 2.34 ev (the energy of a photon at 532 nm) (see figure 6c). For 15 nm nps and excitation with 5 ns laser pulses with 0.15 j cm intensity, this predicts that not more than three primary hot electrons are available within an energy interval of 0.1 ev around the level at any time during the laser pulse, assuming that all levels are equally populated and taking into account that the density of states for gold has an essentially constant value in the relevant energy range around ef . This is significantly less than the number of hot (equilibrated) electrons available at the maximum electron temperature, which was estimated to be on the order of 260 for the same energy interval (see above). Thus, we can conclude that it is indeed the hot electrons which are responsible for the observed o2 photogeneration when using short laser pulses for excitation . Similar results to those obtained here using pulsed irradiation have recently been reported for spherical gold nps with 40 nm diameter . Although a significantly lower laser pulse energy density (0.03 j cm, compared to 0.15 j cm here) was used under otherwise similar experimental conditions, slightly faster o2 photogeneration was observed in this study, with the dpbf absorbance decreasing by ca . 24% decrease observed here in the first 10 min (figure 4a). In this context, it is interesting to note that in spite of the lower laser pulse energy density the nps are heated to almost the same electron temperature as in our experiments (see figure 5). This is largely due to (i) heat dissipation from larger nps being slower and (ii) the use of 80/20 etoh / water as solvent in ref (24), which has slower heat transport than a 50/50 etoh / water mixture . An explicit calculation of the number of hot electrons available at the energy of the oxygen state for the two experiments is given in the supporting information; together with a detailed consideration of all other experimental differences, these numbers yield very good agreement between the expected and the observed relative o2 photogeneration rates (see the supporting information for details). This provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot electrons of gold nanoparticles under nanosecond - pulsed laser irradiation . Compared to the effect of pulsed irradiation, the rate of o2 photogeneration is much smaller when using cw light of comparable intensity . Whereas ca . 24% of the dye is bleached after only 10 min of irradiation of spherical nps with 15 nm diameter with 5 ns laser pulses at an average power of 150 mw (figure 4), only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power (1 w) (figure 2). In a previous publication, significantly higher rates of dpbf photobleaching had been reported under conditions which appear to be similar to the ones used here . In this context, we note that we also observed such significantly higher rates of bleaching, but only when the sample preparation protocol described in the experimental section was not followed accurately; for example, the use of dye solution that had not been freshly prepared or of a cuvette that had not been cleaned thoroughly and rinsed multiple times with mq water or the use of a cell that was not sealed during irradiation, leading to some loss of ethanol from the solution, all resulted in larger and highly irreproducible bleaching of dpbf under cw irradiation, up to levels comparable to those reported in ref (24), even in the absence of nps . In the following, we will show that photogeneration of o2 under cw irradiation, unlike pulsed irradiation, is mediated by the initially created primary hot electrons; i.e., it occurs during the short time during which the excited electrons have not yet relaxed to a thermal distribution (see figure 6c). Under the cw irradiation conditions used here (1 w, 1.85 mm beam diameter), a spherical np with 15 nm diameter absorbs photons at an average rate of 1.3 10 s, as estimated from the absorption cross section and the beam intensity . This means that after absorption of a photon there is enough time for full relaxation and transfer of the photon energy into the solvent, which occurs in less than 100 ps, before absorption of the next photon . Absorption of one photon by a 15 nm np yields hot electrons at a temperature of 10 k above the surrounding after electron electron equilibration; these hot electrons lose their energy by electron phonon scattering within a few picoseconds to yield a np whose temperature is only 80 mk above the surrounding . Neither of these effects is expected to yield any significant photochemical effects; estimates analogous to those described above predict that for a spherical 15 nm np with an electron temperature of 35 c there are ca . 2.6 10 electrons within an energy interval of 0.1 ev around the oxygen state and the same number of holes around the state energy . This means that there are on the order of 10 times less hot electrons and 10 times less holes available for the photoreaction than under our pulsed laser irradiation conditions, which rules out any significant reaction; this is also confirmed by the fact that these population numbers are less than a factor 2 larger than those for room temperature, where no o2 is generated in the absence of light . This leaves only the primary hot electrons, i.e., those electrons that are excited upon absorption of a photon but have not yet equilibrated by electron electron scattering, as potential cause for photogeneration of o2 . Absorption of a single photon can potentially excite electrons to energies of up to 2.34 ev above the fermi level . If one assumes excitation of only one electron by each photon and equal excitation probability for all available electrons, as shown in figure 6c, on average there will be 0.043 electrons within an energy interval of 0.1 ev around the oxygen state, which is approximately 5000 times less than during a single laser pulse in the pulsed experiments, see above . Taking into account that the same factor also applies to the holes required for the dexter mechanism, but correcting for the lifetime of the excitation (500 fs for primary hot electrons created during cw irradiation, ca . 3 ns for the hot electron distribution induced by a single laser pulse; see figure s6) and the repetition rates (1.3 10 s for single photon absorption during cw irradiation, 10 s for the pulsed laser irradiation), one would predict a rate of o2 photogeneration under our cw irradiation conditions which is smaller than that expected for our pulsed irradiation conditions by a factor on the order of 10 . This is significantly closer to the experimental results (ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020) than any estimate based on the equilibrated hot electrons after the absorption of a single photon . The main discrepancy between the predicted and observed cw results arises from the assumption of direct excitation of single electrons in the above estimate, which is not valid for irradiation at 532 nm, i.e., in the gold np plasmon resonance band, since this leads to the coherent excitation of many electrons which rapidly dephases without the electrons exchanging energy . Thus, absorption of a single photon yields more than one primary hot electron, with the photon s energy distributed over all of them . Consequently, the energy distribution even of the primary hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi level, thus increasing the population of states around the oxygen energy and hence the yield of o2 . A more quantitative estimate of this effect is beyond the scope of this paper . An alternative possibility for the mechanism of o2 photogeneration by gold nps could be envisaged, which is based on increased direct photoexcitation of oxygen due to the well - known local electric field enhancement in the vicinity of metal nps by the plasmon electrons . However, significant field enhancement extends to distances comparable to the dimensions of the nanoparticle, so that this mechanism is in disagreement with our observation that a peg - oh capping layer, which has a thickness of only 2 nm, completely inhibits o2 photogeneration (figure 3). Dexter - type electron exchange coupling, on the other hand, is known to be of significance only over distances of less than 1 nm, as discussed above, and thus is further supported by this observation . In conclusion, cw irradiation is less efficient than pulsed laser irradiation in photogenerating o2 since the (equilibrated) hot electrons, which are the main mediator of the photochemistry in the case of pulsed irradiation, do not have sufficient energy / temperature to drive o2 photogeneration . Nevertheless, our results confirm that cw irradiation of spherical gold nps produces detectable amounts of o2 . The absence of significant amounts of (equilibrated) hot electrons means that o2 photogeneration proceeds via a different mechanism under cw irradiation compared to pulsed laser generation; the above estimates indicate that it is the primary hot electrons, i.e., the directly photoexcited electrons, which are responsible for the photochemistry here and that the photochemical reaction must occur before these equilibrate by electron electron thermalization . Photodynamic therapy holds great promises for medical applications, such as the treatment of cancer, because of the ability to selectively affect diseased tissue only . However, wider use of photodynamic cancer therapy is currently prevented by several limitations imposed by the available photosensitizers . These limitations include toxicity, poor stability and photostability, poor selectivity for cancer tissue, and the need of using visible light with poor tissue penetration . All of these limitations, in principle, can be overcome by the use of gold nanoparticles, which are nontoxic, have excellent stability even under irradiation, can target cancer tissue either passively by the enhanced penetration and retention (epr) effect or by active targeting, have extinction coefficients that are larger than those of dye molecules by several orders of magnitude, and can be tuned to absorb in the near - infrared spectral region for maximum tissue penetration . Gold nanoparticles have been reported to have three potential modes of operation for inducing cell death by irradiation, namely (i) hyperthermia, which is based on the rapid conversion of the absorbed light energy into heat, (ii) np - assisted photodynamic therapy, in which the efficiency of a standard sensitizer is amplified by the np plasmon field enhancement effect, or (iii) a direct photochemical mechanism without involvement of a photosensitizer . The feasibility of photothermal therapy has been clearly established by careful experiments in vitro(1113,15,54) and has been shown to work in vivo . However, it should be noted that most studies reporting successful photothermal therapy made no attempt to either confirm significant heating or rule out photochemical effects, which means that some of these reported results could in fact arise from photochemical rather than photothermal effects or from a synergistic combination of photochemical and photothermal effects . As has been pointed out recently, another problem with many reports on photoinduced hyperthermia using nps is the relatively high light intensity required to reach sufficient temperatures, which often were well above the generally accepted skin tolerance threshold . The presence of gold nps can also lead to increased photogeneration of o2 by traditional photodynamic sensitizers, which can be ascribed to the local electric field enhancement in the vicinity of metal nps by the plasmon electrons, similar to the well - studied sers (surface - enhanced raman spectroscopy) effect . This approach might help to alleviate some of the drawbacks of traditional photodynamic therapy but does not directly overcome them, since it still requires the presence of a sensitizer . In fact, the requirement for two active components, gold np and sensitizer, which must be colocalized, introduces an additional complication, and care must be taken to avoid a reduction of the photosensitizer effect due to quenching of its excited state by the metal np . In an alternative approach, cell death has been demonstrated to occur following irradiation of intracellular (endocytosed) gold nps even at irradiation levels that are not high enough to cause significant heating . This photochemical effect has been related to the observation that irradiation of nps in vitro results in the formation of singlet oxygen, which is the active species in traditional photodynamic therapy . Because of the short lifetime of o2 (3.4 s in water), its action is highly localized an oxygen molecule only diffuses over the length scale of 100 nm in this time . For this reason, only intracellular nps are expected to trigger cell death by the photochemical route, although they may initially be located inside endosomes which are known to be broken up by nps under cw irradiation . On the other hand, the localization of the photochemical effects within individual cells also means that in situations where different types of cells coexist in close vicinity, selective targeting of particular cells and minimization of collateral damage should be achievable . This is different than photothermally induced cell death, which affects all cells within the irradiated volume more or less indiscriminately, as long as some of them contain nps, because of the fast diffusion of heat over the relevant length scales . The results of the experiments described here provide more insight into the direct photochemical mechanism and allow some important conclusions to be made for the further development of practical applications . They show that detectable amounts of o2 are generated by irradiation of nps with short laser pulses or cw light even in the absence of a photodynamic sensitizer, albeit with low quantum yield . Short laser pulses are significantly more efficient at this process, since they can heat a significant fraction of the np conduction band electrons to high enough temperatures to excite oxygen to the singlet state . However, this requires pulse energy densities that are well above generally accepted safe levels for the irradiation of skin with pulsed laser light . Since the effect requires the absorption of many photons by a np during one laser pulse, it is highly nonlinear with respect to irradiation intensity, and thus it will not be possible to compensate lower irradiation levels by longer irradiation times . Furthermore, pulsed irradiation at the required intensities also can cause other effects, such as np fragmentation or bubble formation, and it is not clear what consequences these effects may have when occurring in tissue . Although the use of femtosecond laser pulses, as compared to the nanosecond pulses used here, might alleviate some of these problems to some extent, they would require even more sophisticated equipment which may not be suitable for a clinical environment . The use of cw light, on the other hand, is straightforward and does not even require a laser but can be achieved with simple lamps . In spite of the lower quantum yield of o2 photogeneration by nps under cw irradiation which is reported here, cell death induced by cw light in the presence of nps has been reported in vitro and in vivo . Although the experiments described here, which did not involve any biological material, were undertaken at light intensities that are above safe irradiation levels, the mechanism of o2 photogeneration by nps under cw illumination is shown to be based on the absorption of single photons . This means that lower light intensities can be compensated for by longer irradiation times, allowing one to reduce the intensity to safe levels for biological or medical applications without affecting the amount of o2 generated . O2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation on the minute time scale at intensities below safe levels and using nonlaser light sources . The results presented here indicate another design criterion which needs to be fulfilled for successful implementation of the photochemical route of inducing cell death by irradiation of nps, namely that the nps must not possess a dense capping layer . Even the thin capping layer formed by peg - oh, with a thickness of only 2 nm, is sufficient to completely inhibit o2 photogeneration (figure 3). This is in full agreement with the suggestion that energy exchange between the np and the oxygen molecule occurs via the dexter (two electron exchange) mechanism, which is limited to distances of less than 1 nm . Similar effects have been reported for o2 photogeneration by si nanocrystals, which is significantly affected by a thin oxide layer . It is important to point out that the assay used here for o2 detection (bleaching of dpbf) only reports on singlet oxygen found outside the np capping layer; thus, it cannot be ruled out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with the np ligands and hence is quenched . On the other hand, such singlet oxygen would not be of any direct use for practical applications, such as the induction of cell death, so that the assay results in fact report the effects relevant for such applications . Uncapped (citrate) nps are rapidly covered by a protein corona after they have been taken up into live cells, but it appears that this corona is permeable enough for oxygen to not completely prevent singlet oxygen formation, as evidenced by fact that cell death by the photochemical route has been observed with such nps; it may be that because of their size proteins are not able to form a capping layer (corona) of similar density as the smaller ligands used here . Similarly, a lipid bilayer appears to allow oxygen access to the surface of gold nanorods, whereas a dense pentapeptide (calnn) capping layer on spherical gold nanoparticles is sufficient to suppress the photochemical mechanism of cell death . It also seems likely that the thick peg capping layer present on the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation in either the transversal or the longitudinal plasmon resonance band that was observed here . Because of their longitudinal plasmon resonance band, which is in the near - ir spectral region with high tissue penetration, nanorods are more suitable for practical photodynamic applications in tissue . However, standard synthesis protocols yield nanorods within a bilayer of cytotoxic ctab, so that ligand exchange is required before any biological or medical application, and care will need to be taken to choose suitable ligands to allow access of oxygen to the nanorod surface . Potential examples for these are poly(vinylpyrrolidone), lipid bilayers, or mesoporous silica, all of which have been used successfully in experiments showing photochemically induced cell death using gold nanorods . Dpbf has been used successfully to detect singlet oxygen that is formed when spherical nps are irradiated at 532 nm, either with cw or pulsed laser irradiation . Singlet oxygen generation by pulsed laser irradiation has been shown to act via the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse; cw irradiation, on the other hand, can act only via the directly excited primary hot electrons, which rapidly lose their energy by electron electron equilibration, and hence is significantly less efficient for the formation of singlet oxygen . Nevertheless, even cw irradiation can produce enough singlet oxygen for photodynamic therapy applications and will allow practical applications of the effect at safe irradiation levels . Photodynamic therapy using gold nanoparticles will also require careful design of the nanoparticles with respect to size, shape, and capping layer and will require internalization of the nps, not just attachment to the cell surface, which is sufficient for photothermal therapy.
Supernumerary teeth are a relatively frequent disorder of odontogenesis characterized by an excess number of teeth, with mesiodens being the most frequent.1 the term of mesiodens is used to refer to an unerupted supernumerary tooth in the midline of maxilla, between the central incisors.2,3 the incidence of mesiodens among caucasians is 0.153% for permanent teeth,4,5 and is found two times more frequently in males, compared to females.46 a mesiodens can manifest bilaterally,46 or in an inverted (upward) position.4,5 the frequency of inverted mesiodens constitutes to approximately 967% of all reported cases.711 to date, a total of 278 single inverted mesiodentes have been reported (table 1). The mesiodens may present a rudimentary morphology with a cone - shaped crown, generally smaller in size than the adjacent normal teeth . It may also be found to mimic a natural tooth shape . The root is often totally formed, and may be curved or globular.12 mesiodens is frequently associated with several craniofacial disturbances, including cleft lip and palate13 and cleidocranial dysostosis;14 and to a lesser extent with gardner s syndrome or chondorectodermal dysplasia.15 only 25% of all mesiodentes spontaneously erupt into the oral cavity.6 in general, they remain impacted and asymptomatic; and are commonly discovered during routine radiographic examination.16 the most common complications of mesiodentes are the delay or prevention of eruption (2652%) and displacement / rotation (2860%) of maxillary permanent incisors . Relatively less common complications include crowding, diastema, dilaceration of permanent teeth, cyst formation and eruption into the nasal cavity.6 this case reports presents radiographic features of a unique occurrence of two impacted, inverted mesiodentes in a child patient; and the 24-month radiographic and clinical follow - up after surgical removal . An 8-year - old boy was referred to the department of pediatric dentistry with a chief complaint of crowding in the maxillary anterior region . Being born to non - consanguineous parents, the patient s health status was excellent . Panoramic radiograph revealed the presence of two supernumerary teeth, located between the roots of the permanent central incisors (figure 2). Occlusal and periapical radiographs of the region showed that these two teeth were inverted mesiodentes (figure 3). Apparently, the roots of the permanent central incisors had migrated distally, owing to the presence of the supernumerary teeth . The lateral cephalogram showed that the mesiodentes were located buccally to the apices of the central incisors (figure 4). The treatment plan consisted of surgical removal the inverted mesiodentes . The teeth were removed under general anaesthesia without any complication (figure 5). Following removal of sutures one week later, the patient was scheduled for bimonthly recalls . At the second - month recall, the extraction cavities were completely filled with bone (figure 6) and the permanent central incisors responded favorably to thermal and electrical pulp tests . Removal of the inverted mesiodentes had slightly aided to self - correction of the axial rotations of the central incisors . Although mesiodentes are often located palatally,2 the present case demonstrates the possibility of their occurrence in a more vestibular location . In addition to routine ortopantomography or periapical / occlusal radiographs which can rapidly solve the initial diagnostic phase of mesiodentes, further techniques may be required to better define their position in relation to the adjacent teeth and anatomic structures . In the present case, the exact route (i.e., buccal / palatal) for surgical removal of the inverted mesiodentes undoubtedly, greater detail concerning their location could be obtained through computerized axial tomography (cat),17 which also can help evaluate their relationship with the adjacent tooth structures . In this regard, periapical and occlusal radiographs were used in conjunction with the lateral cephalogram, since cat was not available in our centre . Extraction is not the only treatment choice for impacted mesiodentes . If the mesiodens remains in place without symptoms and does not adversely affect the adjacent teeth, it may be left in place and observed periodically.16 in the present case, surgical removal of the mesiodentes was judged necessary, since these teeth had caused axial rotation of the permanent central incisors . There are no precise indications concerning the ideal time for surgical removal of impacted mesiodens . According to atwan,18 a mesiodens can be best removed when the permanent central incisors start to erupt, but this may not be always possible . Contrary, primosh19 discourages early extraction of mesiodentes due to the risk of iatrogenic damage to the developing adjacent permanent teeth . In the present case, the timing for surgical removal of the inverted mesiodentes was judged to be suitable, since both maxillary central incisors had totally erupted, showing complete root formation and advanced apical closure . The case presented herein was considered to be extremely rare, since inversion of two mesiodentes in a single patient has only been reported once20 (table 1). The clinical significance of the inversion is questionable, since both mesiodentes remained intact; i.e., did not erupt ectopically . However, the occurrence of two mesiodentes was definitely regarded as a contributory factor to the crowding in the anterior maxillary region, as evidenced by the deviation in the vertical axis of both central incisor teeth . Fortunately, the post - surgical phase was uneventful, and did not affect commencement of fixed orthodontic therapy after a relatively short follow - up period.
The whole school, whole community, whole child (wscc) model (figure 1), developed by ascd (formerly the association for supervision and curriculum development) and the us centers for disease control (cdc), provides a framework and a call for the health and education sectors to work toward greater alignment and coordination of policy, process, and practice . The wscc model, released in 2014, was developed to ensure that school - community and education - health sector alignments are front and center . The model expands the traditional 8-component coordinated school health (csh) approach and deliberately places it inside the larger community in order to emphasize this connection and role . This includes not only an increase in the number of school health components from 8 to 10 but also the alignment of an education focus with a health focus . The new model calls for a greater collaboration across the community, school, and health and education sectors to meet the needs and support the full potential of each child.8 whole school, whole community, whole child (wscc) model . The benefits of collaboration, alignment, and integration between health and education can best be viewed in 3 key areas: leveraging resources, utilizing resources efficiently, and improving both health and education outcomes . A 2010 report from the coalition of community schools cited 5 key ways that schools can efficiently leverage resources.9 this includes collaborative efforts to strengthen the core instructional mission of schools, leveraging community - wide financial resources, developing collaborative leadership structures and ownership, enhancing public / private partnerships, and providing coordination between and across systems . In terms of more efficient utilization, increased integration of health and education can improve usage of facilities and resources . Leveraging resources from across the community, as illustrated in the wscc model, can aid both the establishment of school - community connections and health and educational outcomes . Examples include shared use agreements that can lead to improved community access to facilities for physical activity10,11 and school - based health centers that have been shown to reduce a community's inappropriate emergency room use, increase use of primary care, and result in fewer hospitalizations among regular users.1214 finally, strengthening the integration and collaboration between health and education benefits both sectors because research suggests that educational attainment is important to achieving better health outcomes,15,16 and health is key to better educational outcomes.2,3 operationalizing wscc requires moving from theoretical model to practical implementation through the development of state and local school policies and practices . Although the model is new, a number of states and local school districts have made significant strides over the last few years in their efforts to better align health and education by building a collaborative, integrated approach . Arkansas, kentucky, colorado, maine regional school unit #22, and denver public schools (dps) all provide valuable examples of states and local districts that embraced wscc's core vision of aligning health and education through the adoption of integrated policies and initiatives . Their trailblazing efforts demonstrate both the feasibility of implementing the wscc model and the visionary leadership required to do so . We conducted a search of the health and education department's websites in arkansas, colorado, and kentucky, and the school districts' websites in maine regional school unit #22, and dps for materials demonstrating adoption and implementation of csh and its respective components or the wscc model . We also conducted a search of the respective state legislative databases, state health and education department websites, and local school district websites for adopted policy relevant to csh and the wscc model . We searched pubmed, web of science, and academic search premier and for relevant peer - reviewed articles around the implementation of aspects of csh or wscc in the 4 states and 2 local school districts . In addition, we searched online for news articles and gray literature highlighting the implementation of csh or the wscc model, or aspects of them, in each state and local school district . Secondarily, we conducted informal reinforcing interviews via phone with 1 person in a key school health leadership role related to the promotion and implementation of the wscc model in each district and state: arkansas, kentucky, and colorado, maine regional school unit #22 and dps . In arkansas, the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . The 2 agencies have jointly operated the coordinated school health program (cshp) for over 2 decades, working together to expand csh in school districts across the state . They have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . In 2003, the arkansas legislature adopted act 1220, landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index (bmi) screening in schools; the establishment of a state - level child health advisory committee (chac) to develop rules and regulations for school nutrition, physical education (pe), and physical activity, and to make recommendations to the arkansas state board of education and board of health; and the establishment of a wellness committee in each local school district . Act 1220 also created positions for regional community health promotion specialists (chps) specifically to assist schools and led to new standards for competitive foods and physical activity in schools, created by the arkansas chac . Act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements, provide a parental opt - out clause, and require the department of health to assign regional community health nurse specialists (chns) to work with schools to ensure appropriate protocols and follow up . Act 317 (2007) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009, act 180 (tobacco excise tax) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education (ade), the arkansas department of health (adh), and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . Act 180 also provided funding for an annual school - based health center grant, a collaboration among ade, adh, the arkansas department of human services, and medicaid in the schools . This funding led to the establishment of 22 school - based health centers across the state, providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better ., post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments, policies, and practices, with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state, including reduced access to less healthy competitive foods and beverages and increased access to healthier options, and stronger policies to promote physical activity and recess.20,21,2327 the chps, employed by the state health department but housed in regional educational cooperatives, have worked together with chns to support local school districts, including assisting with the creation of wellness committees and policies, administering the school health index, and developing strategies to improve nutrition, physical activity, and health environment policies and programs . The state health and education departments have worked collaboratively, with initiatives guided by a school health services team comprised of staff from various areas in each agency, including school health services, human immunodeficiency virus infection / acquired immune deficiency syndrome, mental health, arkansas medicaid in the schools, the state school nurse consultant, act 1220, and csh . Team members provide ongoing support, training, and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index . Kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . Kentucky's state health and education departments have enjoyed a strong, collaborative relationship in recent years . Together, staff from each agency have worked together to increase awareness of csh among education leaders across the state, including superintendents, principals, and school board members . They have collaborated with state associations including the kentucky association of school councils, kentucky school nutrition association, kentucky family resource youth service centers, and kentucky association of health, physical education, recreation, and dance to promote understanding, adoption, and implementation of csh at the local level . The state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement, discipline, and attendance . This foundation, along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association, has facilitated the adoption of education policies that support healthier schools and students . An example of this can be seen in kentucky's program review process . In 2009, the kentucky legislature adopted senate bill 1, which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement, the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . The program review includes a practical living / career studies component, encompassing health education and pe . The state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component, setting a high baseline to support and strengthen the integration of health into school - level policies and practices . Schools receive one of 4 designations during the program review process: no implementation, needs improvement, proficient, and distinguished . To achieve the proficient level for health education in the practical living / career studies program review, a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment (table 1). Similarly, to achieve proficient in the practical living / administrative leadership / support and monitoring component, a school must convene its csh committee at least twice a year, implement the district - level wellness policy via a school - level wellness policy reviewed annually, and include goals for school wellness in the comprehensive school improvement plan (csip). For pe, the csh committee of a proficient school must utilize a comprehensive school physical activity program (cspap) to increase quality pe and physical activity opportunities, and integrate the pe curriculum, providing opportunities for cross - disciplinary connections (table 1). Whereas schools may go beyond these standards to achieve the distinguished level, setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state, creation of school - level wellness policies, integration of wellness goals into the school improvement plans, and expansion of cspap . Using a common sense approach, kentucky has intentionally and thoughtfully woven health into education measures, and in doing so, has taken a major step toward ensuring better integration of health and education at the local level . Examples from kentucky practical living / career studies (plcs) program review rubric * adapted from the kentucky department of education . In colorado, strong collaboration and leadership between health and education partners, both public and private, have been instrumental in advancing the integration of health and education across the state . Cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . In addition to a solid working partnership between the state health and education agencies, the state has benefitted from the strategic support of local foundations and organizations including: the colorado education initiative, rmc health, the colorado health foundation, and kaiser permanente . In 2010, these partners came together to develop and disseminate the healthy school champion score card, a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools (tables 2 and 3). Whereas the design is based on the school health index, it also takes into consideration colorado legislation and policies . The use of the score card grew significantly from its initiation in 2010, when 100 schools participated . In the 20132014 school year, 216 schools, covering over 100,000 students, participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . Score card overview . Colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices, called healthy schools smart source . This effort is funded by kaiser permanente, in partnership with the colorado department of education, the colorado department of public health and the environment, the colorado education initiative, and the colorado coalition for healthy schools . The goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments, streamlining data collection and submission for schools.31 to create the assessment, partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . A pilot of smart source was launched in the 20142015 school year with 90 schools . By the 20162017 school year, smart source will collect and synthesize data about school and district health policies and practices into a statewide system . Smart source will provide schools with school - level reports that offer objective, meaningful comparisons to the aggregated results from all respondents statewide . These reports are designed to provide useful, actionable data as well as recommendations for implementing best practices related to health and wellness (table 4). Reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior, informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . In addition to the smart source initiative, collaborating partners have also launched the colorado healthy schools collective impact (hsci), a purposeful and strategic effort to define healthy schools, prioritize school health - related work in the state, minimize duplication of initiatives, and better leverage resources and funding for healthy schools . Healthy schools collective impact members include 20 foundations and nearly 100 community organizations, state agencies, school districts and schools, and nonprofits that are all interested in strengthening the school health environment . The 4 focus areas, each represented by a work group that meets monthly, are pe and physical activity, nutrition, behavioral health (social, emotional, and mental), and student health services . Healthy schools collective impact's objectives are to (1) coordinate efforts and ensure resources are allocated based on identified needs; (2) engage those impacted by the work, including districts and schools, parents, students, funders, and organizations that champion healthy schools; (3) gather consistent data by coordinating surveys and systems to collect data from schools; (4) use consistent communication by creating a shared definition of a healthy school, possibly producing a one - stop shop for data and resources related to priority school health strategies; (5) support core capacity for schools and districts; and (6) develop shared policy priorities.33 at the local level, many school districts nationally have embraced the importance of addressing the whole child through better alignment and integration of health and education . Two districts that provide strong examples of application and implementation of coordinated school health and/or the wscc model include maine regional school unit (rsu) #22 and denver public schools in colorado . Both have benefited from the leadership of superintendents and local school boards that have embraced the importance of educating the whole child . Maine regional school unit #22, located in the hampden area, began to intentionally focus on the health and wellness of students and staff in 1991 . The vision to strengthen school health and wellness has been driven by rick lyons, district superintendent since 1992 . The district began by forming a wellness team long before the concept had gained widespread popularity . The wellness team, comprised administrators, faculty, support staff, and community members, has been a key factor behind the district's school health achievements since its establishment . In 2001, the district also founded a school health advisory council (shac) comprised of administrators, healthcare professionals, and community members . The shac oversees the cshp, which is headed by a school health coordinator employed by the district . Given rsu #22's small size (approximately 2,200 students and 375 employees), the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . Over the last 20 years, rsu #22 has taken many proactive steps toward strengthening school health in each of its 6 schools . It increased the number of positions and staffing time for health education teachers, nurses, and guidance counselors and forged partnerships with behavioral health providers . The district promotes active transport to school with a walking school bus, works with the hampden town council to repair bike lanes and install street lights, and partners with the hampden recreation department to provide before- and after - school programming . Regional school unit #22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities (such as weight training, aerobics, bicycling, and running) along with activities such as skiing, tennis, snowshoeing, and swimming and uses recess rocks in k2 classrooms . In 2002, the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . Regional school unit #22's schools offer salad bars at lunch, and the food service director strives to use locally sourced foods as much as possible . In 2010, parents, students, the district's food service director, wellness coordinator, and community partners collaborated to plant apple orchards and a vegetable garden . Leadership from school board members and the superintendent has been key in rsu #22's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . The shac, wellness team, and school health coordinator have also been integral to the process . They have worked together to facilitate the integration of health and education in numerous areas, including the district's strategic plan and the interview process for prospective employees, which includes questions about their thoughts on the district's wellness priorities . The district has focused on improving student and staff health and dedicated significant financial resources to both . The local school board allocates approximately $40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year, and 60% of staff members have qualified for this reward . Regional school unit #22 promotes periodic wellness challenges for staff and a 7-week total health track through adult education . In 2012, it opened a new high school that includes a fitness center, track, and tennis courts open to students, staff, and the community . As a result of these efforts, the wellness council of america awarded the district their gold level workplace wellness award . Superintendent leadership also has been critical to the success of dps' strategic inclusion of student health in the district's overall priorities and plan . Since being appointed to the position by the board of education in 2009, superintendent tom boasberg has managed the district's 183 schools, which serve a diverse population of over 90,000 children . Seventy - two percent of students qualify for free or reduced - price lunch, and 41% speak a language other than english at home.34 the district has also committed significant resources to student health, and made it a part of the district's strategic planning process . In 2010, boasberg oversaw the denver school health advisory council's creation of the dps health agenda 2015, which outlines the districts' health priorities.35 the district has since invested over $22 million toward its health goals, including the addition of 6 full - time district - funded employees . Some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell; adding pe teachers to elementary schools, which has led to an additional 22 minutes per week of pe classes; and increasing the number of school counselors, which has corresponded with a decrease in expulsion and suspension rates . Denver public school has also added a school - based health center every year since 2010, with a fifth scheduled to open in 2015 . In august 2014, dps released the current version of its 5-year strategic plan, denver plan 2020 . This version, which was developed with the input of nearly 3,000 educators, parents, students, community partners, and city leaders, includes support for the whole child as one of 5 goals . Using the wscc model as a backbone, an advisory committee is assessing dps' activities, and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda, dps health agenda 2020 . The district has solicited input from students, parents, and community members through a widely disseminated survey about health issues that most impact students . Survey topics include asthma, vision, oral health, nutrition, physical activity, social / emotional health, substance abuse, teen pregnancy, and school culture . Denver public school plans to use the results of this survey to improve student health and wellness going forward . In arkansas, the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . The 2 agencies have jointly operated the coordinated school health program (cshp) for over 2 decades, working together to expand csh in school districts across the state . They have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . In 2003, the arkansas legislature adopted act 1220, landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index (bmi) screening in schools; the establishment of a state - level child health advisory committee (chac) to develop rules and regulations for school nutrition, physical education (pe), and physical activity, and to make recommendations to the arkansas state board of education and board of health; and the establishment of a wellness committee in each local school district . Act 1220 also created positions for regional community health promotion specialists (chps) specifically to assist schools and led to new standards for competitive foods and physical activity in schools, created by the arkansas chac . Act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements, provide a parental opt - out clause, and require the department of health to assign regional community health nurse specialists (chns) to work with schools to ensure appropriate protocols and follow up . Act 317 (2007) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009, act 180 (tobacco excise tax) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education (ade), the arkansas department of health (adh), and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . Act 180 also provided funding for an annual school - based health center grant, a collaboration among ade, adh, the arkansas department of human services, and medicaid in the schools . This funding led to the establishment of 22 school - based health centers across the state, providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better ., post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments, policies, and practices, with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state, including reduced access to less healthy competitive foods and beverages and increased access to healthier options, and stronger policies to promote physical activity and recess.20,21,2327 the chps, employed by the state health department but housed in regional educational cooperatives, have worked together with chns to support local school districts, including assisting with the creation of wellness committees and policies, administering the school health index, and developing strategies to improve nutrition, physical activity, and health environment policies and programs . The state health and education departments have worked collaboratively, with initiatives guided by a school health services team comprised of staff from various areas in each agency, including school health services, human immunodeficiency virus infection / acquired immune deficiency syndrome, mental health, arkansas medicaid in the schools, the state school nurse consultant, act 1220, and csh . Team members provide ongoing support, training, and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index . Kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . Kentucky's state health and education departments have enjoyed a strong, collaborative relationship in recent years . Together, staff from each agency have worked together to increase awareness of csh among education leaders across the state, including superintendents, principals, and school board members . They have collaborated with state associations including the kentucky association of school councils, kentucky school nutrition association, kentucky family resource youth service centers, and kentucky association of health, physical education, recreation, and dance to promote understanding, adoption, and implementation of csh at the local level . The state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement, discipline, and attendance . This foundation, along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association, has facilitated the adoption of education policies that support healthier schools and students . An example of this can be seen in kentucky's program review process . In 2009, the kentucky legislature adopted senate bill 1, which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement, the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . The program review includes a practical living / career studies component, encompassing health education and pe . The state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component, setting a high baseline to support and strengthen the integration of health into school - level policies and practices . Schools receive one of 4 designations during the program review process: no implementation, needs improvement, proficient, and distinguished . To achieve the proficient level for health education in the practical living / career studies program review, a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment (table 1). Similarly, to achieve proficient in the practical living / administrative leadership / support and monitoring component, a school must convene its csh committee at least twice a year, implement the district - level wellness policy via a school - level wellness policy reviewed annually, and include goals for school wellness in the comprehensive school improvement plan (csip). For pe, the csh committee of a proficient school must utilize a comprehensive school physical activity program (cspap) to increase quality pe and physical activity opportunities, and integrate the pe curriculum, providing opportunities for cross - disciplinary connections (table 1). Whereas schools may go beyond these standards to achieve the distinguished level, setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state, creation of school - level wellness policies, integration of wellness goals into the school improvement plans, and expansion of cspap . Using a common sense approach, kentucky has intentionally and thoughtfully woven health into education measures, and in doing so, has taken a major step toward ensuring better integration of health and education at the local level . Examples from kentucky practical living / career studies (plcs) program review rubric * adapted from the kentucky department of education . In colorado, strong collaboration and leadership between health and education partners, both public and private, have been instrumental in advancing the integration of health and education across the state . Cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . In addition to a solid working partnership between the state health and education agencies, the state has benefitted from the strategic support of local foundations and organizations including: the colorado education initiative, rmc health, the colorado health foundation, and kaiser permanente . In 2010, these partners came together to develop and disseminate the healthy school champion score card, a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools (tables 2 and 3). Whereas the design is based on the school health index, it also takes into consideration colorado legislation and policies . The use of the score card grew significantly from its initiation in 2010, when 100 schools participated . In the 20132014 school year, 216 schools, covering over 100,000 students, participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . Score card overview . Colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices, called healthy schools smart source . This effort is funded by kaiser permanente, in partnership with the colorado department of education, the colorado department of public health and the environment, the colorado education initiative, and the colorado coalition for healthy schools . The goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments, streamlining data collection and submission for schools.31 to create the assessment, partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . A pilot of smart source was launched in the 20142015 school year with 90 schools . By the 20162017 school year, smart source will collect and synthesize data about school and district health policies and practices into a statewide system . Smart source will provide schools with school - level reports that offer objective, meaningful comparisons to the aggregated results from all respondents statewide . These reports are designed to provide useful, actionable data as well as recommendations for implementing best practices related to health and wellness (table 4). Reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior, informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . In addition to the smart source initiative, collaborating partners have also launched the colorado healthy schools collective impact (hsci), a purposeful and strategic effort to define healthy schools, prioritize school health - related work in the state, minimize duplication of initiatives, and better leverage resources and funding for healthy schools . Healthy schools collective impact members include 20 foundations and nearly 100 community organizations, state agencies, school districts and schools, and nonprofits that are all interested in strengthening the school health environment . The 4 focus areas, each represented by a work group that meets monthly, are pe and physical activity, nutrition, behavioral health (social, emotional, and mental), and student health services . Healthy schools collective impact's objectives are to (1) coordinate efforts and ensure resources are allocated based on identified needs; (2) engage those impacted by the work, including districts and schools, parents, students, funders, and organizations that champion healthy schools; (3) gather consistent data by coordinating surveys and systems to collect data from schools; (4) use consistent communication by creating a shared definition of a healthy school, possibly producing a one - stop shop for data and resources related to priority school health strategies; (5) support core capacity for schools and districts; and (6) develop shared policy priorities.33 in arkansas, the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . The 2 agencies have jointly operated the coordinated school health program (cshp) for over 2 decades, working together to expand csh in school districts across the state . They have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . In 2003, the arkansas legislature adopted act 1220, landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index (bmi) screening in schools; the establishment of a state - level child health advisory committee (chac) to develop rules and regulations for school nutrition, physical education (pe), and physical activity, and to make recommendations to the arkansas state board of education and board of health; and the establishment of a wellness committee in each local school district . Act 1220 also created positions for regional community health promotion specialists (chps) specifically to assist schools and led to new standards for competitive foods and physical activity in schools, created by the arkansas chac . Act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements, provide a parental opt - out clause, and require the department of health to assign regional community health nurse specialists (chns) to work with schools to ensure appropriate protocols and follow up . Act 317 (2007) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009, act 180 (tobacco excise tax) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education (ade), the arkansas department of health (adh), and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . Act 180 also provided funding for an annual school - based health center grant, a collaboration among ade, adh, the arkansas department of human services, and medicaid in the schools . This funding led to the establishment of 22 school - based health centers across the state, providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better ., post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments, policies, and practices, with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state, including reduced access to less healthy competitive foods and beverages and increased access to healthier options, and stronger policies to promote physical activity and recess.20,21,2327 the chps, employed by the state health department but housed in regional educational cooperatives, have worked together with chns to support local school districts, including assisting with the creation of wellness committees and policies, administering the school health index, and developing strategies to improve nutrition, physical activity, and health environment policies and programs . The state health and education departments have worked collaboratively, with initiatives guided by a school health services team comprised of staff from various areas in each agency, including school health services, human immunodeficiency virus infection / acquired immune deficiency syndrome, mental health, arkansas medicaid in the schools, the state school nurse consultant, act 1220, and csh . Team members provide ongoing support, training, and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index . Kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . Kentucky's state health and education departments have enjoyed a strong, collaborative relationship in recent years . Together, staff from each agency have worked together to increase awareness of csh among education leaders across the state, including superintendents, principals, and school board members . They have collaborated with state associations including the kentucky association of school councils, kentucky school nutrition association, kentucky family resource youth service centers, and kentucky association of health, physical education, recreation, and dance to promote understanding, adoption, and implementation of csh at the local level . The state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement, discipline, and attendance . This foundation, along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association, has facilitated the adoption of education policies that support healthier schools and students . An example of this can be seen in kentucky's program review process . In 2009, the kentucky legislature adopted senate bill 1, which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement, the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . The program review includes a practical living / career studies component, encompassing health education and pe . The state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component, setting a high baseline to support and strengthen the integration of health into school - level policies and practices . Schools receive one of 4 designations during the program review process: no implementation, needs improvement, proficient, and distinguished . To achieve the proficient level for health education in the practical living / career studies program review, a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment (table 1). Similarly, to achieve proficient in the practical living / administrative leadership / support and monitoring component, a school must convene its csh committee at least twice a year, implement the district - level wellness policy via a school - level wellness policy reviewed annually, and include goals for school wellness in the comprehensive school improvement plan (csip). For pe, the csh committee of a proficient school must utilize a comprehensive school physical activity program (cspap) to increase quality pe and physical activity opportunities, and integrate the pe curriculum, providing opportunities for cross - disciplinary connections (table 1). Whereas schools may go beyond these standards to achieve the distinguished level, setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state, creation of school - level wellness policies, integration of wellness goals into the school improvement plans, and expansion of cspap . Using a common sense approach, kentucky has intentionally and thoughtfully woven health into education measures, and in doing so, has taken a major step toward ensuring better integration of health and education at the local level . Examples from kentucky practical living / career studies (plcs) program review rubric * adapted from the kentucky department of education . In colorado, strong collaboration and leadership between health and education partners, both public and private, have been instrumental in advancing the integration of health and education across the state . Cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . In addition to a solid working partnership between the state health and education agencies, the state has benefitted from the strategic support of local foundations and organizations including: the colorado education initiative, rmc health, the colorado health foundation, and kaiser permanente . In 2010, these partners came together to develop and disseminate the healthy school champion score card, a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools (tables 2 and 3). Whereas the design is based on the school health index, it also takes into consideration colorado legislation and policies . The use of the score card grew significantly from its initiation in 2010, when 100 schools participated . In the 20132014 school year, 216 schools, covering over 100,000 students, participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . Colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices, called healthy schools smart source . This effort is funded by kaiser permanente, in partnership with the colorado department of education, the colorado department of public health and the environment, the colorado education initiative, and the colorado coalition for healthy schools . The goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments, streamlining data collection and submission for schools.31 to create the assessment, partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . A pilot of smart source was launched in the 20142015 school year with 90 schools . By the 20162017 school year, smart source will collect and synthesize data about school and district health policies and practices into a statewide system . Smart source will provide schools with school - level reports that offer objective, meaningful comparisons to the aggregated results from all respondents statewide . These reports are designed to provide useful, actionable data as well as recommendations for implementing best practices related to health and wellness (table 4). Reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior, informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . In addition to the smart source initiative, collaborating partners have also launched the colorado healthy schools collective impact (hsci), a purposeful and strategic effort to define healthy schools, prioritize school health - related work in the state, minimize duplication of initiatives, and better leverage resources and funding for healthy schools . Healthy schools collective impact members include 20 foundations and nearly 100 community organizations, state agencies, school districts and schools, and nonprofits that are all interested in strengthening the school health environment . The 4 focus areas, each represented by a work group that meets monthly, are pe and physical activity, nutrition, behavioral health (social, emotional, and mental), and student health services . Healthy schools collective impact's objectives are to (1) coordinate efforts and ensure resources are allocated based on identified needs; (2) engage those impacted by the work, including districts and schools, parents, students, funders, and organizations that champion healthy schools; (3) gather consistent data by coordinating surveys and systems to collect data from schools; (4) use consistent communication by creating a shared definition of a healthy school, possibly producing a one - stop shop for data and resources related to priority school health strategies; (5) support core capacity for schools and districts; and (6) develop shared policy priorities.33 at the local level, many school districts nationally have embraced the importance of addressing the whole child through better alignment and integration of health and education . Two districts that provide strong examples of application and implementation of coordinated school health and/or the wscc model include maine regional school unit (rsu) #22 and denver public schools in colorado . Both have benefited from the leadership of superintendents and local school boards that have embraced the importance of educating the whole child . Maine regional school unit #22, located in the hampden area, began to intentionally focus on the health and wellness of students and staff in 1991 . The vision to strengthen school health and wellness has been driven by rick lyons, district superintendent since 1992 . The district began by forming a wellness team long before the concept had gained widespread popularity . The wellness team, comprised administrators, faculty, support staff, and community members, has been a key factor behind the district's school health achievements since its establishment . In 2001, the district also founded a school health advisory council (shac) comprised of administrators, healthcare professionals, and community members . The shac oversees the cshp, which is headed by a school health coordinator employed by the district . Given rsu #22's small size (approximately 2,200 students and 375 employees), the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . Over the last 20 years, rsu #22 has taken many proactive steps toward strengthening school health in each of its 6 schools . It increased the number of positions and staffing time for health education teachers, nurses, and guidance counselors and forged partnerships with behavioral health providers . The district promotes active transport to school with a walking school bus, works with the hampden town council to repair bike lanes and install street lights, and partners with the hampden recreation department to provide before- and after - school programming . Regional school unit #22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities (such as weight training, aerobics, bicycling, and running) along with activities such as skiing, tennis, snowshoeing, and swimming and uses recess rocks in k2 classrooms . In 2002, the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . Regional school unit #22's schools offer salad bars at lunch, and the food service director strives to use locally sourced foods as much as possible . In 2010, parents, students, the district's food service director, wellness coordinator, and community partners collaborated to plant apple orchards and a vegetable garden . Leadership from school board members and the superintendent has been key in rsu #22's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . The shac, wellness team, and school health coordinator have also been integral to the process . They have worked together to facilitate the integration of health and education in numerous areas, including the district's strategic plan and the interview process for prospective employees, which includes questions about their thoughts on the district's wellness priorities . The district has focused on improving student and staff health and dedicated significant financial resources to both . The local school board allocates approximately $40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year, and 60% of staff members have qualified for this reward . Regional school unit #22 promotes periodic wellness challenges for staff and a 7-week total health track through adult education . In 2012, it opened a new high school that includes a fitness center, track, and tennis courts open to students, staff, and the community . As a result of these efforts, the wellness council of america awarded the district their gold level workplace wellness award . Superintendent leadership also has been critical to the success of dps' strategic inclusion of student health in the district's overall priorities and plan . Since being appointed to the position by the board of education in 2009, superintendent tom boasberg has managed the district's 183 schools, which serve a diverse population of over 90,000 children . Seventy - two percent of students qualify for free or reduced - price lunch, and 41% speak a language other than english at home.34 the district has also committed significant resources to student health, and made it a part of the district's strategic planning process . In 2010, boasberg oversaw the denver school health advisory council's creation of the dps health agenda 2015, which outlines the districts' health priorities.35 the district has since invested over $22 million toward its health goals, including the addition of 6 full - time district - funded employees . Some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell; adding pe teachers to elementary schools, which has led to an additional 22 minutes per week of pe classes; and increasing the number of school counselors, which has corresponded with a decrease in expulsion and suspension rates . Denver public school has also added a school - based health center every year since 2010, with a fifth scheduled to open in 2015 . In august 2014, dps released the current version of its 5-year strategic plan, denver plan 2020 . This version, which was developed with the input of nearly 3,000 educators, parents, students, community partners, and city leaders, includes support for the whole child as one of 5 goals . Using the wscc model as a backbone, an advisory committee is assessing dps' activities, and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda, dps health agenda 2020 . The district has solicited input from students, parents, and community members through a widely disseminated survey about health issues that most impact students . Survey topics include asthma, vision, oral health, nutrition, physical activity, social / emotional health, substance abuse, teen pregnancy, and school culture . Denver public school plans to use the results of this survey to improve student health and wellness going forward . Maine regional school unit #22, located in the hampden area, began to intentionally focus on the health and wellness of students and staff in 1991 . The vision to strengthen school health and wellness has been driven by rick lyons, district superintendent since 1992 . The district began by forming a wellness team long before the concept had gained widespread popularity . The wellness team, comprised administrators, faculty, support staff, and community members, has been a key factor behind the district's school health achievements since its establishment . In 2001, the district also founded a school health advisory council (shac) comprised of administrators, healthcare professionals, and community members . The shac oversees the cshp, which is headed by a school health coordinator employed by the district . Given rsu #22's small size (approximately 2,200 students and 375 employees), the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . Over the last 20 years, rsu #22 has taken many proactive steps toward strengthening school health in each of its 6 schools . It increased the number of positions and staffing time for health education teachers, nurses, and guidance counselors and forged partnerships with behavioral health providers . The district promotes active transport to school with a walking school bus, works with the hampden town council to repair bike lanes and install street lights, and partners with the hampden recreation department to provide before- and after - school programming . Regional school unit #22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities (such as weight training, aerobics, bicycling, and running) along with activities such as skiing, tennis, snowshoeing, and swimming and uses recess rocks in k2 classrooms . In 2002, the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . Regional school unit #22's schools offer salad bars at lunch, and the food service director strives to use locally sourced foods as much as possible . In 2010, parents, students, the district's food service director, wellness coordinator, and community partners collaborated to plant apple orchards and a vegetable garden . Leadership from school board members and the superintendent has been key in rsu #22's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . The shac, wellness team, and school health coordinator have also been integral to the process . They have worked together to facilitate the integration of health and education in numerous areas, including the district's strategic plan and the interview process for prospective employees, which includes questions about their thoughts on the district's wellness priorities . The district has focused on improving student and staff health and dedicated significant financial resources to both . The local school board allocates approximately $40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year, and 60% of staff members have qualified for this reward . Regional school unit #22 promotes periodic wellness challenges for staff and a 7-week total health, it opened a new high school that includes a fitness center, track, and tennis courts open to students, staff, and the community . As a result of these efforts, the wellness council of america awarded the district their gold level workplace wellness award . Superintendent leadership also has been critical to the success of dps' strategic inclusion of student health in the district's overall priorities and plan . Since being appointed to the position by the board of education in 2009, superintendent tom boasberg has managed the district's 183 schools, which serve a diverse population of over 90,000 children . Seventy - two percent of students qualify for free or reduced - price lunch, and 41% speak a language other than english at home.34 the district has also committed significant resources to student health, and made it a part of the district's strategic planning process . In 2010, boasberg oversaw the denver school health advisory council's creation of the dps health agenda 2015, which outlines the districts' health priorities.35 the district has since invested over $22 million toward its health goals, including the addition of 6 full - time district - funded employees . Some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell; adding pe teachers to elementary schools, which has led to an additional 22 minutes per week of pe classes; and increasing the number of school counselors, which has corresponded with a decrease in expulsion and suspension rates . Denver public school has also added a school - based health center every year since 2010, with a fifth scheduled to open in 2015 . In august 2014, dps released the current version of its 5-year strategic plan, denver plan 2020 . This version, which was developed with the input of nearly 3,000 educators, parents, students, community partners, and city leaders, includes support for the whole child as one of 5 goals . Using the wscc model as a backbone, an advisory committee is assessing dps' activities, and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda, dps health agenda 2020 . The district has solicited input from students, parents, and community members through a widely disseminated survey about health issues that most impact students . Survey topics include asthma, vision, oral health, nutrition, physical activity, social / emotional health, substance abuse, teen pregnancy, and school culture . Denver public school plans to use the results of this survey to improve student health and wellness going forward . The wscc model provides a framework for the health and education sectors at the state and local levels to work toward stronger alignment, integration, and collaboration . As lloyd kolbe stated, the wscc model provides a new opportunity, impetus, and sense of purpose for both sectors: if we prudently could unite the expertise, perspectives, resources, communication, visibility, and political support of national education, health, and related organizations to help schools and colleges implement a wscc approach, we synergistically could increase the impact of each sector, each organization involved; we could build a lasting national base for new means to improve both education and health; we could accomplish that which we otherwise cannot.37 whereas the wscc model is new, having been launched in march 2014, many states and local school districts are already in the process of adapting and adjusting practices and processes to suit the expanded version of csh . The 3 states and 2 school districts highlighted all provide strong examples of implementation of various aspects of the wscc model . They demonstrate that it is possible to prioritize student health within the education system, for the purpose of better addressing the needs of the whole child . They also demonstrate the importance of strategic leadership at the state and local level, including support from policymakers, superintendents, local school boards, and community organizations that recognize and seize opportunities to integrate and better align the health and education sectors . Moving forward, state and local health and education agencies should focus greater attention toward ensuring the adoption and implementation of policies that serve both sectors and students, and promote the formation of collaborative partnerships . Policymakers need to focus on adoption and implementation of policies and practices that build on the connection between the school health components and educational outcomes, both academic and behavioral . When those in decision - making roles understand and embrace the wscc model, they are able to create programs and policies that lead to collaboration and more effective use of resources, creating healthier school environments that address the needs of the whole child . Information contained in this article was not deemed to involve human subjects, and therefore, was considered exempt from institutional review board examination . Information contained in this article was not deemed to involve human subjects, and therefore, was considered exempt from institutional review board examination.
Juvenile ossifying fibroma (jof) is an actively growing aggressive lesion containing cells rich in the fibrous stroma containing cellular osteoid, trabeculae of woven bone without osteoblastic lining, with or without clusters of giant cells . El - mofty first identified the two histopathological variants of jof: trabecular and psammomatoid . Rare cases of mandibular jof have been reported . Clinical examination reveals it as asymptomatic slow growing swelling causing facial asymmetry, extending to a substantial size and behaving as an aggressive lesion . It has higher male predominance with the mean age of occurrence ranging from 2 to 15 years . The radiographic features show unilocular or multilocular radiolucency with well - delineated borders and few opacification in the center . The suggestive features of the aggressive variant are the presence of the perforation, cortical thinning, tooth displacement, and root resorption . The advanced imaging studies show more invasive and destructive features apart from conventional radiographic features . Histopathologically both the entities are nonencapsulated, well demarcated from the surrounding bone consisting of neoplastic cellular stroma formed by spindle shape fibroblast . In contrast, the psammomatoid pattern forms concentrically laminated ossicles that vary in shape and having peripheral eosinophilic osteoid rims with basophilic centers typically . The overall clinical features, histopathological features, and advanced imaging were indispensable to provide the diagnosis as jof . A nonaggressive form of this lesion is managed by curettage and local excision whereas aggressive form is managed by complete surgical excision, en - bloc resection or hemimaxillectomy to prevent recurrences . Reported here is a case of a trabecular variant of jof in a 17-year - old female patient . A 17-year - old female represented with a chief complaint of pain and slowly progressive swelling of the left side of face along with reduced mouth opening for the last 2 months . Initially, the swelling was smaller in size and increased progressively to attain final size causing facial asymmetry with reduced mouth opening . Extraoral examination revealed an ill - defined, diffuse, and bony hard swelling of approximately measuring about 6 cm 4 cm on the left side of the face, extending superior - inferiorly from left zygomatic arch till angle of the mandible and anteroposteriorly from nasolabial fold till tragus of the left ear with normal overlying skin [figure 1a and b] intraorally, swelling was solitary, slightly tender with the expansion of buccal and lingual cortical plates with no pathological changes to the overlying mucosa . There was reduce mouth opening measuring about 15 mm in the greatest dimension [figure 2a]. Orthopantomogram revealed a large well - bordered, well - defined unilocular, circular, mixed lesion (calcification specks) approximately, 2 cm in size involving the ramus, coronoid, and angle of the mandible [figure 3] posteroanterior view of mandible showed a well - defined, mixed lesion involving the coronoid process, ramus and the angle of the left side of the mandible [figure 4]. Axial and sagittal view of computed tomography confirmed the routine radiographic features and further showed a heterogeneously enhancing mixed lytic - sclerotic lesion with a sclerotic rim extending into adjacent soft tissue measuring 5.12 cm 3.83 cm with exterior and superior extension into the left infratemporal fossa and medial extension up to pterygoid muscle [figure 5a and b]. Biopsy was taken from the involved site, and the h and e section showed the presence of trabeculae of fibrillar osteoid and woven bone . In few areas, multinucleated giant cells were also noted . Based on clinical, radiographic, advanced imaging studies and histopathological report provided the diagnosis of the trabecular variant of jof affecting left mandible with differential diagnosis of cement - ossifying fibroma, osteoblastoma, and osteogenic sarcoma . The lesion was excised by segmental resection using transmandibular approach and reconstruction was done [figure 7a and b]. Postoperative healing was uneventful; mouth opening was increased to the value of 30 mm [figure 2b]. Postoperatively, the esthetics, oral function of the patient such as speaking, and chewing were intact and normal [figure 8a and b]. The patient is still under regular follow - up as the lesion has high recurrence rate . (a and b) preoperative extraoral view of the patient showing swelling on the left side of face (a) preoperative mouth opening, (b) postoperative mouth opening preoperative panoramic view of the patient preoperative posteroanterior view of the patient (a) axial view of computed tomography, (b) sagittal view of computed tomography histopathological section (a) postoperative panoremic view of the patient, (b) postoperative posteroanterior view of the patient (a and b) postoperative extraoral view of the patient with intact facial symmetry jof mostly strikes children with slightly male predominance with a mean age of 11 years . Clinically, it is asymptomatic, however rarely it is aggressive and symptomatic . In the present case, both maxilla and mandible are affected with more prevalence for maxilla . In the present case, the duration of swelling noted was 2 months . The characteristic radiographic features reveal a central opacification with well - defined unilocular to multilocular pattern giving it a ground glass appearance . The 50% of cases of jof are recorded in multiple sinuses with few occurring in the single sinus . The duration, clinical and radiographic features and extension of the lesion into the adjacent soft tissue favors the aggressive type of jof as previously reported . The features like resorption and perforation of cortices, tooth displacement and aggressive growth of the lesion favor the diagnosis as an aggressive variety of jof . In this case, except for tooth resorption all other features like perforation of the cortices and extension into the surrounding tissue was noted . Because of the high recurrence rate, there is no follow - up protocol mentioned in the literature and immediate reconstruction is not advised . Resection is considered where there is recurrence, invasion into surrounding tissues, or when preserving the inferior border is not feasible . Jof is an uncommon clinical entity, because of its aggressiveness and high recurrence rate it is necessary to arrive at an early diagnosis, applying the appropriate clinical, diagnostic approaches, and appropriate treatment for effective results and long - standing follow - up . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed there are no conflicts of interest . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed
Mr urography (mru) can be performed with two different imaging strategies: unenhanced mr urography (t2w - mru), based on heavily t2-weighted fse sequences and contrast - enhanced excretory mr urography (ce - mru), which is performed with a 3d ge t1-weighted sequences following intravenous gadolinium administration . A heavily t2-weighted sequence, utilizing the single - shot fast spin - echo (ssfse) sequence, provides static hydrographic images of fluid inside the urinary tract . It is known to be mainly useful for only evaluating dilated systems,, because of its inability to visualize entirely non - dilated ureters . So that, in routine practice, excretory ce - mru is the technique most commonly used to evaluate the non - dilated collecting system . Low - dose (520 mg) intravenous furosemide administration has been recommended for excretory ce - mru to improve the visualization of the non - dilated upper urinary tract, . To the best of our knowledge, no recent paper has explored the potential of the repetition of this simple breath hold t2w - mru sequence . Our hypothesis was that by using low - dose of furosemide and additional multiple rotations around the ureter may improve the detection of the whole non - dilated ureter as well as mru with intravenous administration of contrast medium and provides a pertinent information . The purpose of this prospective study was to compare those two types of mru in terms of ureteral visualization in patients with non - dilated ureters, using diuretic administration by means of a direct confrontation of each technique in the same patient . To the best of our knowledge, there is no paper in the literature concerning this evaluation . This prospective, single - institution study received approval by our local ethical committee and no written informed consent was necessary . However all patients were orally informed just before the examination of this additional exploration of their urinary tract regarding to the use of a supplementary drug . During a period of 12 months from april 2012 through april 2013, among a cohort of 155 patients referred to our mr department for a pelvic with retroperitoneal mr exploration, we have selected 126 patients (extensive endometriosis: 38 patients, various pelvic carcinoma staging: 54 patients, suspicion for retroperitoneal fibrosis: 15 patients, lymph nodes or retroperitoneal masses recurrency: 19 patients) with bilateral non - dilated ureter who underwent both a 2d t2w - mru immediately followed by an excretory 3d ce - mru . We have excluded from the study the patients with urinary tract dilatation (n = 6) or urinary symptoms (n = 2), moderate renal deficiency or known allergic reaction to medication (n = 6) and those who had undergone a previous pelvic surgery (n = 15) (fig . Mr examinations were performed on a 3.0 t mr unit (achieva, philips medical systems, best, the netherlands) by using the phased array dedicated coil for pelvic and lower abdomen exploration . The field of view was large enough to cover entirely the retroperitoneum with the exploration of the whole ureter from the uretero pelvic junction to the bladder neck . Each patient was explored in addition to our routine protocol for the clinical context with two breath - hold mru sequences in coronal orientation . Patients were asked to void their bladder before mr examination and received an intravenous antispasmodic medication as glucagon at the dose of 1 mg (glucagen lab novo nordisk pharmaceutics sa) at the beginning of the exploration . If no urinary dilatation was observed on the first non - injected routine sequences, the patient received an intravenous injection of furosemide at the dose of 20 mg (lasilix lab sanofi - aventis) (fig . Patients were orally informed of potential side effects such as increased urination at the end of the exploration . Five minutes later a 2d coronal single shot turbo fast se t2-weighted static fluid mru (t2w - mru) was performed localized on the right and the left urinary tract with an acquisition time of 5 s. each sequence was repeated nine times with coronal rotations and cine loop registration on each urinary tract . At the end of this first mru sequence, an intravenous injection of 0.2 ml / kg body weight gadolinium - based contrast medium, (dotarem, guerbet, roissy cdg, france) followed by a 15 ml saline five minutes later, coronal excretory phase mr urogram was obtained by means of a 3d breath - hold t1-weighted ge sequence with an acquisition time from 15 to 18 s (table 1). Maximum - intensity - projection (mip) images were processed from the original source images of this excretory mru . The two types of mru, including original source images and mip reformations available from the workstation, were retrospectively reviewed . Each sequence was evaluated separately and independently by two radiologists (phh, ga) with five and three years of experience in abdominal mr imaging, respectively . Disagreements were checked by consensus with a third radiologist (cr) with twenty years experienced in that field to find a consensus . The readings were done in several sessions and in each session; it was given to each reviewer a random combination of isolated mru with native images and mip . Readers were neither informed of the clinical context nor the radiological report done by the radiologist who was in charge of the examination . Readers evaluated the whole technical image quality with the precise criterion of the presence of disturbing artifacts such as ghosting or pulsation artifacts using a four points subjective scale (1 poor, 2 fair, 3 good, 4 excellent). The distension of the ureter on ce - mru was compared with that of obtained on t2w - mru and quoted as higher or equal . Based on visual assessment, readers were asked to score four segments of each ureter . The lumbar ureter was divided in two segments: a proximal part (from the pelvic junction to the level of the lower pole of the kidney) (pl) and a distal part (from the lower pole of the kidney and iliac crest) (dl). The pelvic ureter was also checked with two segments: an upper portion from iliac vessels to mid pelvic cavity (pp) and an inferior portion from mid pelvic cavity to the bladder (dp)., readers were asked to evaluate independently one rotation (chosen as strictly anteroposterior view), 6 rotations and finally the entire sequence (9 rotations). For ce - mru, they were asked to quote the number of entire ureter identified on mip images . The kappa statistic was used to measure the degree of interobserver agreement for qualitative assessment . Strength of agreement was classified as poor (value less than or equal to 0.20), fair (value between 0.21 and 0.40), moderate (value between 0.41 and 0.60), substantial (value between 0.61 and 0.80), or excellent (value between 0.81 and 1.0). We used the wilcoxon test to compare the scoring of the segmental analysis of our two mru techniques . A p value less than 0.05 was considered to be statistically significant . This prospective, single - institution study received approval by our local ethical committee and no written informed consent was necessary . However all patients were orally informed just before the examination of this additional exploration of their urinary tract regarding to the use of a supplementary drug . During a period of 12 months from april 2012 through april 2013, among a cohort of 155 patients referred to our mr department for a pelvic with retroperitoneal mr exploration, we have selected 126 patients (extensive endometriosis: 38 patients, various pelvic carcinoma staging: 54 patients, suspicion for retroperitoneal fibrosis: 15 patients, lymph nodes or retroperitoneal masses recurrency: 19 patients) with bilateral non - dilated ureter who underwent both a 2d t2w - mru immediately followed by an excretory 3d ce - mru . We have excluded from the study the patients with urinary tract dilatation (n = 6) or urinary symptoms (n = 2), moderate renal deficiency or known allergic reaction to medication (n = 6) and those who had undergone a previous pelvic surgery (n = 15) (fig . Mr examinations were performed on a 3.0 t mr unit (achieva, philips medical systems, best, the netherlands) by using the phased array dedicated coil for pelvic and lower abdomen exploration . The field of view was large enough to cover entirely the retroperitoneum with the exploration of the whole ureter from the uretero pelvic junction to the bladder neck . Each patient was explored in addition to our routine protocol for the clinical context with two breath - hold mru sequences in coronal orientation . Patients were asked to void their bladder before mr examination and received an intravenous antispasmodic medication as glucagon at the dose of 1 mg (glucagen lab novo nordisk pharmaceutics sa) at the beginning of the exploration . If no urinary dilatation was observed on the first non - injected routine sequences, the patient received an intravenous injection of furosemide at the dose of 20 mg (lasilix lab sanofi - aventis) (fig . Patients were orally informed of potential side effects such as increased urination at the end of the exploration . Five minutes later a 2d coronal single shot turbo fast se t2-weighted static fluid mru (t2w - mru) was performed localized on the right and the left urinary tract with an acquisition time of 5 s. each sequence was repeated nine times with coronal rotations and cine loop registration on each urinary tract . At the end of this first mru sequence, an intravenous injection of 0.2 ml / kg body weight gadolinium - based contrast medium, (dotarem, guerbet, roissy cdg, france) followed by a 15 ml saline flush was performed . Five minutes later, coronal excretory phase mr urogram was obtained by means of a 3d breath - hold t1-weighted ge sequence with an acquisition time from 15 to 18 s (table 1). Maximum - intensity - projection (mip) images were processed from the original source images of this excretory mru . The two types of mru, including original source images and mip reformations available from the workstation, were retrospectively reviewed . Each sequence was evaluated separately and independently by two radiologists (phh, ga) with five and three years of experience in abdominal mr imaging, respectively . Disagreements were checked by consensus with a third radiologist (cr) with twenty years experienced in that field to find a consensus . The readings were done in several sessions and in each session; it was given to each reviewer a random combination of isolated mru with native images and mip . Readers were neither informed of the clinical context nor the radiological report done by the radiologist who was in charge of the examination . Readers evaluated the whole technical image quality with the precise criterion of the presence of disturbing artifacts such as ghosting or pulsation artifacts using a four points subjective scale (1 poor, 2 fair, 3 good, 4 excellent). The distension of the ureter on ce - mru was compared with that of obtained on t2w - mru and quoted as higher or equal . Based on visual assessment, readers were asked to score four segments of each ureter . The lumbar ureter was divided in two segments: a proximal part (from the pelvic junction to the level of the lower pole of the kidney) (pl) and a distal part (from the lower pole of the kidney and iliac crest) (dl). The pelvic ureter was also checked with two segments: an upper portion from iliac vessels to mid pelvic cavity (pp) and an inferior portion from mid pelvic cavity to the bladder (dp)., readers were asked to evaluate independently one rotation (chosen as strictly anteroposterior view), 6 rotations and finally the entire sequence (9 rotations). For ce - mru, they were asked to quote the number of entire ureter identified on mip images . The kappa statistic was used to measure the degree of interobserver agreement for qualitative assessment . Strength of agreement was classified as poor (value less than or equal to 0.20), fair (value between 0.21 and 0.40), moderate (value between 0.41 and 0.60), substantial (value between 0.61 and 0.80), or excellent (value between 0.81 and 1.0). We used the wilcoxon test to compare the scoring of the segmental analysis of our two mru techniques . No patient suffered from any serious side effects of furosemide, especially no sign of allergic reaction was recorded . A total of 252 ureters were evaluated and 1008 segments were checked . For the assessment of image quality, readers judged both sequences good or excellent for all segments (score 3: 35/252; 51/252; score 4: 217/252; 201/252 for t2w - mru and ce - mru, respectively). Comparison of the two sets of images revealed no significant difference with respect to overall image quality (3.8 0.5 vs 3.6 0.7, p value: 0.13). Statistically, the interobserver agreement was excellent with a correlation coefficient as high as 0.89 for t2w - mru and 0.92 for ce - mru, respectively . One rotation of t2w - mru was found clearly inaccurate to assess the non - dilated ureter . Increasing the number of rotations, significantly improved the visualization of all segments . With 9 rotations and regard to identification, there were no statistically significant differences in the visualization of the ureteral segments between the two types of mru . The lumbar distal portion of the ureter was less pertinent for identification on t2w - mru with a sensitivity of 71% vs 78% for ce - mru (table 2). Both segments up and down the pelvic ring have less than excellent identification in both mru techniques by comparison with two others . A bilateral entirely opacified ureteral column until the vesicoureteral junction was found in 236/252 cases with ce - mru, simulating a mild obstruction . On the contrary, on one rotation of t2w - mru, it was constantly found at least one segment, indicating that the others were collapsed . The ureter was visualized as a linear thicker structure than on t2w - mru (fig . Deviation of the pelvic ureter due to the genital tract was found in 118 ureteral segments equally recognized on both sequences (fig . Deviations due to ovarian cysts and follicules were easier to diagnose on t2w - mru than on standard t2 sequence . Mru permits an evaluation of the urinary tract without the exposure to ionizing radiation and iodinated contrast medium . T2w - mru was the first sequence to be proposed to study excretory urinary tract . Its initial relatively low resolution has been improved by the development of heavily t2-weighted turbo spin - echo sequences such as rapid acquisition with relaxation enhancement (rare) utilizing multiple thin - slices with mip or a thick - slice imaging technique and half - fourier acquisition single - shot turbo spin - echo (haste) sequences . High - resolution thick - slice images can be achieved during a single breath - hold . However, in the literature, t2w - mru is known to have several limitations . These include the superimposition of hyperintense extraurinary fluid collections on thick slice mru, no functional information and undetectable small stones . The inability of t2-w mru to fully visualize complete non - dilated ureters is cited as a major negative point in many papers,,, . Finally, t2w - mru has been shown to be a rapid and non - invasive imaging technique which is useful and reliable to reveal hydronephrosis and perirenal high - intensity signals in acute urinary obstruction,,, . The concept of ce - mru has been developed later . In his initial experimental study, nolte - ernsting et al . Presented the advantages of this imaging technique in retrieving high - spatial - resolution images in non - obstructed urinary tracts . The use of a paramagnetic contrast agent permits the evaluation of renal excretory function and better visualization of the non - dilated urinary tract . Several papers,,, have advocated the need for a diuretic to optimize the endoluminal concentration of gadolinium and to produce accelerated distention . These authors stated that furosemide initially induces an increase in urine volume resulting in mild distention of the urinary tract, and then there is a dilution effect on the excreted gadolinium . The limitation of ce - mru is for cases with delayed excretion due to severe obstruction, the long duration of examination for revealing the underlying abnormality increasing patient and personnel inconvenience, . As several previous papers have shown the superiority of enhanced over unenhanced imaging, ce - mru is becoming in clinical routine practice the technique most commonly used to evaluate the non - dilated collecting system . To our knowledge, in the previous recent literature in adult, there are no clinical studies comparing those two mru techniques in the same patient . Additionally, our evaluation has been focused on the non - dilated ureter, which is considered as being the most difficult task to obtain a visualization of all segments . As the use of low - dose (520 mg) intravenous furosemide administration is essential to perform ct urography; we have also used this medication at the dose of 20 mg to adequately distend the non - dilated ureter . In addition, it causes no real additional cost as it has a very low price . This t2w - mru sequence, with its short acquisition time, permits repeated series of breath - hold images in several coronal oblique orientations, thus providing obvious improvement to the use of this sequence previously described by other investigators . Our results attest that furosemide alone is sufficient to distend and visualize all parts . In identifying ureteral segments our results with t2w - mru were quite similar to those of ce - mru, except for the lower part of abdominal ureter . Image quality was excellent in all patients, and, unlike other investigators, we never have difficulty in removing superimposed fluid - filled bowel loops by changing the orientation by rotations . A potential advantage of t2w - mru over ce - mru is that it can reveal an ovarian cyst or follicles which cause a deviation of the pelvic ureter . Another advantage might be the evaluation of an inflammatory process, due to the superiority of t2-weighted sequences in revealing edema and a small amount of ascites . The ability to identify one or two different segment by rotation is an indicator for normal contractions of some parts of the ureter . This visualization of the peristalsis is a good indicator to affirm that there is no ureteral repercussion for instance of a pelvic mass . In contrary when using ce - mru, we have commonly found the entire column simulation mild obstruction . The size of ureter on ce - mru was larger than on t2w - mru with the same dose of diuretic . We suggest that the contrast medium itself is a significant factor to distend the ureter and produces rapidly an over distended bladder with a risk of false positive diagnosis of mild obstruction . We could not attribute this aspect to our protocol firstly because patients were asked to void their bladder just before the beginning of the mr examination, secondly because the two sequences were performed without any supplementary delay between them (5 min to obtain the excretory phase after gadolinium administration). In addition, t2w - mru has the advantage of obtaining high quality images in a few seconds, without need for multiplanar and mip reconstructions in the contrary of ce - mru . As breath - hold t2w - mru with intravenous diuretic and multiple rotations is sufficient to produce a qualitative study of non - dilated ureter with identification of the peristaltism, we can speculate that it could be an adequate sequence to detect an initial ureteral involvement before hydronephrosis occurs . The identification of the same missing segment on all rotations with a persistent non - dilated column above could reflect an abnormal peristaltism indicating a thorough analysis of this localization . This can be potentially very useful in the cases of extrinsic ureteral involvement such as in endometriosis which is progressive and clinically silent or can be confuse with other symptoms of the disease . The identification of the same segment on all rotations can be a clue for this initial diagnosis, before the classical appearance of persistent column with gradual tapering or filling defect at the level of obstruction . However, it needs further investigation to prove that t2-mru can detect initial obstruction, prior to clinical presentation of hydronephrosis . It could also be used to evaluate potential renal donors or in patients unable to receive gadolinium . Even if our material has an important number of cases, this study has some limitations . First, there is no data regarding the relative performance of the techniques in a clinical context, but we would like to focus on the ability of t2w - mru to visualize the entire ureter . Second, even if our field of view was large enough to obtain an evaluation of the whole ureter it does not permit a constant evaluation of the renal pelvic cavities, especially for our tallest patients . However a precise caliceal anatomy or pathology was not the purpose of our mr examinations . Of course, equally to ce - mru, the major drawback of t2w - mru remains its poor sensitivity in detecting calcifications . The t2w - mru sequence performed with multiple coronal orientations and diuretic administration is sufficient to identify entirely the non - dilated ureter . Our series was large enough to suggest that pelvic mr imaging combined with t2w - mru allows a complete work - up in a single imaging evaluation, especially if gadolinium injection is not necessary or contraindicated . The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome . The authors confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.
The first case was described by cruveilhier in 1835 and termed as hamartoma . Till date, less than 200 cases have been described with synonyms of brunner gland adenoma, brunner gland hamartoma (bgh) or brunneroma . There is no sex predilection and patients present in the fifth to sixth decades of life . They are usually seen as incidental findings on imaging with duodenal bulb as the most common location . However, it may present with a variety of clinical presentations depending on location and tumor size such as abdominal pain, dyspepsia, nausea, vomiting, upper gastrointestinal (gi) bleeding and occasionally obstructive symptoms with associated chronic pancreatitis . A 42-year - old man presented with history of pain epigastrium since one year and recurrent vomiting within half an hour of taking food since one month . A biopsy of duodenum was performed by the same procedure which was diagnosed as well differentiated adenocarcinoma . The patient was further subjected to abdominal computed tomography (ct) which showed circumferential thickening of the second part of the duodenum (15.2 mm maximum thickness) abutting head of pancreas with loss of fat planes . Laboratory investigations showed normal blood counts, liver, and kidney function tests but elevated serum amylase and lipase levels . This patient underwent radical surgery (whipple s procedure) at our institute with clinical diagnosis of carcinoma periampullary region and the specimen was sent for histopathological examination . Gross examination of the specimen revealed circumferential thickening of the second part of the duodenum and bulky head of the pancreas . Cut section revealed a diffuse grey white area measuring 4x4 cm with few cystic and hemorrhagic areas . Six lymph nodes were isolated from the specimen and a single celiac node was examined which was received separately . Microscopy showed a tumor composed of lobules of brunner s glands with a dilatation of few glands . These lobules were separated by fine fibrous septa and constituted> 50% of the thickness of the duodenal wall (figures 1, 2 and 3). Hyperplasia of brunner s glands spanning> 50% of wall thickness (h&e stain, 4x) lack of features of anaplasia in proliferating brunner s glands (h&e stain, 20x) smooth muscle actin stain highlighting the intervening smooth muscles (ihc, 10x) brunner glands are branched acinotubular glands with submucosal location, found exclusively between the pyloric ring and the papillae of vater . Their major role is the protection of duodenal epithelium from acidic gastric chyme by secretion of neutralizing alkaline fluid composed of viscous mucin . The pathogenesis of bgh has been linked to excessive local irritation from acidic gastric chyme, vagal stimuli, or unidentified antral hormones . Hence, it is associated with conditions related to gastric hyperacidity and changes in gastric emptying such as esophagitis, hiatus hernia, and gastric - peptic ulcer . The association of this tumor with uremia and chronic pancreatitis has also been reported . In a study of patients undergoing pancreaticoduodenectomy, 76% of the patients had diffuse hyperplasia of brunner s gland, raising the question whether this is an adaptation to pancreatic insufficiency . An alternate suggestion for this hyperplasia is that these glands proliferate in reaction to inflammationas evidenced by presence of lymphocytes in the lesion . Support to this hypothesis is not rationalized as lymphocytes may be present in normal submucosa of the gastrointestinal tract . In a study on the role of helicobacter pylori infection in the pathogenesis of bgh, 5 of 7 cases with bgh had concurrent helicobacter pylori infection . A definite relationship has not yet been proven because of the high prevalence of helicobacter pylori and rarity of this lesion in the general population . Initially, the hyperplasia of brunner s gland can be focal, localized, and solitary usually involving a lobule . However, if stimulus persists then the lesion enlarges involving multiple lobules leading to formation of diffuse and annular lesion infiltrating into the wall and circumference of the duodenum . Endoscopically these can be nodular, polypoid or diffuse with thickening of the duodenal wall . Due to this presentation, these lesions can be misdiagnosed as gastrointestinal stromal tumor (gist), lymphoma, carcinoid, peutz jeghers polyp, prolapsed pyloric mucosa, or aberrant pancreatic tissue . Although surface biopsy of this lesion can be misleading as in our case, a deeper biopsy will be more diagnostic . Microscopic diagnosis of hyperplasia requires presence of lobules of brunner s glands within the submucosa in at least 50% of the length of the biopsy showing no features of dysplasia . Adenoma has been used for lesions> 1 cm and the term hamartoma if it contains mesenchymal elements also . Conflicts regarding this terminology exist as some authors suggest that mere change in size indicate progression of hyperplasia and do not imply neoplastic behavior . Ductal malignancy arising from brunner gland adenoma has been reported in one case which is a very rare circumstance (0.5%). Brunner gland adenoma in the duodenal wall with a focus of atypical cells supported by mib-1 and p53 positivity has also been reported . The treatment of bgh is done with surgical or endoscopic removal in symptomatic patients taking into account the size and gross appearance . Although endoscopic techniques of removal are more helpful, this procedure can be limited by difficult anatomical sites . Recurrence in such a lesion is not known . Even though diagnosis of bgh can be made by various imaging studies, in our case hence, these possibilities should be kept in mind while dealing with duodenal masses.
Metastasis of transitional cell carcinoma (tcc) of the renal pelvis to the eye is a very rare event and has only been reported in one case in the literature . Once diagnosed, these cases usually show widespread metastases and carry a very poor prognosis . We present a case report of a male patient with unilateral choroidal metastasis secondary to tcc of the renal pelvis . Review of the relevant literature will be addressed to highlight the involvement of the choroid in cases of metastatic disease, the unusual presentation of tcc, diagnostic methods, and the various therapeutic modalities used to treat these cases . A 61-year - old male, who has a history of congestive heart failure, is a nonsmoker, and has no significant family history of malignancies, presented to the ophthalmology clinic with sudden - onset loss of vision of the left eye associated with intermittent left eye pain, relieved by analgesics . Ophthalmic evaluation revealed a visual acuity of 20/40 in the right eye and 20/400 in the left eye . Funduscopic examination of the left eye showed an amelanotic macular choroidal lesion associated with inferior exudative retinal detachment (fig 1a). A b - scan ocular ultrasound showed a dome - shaped, 2.8-mm choroid thickening with variable internal reflectivity, associated with inferior exudative retinal detachment (fig 1b). Fluorescein fundus angiography revealed early choroid blockage (fig 1c) and late leakage associated with hot spots at tumor margin . All of these clinical features are suggestive of the clinical diagnosis of left choroidal metastasis . Physical examination of the patient also revealed multiple suspicious chest wall nodules . In search for a primary malignancy, imaging studies were done, which revealed multiple pulmonary and liver metastasis, subcutaneous metastasis, multiple lymphadenopathies, and left renal upper pole mass (fig 2). Ct - guided left renal mass biopsy and ultrasound - guided biopsy of the left chest wall subcutaneous mass were performed . They are negative for ck20, pax2, ttf-1, synaphopysin, and s100 (fig 3). External beam radiation therapy (ebrt) is a common mode of treatment and will be considered if there is no improvement of visual symptoms with chemotherapy . Choroidal metastasis is the most common intraocular malignancy, due to its rich vascular supply and high oxygen concentration [4, 5]. Less common primary cancers include the thyroid, prostate, kidney, testicles, pancreas, ovary, and liver . Rarely is it seen in cases of tcc, as there have only been 5 previous reports in the literature involving choroidal metastasis, 3 from tumors involving the bladder, and 2 from other parts of the urinary tract . Choroidal metastasis is commonly associated with disseminated disease and, unfortunately, carries a very poor prognosis . Tcc usually metastasizes to the regional lymph nodes (78%), liver (38%), lung (36%), bone (27%), adrenal gland (21%), and intestine (13%). The heart, brain, kidney, spleen, pancreas, meninges, uterus, ovaries, prostate, and testes have been reported as unusual sites of presentation of tcc . Other less common clinical presentations include flashes (5%) and floaters (5%). Diagnosis is based on clinical findings, which are typically seen as yellow, plateau - shaped lesions associated with subretinal detachment . A - scan and b - scan ultrasonography show irregular internal reflectivity and a solid plateau - shaped mass, respectively . Fluorescein fundus angiography typically shows mottled hypofluorescence in early stage and leakage in late stage . The treatment of choroidal metastasis should be individually tailored, taking into consideration the patient's systemic state, disease activity, and the presence of visual symptoms . Its aim is palliation, to improve the quality of life of the patient and to restore or maintain visual function . Local therapeutic regimens include ebrt, plaque radiotherapy, and complete enucleation for painful, blind eyes [13, 14]. Ebrt delivered at a dose of 40 gy over 20 fractions was effective in improving and maintaining vision in patients with choroidal metastasis [15, 16]. In cases of widespread metastases, systemic chemotherapy alone or in combination with local therapy is an accepted therapeutic option . In patients with choroidal metastasis alone, local therapy has been reported to be safe, maintaining visual functions without the systemic side effects of chemotherapy . Choroidal metastasis related to tcc of the renal pelvis is an extremely rare event and is associated with widespread disease and a very poor prognosis . Systemic chemotherapy with carboplatin and gemcitabine is a palliative treatment option for this patient . Ebrt offers local palliation and will be considered according to the patient's visual response to chemotherapy.
Chronic kidney disease (ckd) is defined as the slow and steady damage of kidney function in an irreversible manner, which ultimately results in end - stage renal disease (esrd) (1, 2). This chronic disorder is a serious health problem with a high prevalence rate in adults and children . It causes mortality and other health complications, and high costs are incurred due to the frequent medical diagnosis and poor prognosis of patients (1, 2). The causes of ckd are very different in children than in adults . In a recent north american pediatric renal transplant cooperative study (naprtcs), congenital causes, including congenital anomalies of the kidney and urinary tract (cakut) (48%) and hereditary nephropathies (10%), were the most common causes of ckd in the children (3). Based on previous studies, such a chronic disorder impairs the quality of life of children due to the development of various clinical symptoms, especially developmental disorders and psychiatric disorders (4 - 6). Ckd clinical manifestations in children are presented as edema, hypertension, hematuria, and proteinuria, and specifically during the neonatal period as weight gain, polyuric dehydration, and urinary tract infection (7). In addition to the above mentioned symptoms, ckd may lead to a type of hyperactivity and central nervous system (cns) dysfunction (especially affecting sympathetic function) (8). Based on the literature, the prevalence of cognitive disorders such as memory disorder, and different psychiatric disorders including anxiety disorders (ads), depression, and adjustment disorders in children with different levels of ckd was significantly higher compared to the group of healthy children and children with very early stages of ckd (9 - 12). Based on the available literature, patients with ckd who develop psychiatric and cognitive disorders face longer hospitalization times, more health complications, and greater mortality compared to other patients at the same stage without these disorders (11, 12). Therefore, proper diagnosis of psychiatric and cognitive disorders in these patients is paramount . More specifically, one disorder whose relationship with ckd in children has not been adequately examined is obsessive - compulsive disorder (ocd). Obsessive - compulsive disorder (ocd) is a chronic disabling illness characterized by repetitive ritualistic behaviors over which the patients have little or no control (13, 14). A review of the literature revealed no study that assessed the association between ocd (based on the obsessive compulsive inventory - child version (oci - cv)) and ckd . The aim of this study was to investigate ocd in children with early stages of ckd and to compare it with healthy children . This case - control study was performed on 160 children in the age bracket of 7 to 17 years old who were referred to the pediatric clinic of amir kabir hospital in arak (iran) in 2015 . The ethics committee approved the study (approval code: 93 - 162 - 1, registration code: 1087). Eighty children with early stages of ckd (stages 1, 2 and 3) comprised the case group, and the control group consisted of 80 healthy children without ckd; children were included in the study based on the inclusion criteria . The sample number was calculated with regards to the prevalence of cognitive disorders due to ckd (= 0.05%, = 0.2%). Ckd was defined as the presence of kidney damage (for example, any structural or functional abnormality involving pathological, laboratory, or imaging findings) for 3 months or a glomerular filtration rate (gfr) <60 ml / minute/1.73 m for 3 months (2). In this study, as per the ckd definition (2), children with early stages of ckd (stage 1, 2 and 3 ckd) who were diagnosed with ckd due to renal and urinary - genital tract anomalies, such as obstructive uropathy, renal dysplasia, or reflux nephropathy, were included in the case group . Those children who were diagnosed with ckd due to reasons other than renal and urinary - genital tract anomalies, or those with stages 4 and 5 stage 1, 2, 3 and 4 ckd were defined as kidney damage with normal or increased gfr (gfr 90 cc / minute/1.73 m), kidney damage with mild decreased gfr (gfr = 60 - 89 cc / minute/1.73 m), kidney damage with moderately decreased gfr (gfr = 30 - 59 cc / minute/1.73 m) and kidney damage with severely decreased gfr (gfr = 15 - 29 cc / minute/1.73 m), respectively (15). Esrd or stage 5 ckd was defined by the amounts of gfr <15 cc / minute/1.73 m which are indicative of the start of dialysis (15 - 17). We included children of both sexes in the age range of 7 to 17 years old, and children with stage 1 to 3 ckd (for at least 6 months). We excluded patients with the following conditions or circumstances: a history of considerable psychiatric disorders; intellectual disabilities, or nervous system disorders; a history of any type of anxiety disorder before developing ckd; congenital and chromosomal abnormalities; a chronic medical condition; a family history of major psychiatric disorders in first - degree relatives; parents not consenting to participating in the study; and not completing the questionnaire . Intellectual disability was defined in terms of the intelligence quotient (iq) of 70 (18). Healthy children were selected from children who had been referred to the hospital as outpatients for minor conditions such as the common cold or abdominal pain . The matching method was used for selecting the healthy children, and children were matched in groups based on age, sex, and socioeconomic status . In this 3-month study (april 2015 to july 2015), a total of 40 children were excluded based on the inclusion and exclusion criteria . Among 22 (100%) patients who were excluded in the case group, 17 (77.27%) and 5 (22.72%) patients were excluded due to parental unwillingness to complete the oci - cv and a history of considerable psychiatric disorders (anxiety disorders before the diagnosis of ckd), respectively . For the control group, the remaining children were excluded due to lack of parental consent . After obtaining informed consent from the children s parents, demographic, clinical, and perinatal data (age, sex, residence, birth weight, current weight, height, body mass index (bmi), mother s age at birth, gestational age, maternal education, household incomes, marital status, type of delivery, age at diagnosis of ckd, and duration of ckd) were recorded . Ocd in children was evaluated using the obsessive compulsive inventory - child version (oci - cv) by a psychologist (consultant). This self - reporting questionnaire has been designed for people aged 7 to 17 years, containing 21 items and 6 subscales, including doubting / checking (5 phrase), obsessing (4 phrase), hoarding (3 phrase), washing (3 phrase), ordering (3 phrase), and neutralizing (3 phrase) (19 - 21). The subjects are supposed to indicate their degree of agreement or disagreement with each item through a 3-point likert scale ranging from never to always . The scoring options on this test were as follows: never = 0, sometimes = 1, and always = 2 . Based on multiple sources of evidence, the oci - cv is considered to be a reliable and valid method for identifying children with ocd . The oci - cv was modestly correlated with obsessive compulsive symptom severity on the children s yale - brown obsessive compulsive scale (cy - bocs), as well as with clinician - reported ocd severity (19, 20). The persian version of the oci - cv questionnaire was tested for reliability in a pilot study by the researchers with 30 patients in each of the case and control groups; the cronbach s alpha was 0.89 . The collected data was analyzed with spss software (statistical package for the social sciences, version 18.0, spss inc . Categorical data are expressed as numbers (percentage) and compared with a chi - square test . The mean weight, height, and bmi of the children were 38.15 6.8 kg, 127.61 8.5 cm, and 17.31 3.52, respectively . Of the 160 subjects, 86 (53.75%) were boys and 74 (46.25%) were girls . In the group comprised of children with ckd, the duration of the disease and the age of diagnosis were 1.48 2.12 and 6.9 5.4 years, respectively . Abbreviations: bmi, body mass index; ckd, chronic kidney disease; cs, caesarean section; nvd, normal vaginal delivery . Household incomes: low mean monthly incomes <5000000 rials; moderate mean incomes between 5,000,000 and 10,000,000 rials; high means incomes> 10,000,000 rials . The mean age (p = 0.211), weight (p = 0.51), height (p = 0.113), gender distribution (p = 0.112), average bmi (0.32), mother s age at childbirth (p = 0.66), and the child s birth weight (p = 0.08) were not significantly different between the children in the case and control groups . Furthermore, the children in both groups were identically distributed in terms of residency status (p = 0.3), family s monthly income (p = 0.376), gestational age (p = 0.12), marital status (p = 0.311) and type of delivery (p = 456). However, the maternal educational level was significantly different between the two groups (p = 0.001), so that 30 (37.5%) and 37 (46.25%) of mothers in the group of the children with ckd had college and high school education, respectively, whereas 16 (20%) and 50 (62.5%) of the mothers in the control group had college and high school education, respectively . The mean scores of doubting / checking (case: 3.52 2.54, control: 2.5 2.32, p = 0.007) and ordering (case: 2.59 1.81, control: 1.5 2.56, p = 0.005) of the children with ckd was significantly higher than the scores of the healthy ones . Moreover, the mean total scores for the oci - cv of the children with ckd at 15.32 7.69 was significantly higher than those of the healthy ones at 11.12 2.54 (p = 0.021). Nevertheless, the mean scores of obsessing (p = 0.11), hoarding (p = 0.117), washing (p = 0.211), and neutralizing (p = 0.41) were not significantly different between the case and control groups (table 2 and figure 1). According to the results of spearman s test, there was significant correlation between the duration of ckd and doubting / checking (p = 0.004, correlation coefficient (cc): 0.4), obsessing (p = 0.06, cc: 0.02), washing (p = 0.031, cc: 0.8), ordering (p = 0.001, cc: 0.2), and the total scores of the oci - cv questionnaire (p = 0.04, cc: 0.4) (table 3). According to the results of the current study, ocd and certain subscales of this disorder are more likely to occur in children with early stages of ckd than in healthy children . In this respect, doubting / checking and ordering had higher rates among children with ckd than among healthy children . Furthermore, the results indicated that the duration of ckd was significantly correlated with the mean total scores of the oci - cv and certain subscales, such as doubting / checking, obsessing, washing, and ordering . On the basis of previous evidence, chronic disorders or diseases in adults and children can be associated with reduced quality of life and social, occupational and educational poor performance (22). In fact, it has been revealed that such an association can in particular cases have a negative effect on the clinical course of the underlying disease (22). According to relevant studies, the risk of mood and anxiety disorders occurring among people with chronic medical illnesses tends to be greater as compared to the corresponding occurrence rates among healthy people in the general population (22). Although there have been a number of studies assessing the psychiatric disorders in children with ckd, no study has been carried out on the relationship between ocd and ckd based on the oci - cv . (9) studied 19 children with ckd who did not need dialysis and 19 others with esrd in order to determine if any of them had a psychiatric disorder . The evidence indicated that 18.4%, 10.3%, 7.7%, 5.1%, and 2.6% of the subjects had adjustment disorder, depression, cognitive disorder, anxiety, and elimination disorder . The total prevalence of the above disorders were calculated at 68.4% and 36.8% in dialysis and non - dialysis groups, respectively . In another study on 30 children with both ckd and continuous peritoneal dialysis (cpd), 30 children who had undergone renal implantation, and 33 healthy children, fukunishi and honda (10) revealed that there was a significant difference in the prevalence of adjustment disorder between the children in the three groups; the dialysis and control groups had the highest and lowest prevalence of this disorder, respectively . With the objective of investigating cognitive function in children with ckd, slickers et al . Studied 29 children aged 7 to 19 simulated through creatinine clearance (crcl). The results showed that the severity of ckd was significantly correlated with decreased iq and impaired memory function . Furthermore, it was revealed that a longer duration of ckd and its onset at a younger age were considerable risk factors for the development of cognitive disorders (11). In another study on children with esrd and healthy children, fukunishi and kudo (23) concluded that the prevalence of anxiety and depression was significantly higher in children with esrd and peritoneal dialysis (pd) than in healthy children . (24) examined 15 children aged 8 to 16 with renal disease (8 children with esrd and 7 children with mild kidney disease as the control group) and showed that the depression rate in the case group was significantly higher than that in the control group . In 2015, yousefichaijan et al . (25) compared the occurrence of attention deficit / hyperactivity disorder (adhd) among 75 children aged 5 - 16 with ckd (stages 1 to 3) and 75 healthy children . The results of this study indicated that the prevalence of adhd (case group: 12%, control group: 16%, p = 0.664) was not significantly different between the two groups . In another study on adhd, yousefichaijan et al . (26) examined 100 children with esrd undergoing peritoneal dialysis and 100 healthy children . They found that the rates of attention deficit (p = 0.01) and hyperactivity (p = 0.002) among children with esrd were significantly higher than the corresponding rates among healthy children . According to previous studies, it can be argued that the risk of various psychiatric disorders in children with ckd at early stages and children with esrd tend to be significantly higher as compared to healthy children . Moreover, such disorders can be found more frequently in children with esrd, particularly in those undergoing dialysis, than in patients experiencing the early stages of ckd . Similarly, the results of this study were indicative of the higher prevalence of ocd as an anxiety disorder in children with ckd as compared to healthy children . Although it can be concluded that psychiatric interventions, particularly in the case of anxiety disorders, can be beneficial for children with varying degrees of ckd, there is an insufficient number of studies exclusively focused on each of the psychiatric disorders (such as ocd in our study) and a lack of evidence concerning the clinical impact of these disorders on the clinical course of ckd . Therefore, it is highly recommended that future studies be conducted so as to yield more definitive conclusions . One limitation of our study involved the lack of cooperation of some parents in completing the oci - cv questionnaire . Although this criterion led to the exclusion of some otherwise eligible children, we tried to encourage the parents by describing the possible usefulness of the study and by offering to help them fill in the questionnaire . According to our findings, the risk of ocd among children with ckd is significantly higher than the risk among healthy children . Although the results suggest that psychiatric interventions could be helpful in treating children with ckd, the limited number of studies on this particular issue suggests that further investigation into this medical condition is required so as to obtain more conclusive results.
Problems with shoe wear have long been recognized as an endemic issue among the geriatric population with a prevalence rate of nearly 80% . Women in particular are more susceptible to these problems than men . Individuals having foot pathology are often severely physically impaired, making it increasingly difficult to perform activities of daily living . Consequentially, this leads to physical inactivity, which is cited as one of the first signs of deterioration and the overall decrease in quality of life . Moreover, some studies have linked physical inactivity to suicide, depression, and increased risk of cardiovascular - related problems . Individuals with diabetes, chronic disease, nondiabetic neuropathy, and inflammatory conditions are at a severe disadvantage . Further complications of foot pathology, which include cellulitis, ulcerations, and difficulty in maintaining balance, have increased the risk of serious injuries and fractures from falls . Studies have shown that adults older than 65 years fall at least once per year on average, some of which are attributed to generalized thinning of skin and fat pad atrophy . The increased risk of falls may highlight the need to promote preventative measures to combat this issue . Unfortunately, little current data or research focuses on the role of footwear in the prevention of inactivity in the elderly patients . Common foot pathologies like corns, hallux valgus (bunions), and hammertoes have been known to increase plantar pressure, cause discomfort, pain, and swelling . Multiple etiologies have been noted to cause foot problems, and studies have indicated ill - fitting shoes as one of the major underlying cause . A prevailing hypothesis is that wearing the appropriate footwear will improve factors such as plantar pressure, thereby mitigating pathology while facilitating balance . This review aims to form a general basis of understanding footwear - associated foot pathologies pertaining to the elderly patients . Elderly individuals with preexisting clinical conditions such as diabetes, neuropathies, and musculoskeletal disorders are at a higher risk of developing foot problems when compared to normal, healthy individuals . Furthermore, factors such as exercise and living conditions contribute to the development of foot problems . Finally, the uneven pressure provoked by ill - fitting footwear has been documented as a key factor that can cause, accelerate, or exacerbate foot - related conditions . Some common foot pathologies include hallux valgus (bunions), corns (callus), and hammertoes . Callus formations, or corns, result from continuous pressure from tight footwear, leading to hyperkeratosis of the skin . If left unattended, callus formations can lead to ulcerations, which are particularly troublesome for diabetic patients . The hallux valgus deformity, or bunion, is also frequently linked to ill - fitting (ie, tight, narrow) footwear . It is caused by the lateral deviation of the great toe, which causes a valgus deformity in the first metatarsophalangeal (mtp) joint . Lesser toe deformities, such as hammertoes, are also associated with ill - fitting footwear . These deformities of the second, third, fourth, and fifth toe are caused by contracture of the phalangeal joints . Clinical presentations include the deformity accompanied by pain over the dorsal surface of the foot, which tends to worsen with ill - fitting footwear . One of the unifying elements to these 3 common forms of foot pathology is their link with ill - fitting footwear and their tendency to go unnoticed in the elderly population . Nonoperative approaches have been employed as the first line of treatment for the most common foot pathologies . Measures are taken to adjust or alleviate pressure from the affected area . As a result, foot inserts (ie, dr scholls) are also used to facilitate balance by improving arch support, which can decrease the width of the foot during weight bearing maneuvers . Furthermore, met pads and splits operative management is considered only when conservative measures fail to relieve progressed, advanced - stage pathology . Usually, symptomatic relief can be achieved with more accommodating shoe wear after the soft tissue envelope around the foot is stabilized . This process of relieving the pressure areas and encouraging natural healing of ulcerations or soft tissue problems to heal before using modified shoe wear and orthotics often involves close follow - up in the office or wound care center . Special consideration is given to diabetic patients, who have a higher risk of developing ulcerations due to peripheral neuropathy . Therefore, routine physical examination is encouraged to detect and address these issues early before they lead to progressive deterioration . Studies have shown that women are more susceptible than men to problems associated with inappropriate footwear in terms of the choice of footwear in the elderly patients, lord et al concluded that individuals had better balance when wearing shoes with higher collars than with lower ones . Wide shoes are effective against bunions, and extra - depth shoes are more appropriate if the individual has hammertoes or mid - foot arthrosis . Low - heeled footwear is known to reduce the risk of falls, likely due to the associated lowering of the center of gravity . While assessing different footwear, athletic and canvas shoes (sneakers) are associated with the lowest risk of a fall when compared to other types of shoes . Moreover, some suggested that sole hardness have a little effect on overall balance in the absence of a specific condition, such as hallux rigidus, which may benefit from application of hard sole . As an extra preventative measure, better fitting footwear with slip - resistant soles is recommended both inside and outside the household to reduce the risk of falls . Taken together, these studies seem to support the use of footwear with low heels, slip - resistant soles and wider frames to ensure comfort, better balance, and reduced risk of injury in the elderly population . In the event of multiple deformities, an individual may benefit from the combination of both extra - depth, wide, and low - heeled footwear to increase foot stability and facilitate balance . The use of appropriate shoe wear in the elderly patients is related to the socioeconomic factors . Millions of elderly individuals live at or below the poverty level . In fact, women older than 75 years are 3 times more likely to become poor . Without established financial programs such as social security, nearly half of all elderly individuals would be considered poor today . Changes in living conditions (ie, community homes, nursing homes, etc) can often worsen the socioeconomic status of elderly patients . Deteriorating health conditions followed by reduced physical activity can lead to a shift in clinical care, one that emphasizes the management of illness . In the face of such socioeconomic constraints, furthermore, retail stores often do not stock the customized shoes (ie, extra wide) needed for elderly customers, making availability an issue for elderly patients . The neglect of appropriate footwear in the elderly has been linked to early amputation, and in rare cases, early demise . To help alleviate the financial burden, elderly patients enrolled in medicare part b may benefit from the medical coverage of certain medical supplies and preventative services . Eligible patients typically have diabetes or other severe foot diseases . Under the medicare part b provision, such patients are entitled to a pair of custom - molded shoes, 3 pairs of inserts, or a pair of extra - depth shoes per calendar year . The patient will be held responsible for only 20% of the cost while medicare covers the rest . . Some of these hazards such as wet pavements and bad weather conditions are capable of causing serious injury . These pathologies, in return, have contributed to the growing risk of even more serious injuries such as falls and fractures . Ultimately, the noticeable decrease in physical activity in the elderly patients has been linked to depression and an overall decline health and quality of life . Clinically, preventative measures such as appropriate footwear should be continually emphasized to stop or delay the progression of some foot problems . While recommending appropriate footwear is a cost - effective approach when compared to other options, it is often hindered by socioeconomic obstacles . Nevertheless, studies have shown that it can be a vital step to ensure prolonged musculoskeletal and overall health among the elderly patients.
During vertebrate development, heart is formed by a highly stereotypic and organized process, and the expression of genes in these processes need be tightly regulated in a spatiotemporal manner (barnett et al ., 2012; wang, 2012). Recent reports suggest that epigenetic regulation modulates genetic program during differentiation and development of various organs including heart and somitic lineages (wamstad et al ., 2012). For instance, modifications of histone substantially alter the accessibility and structure of chromatin, therefore modifying transcriptional activity of specific gene clusters . Histones are known to undergo specific modification such as methylation and acetylation: while methylation and demethylation of histone are coordinated by histone methyltransferases (hmts) and demethylase (hdms), acetylation of histone is modulated by acetyltransferases (hat) and deacetylases (hdac) (bhaumik et al ., 2007; helin and dhanak, 2013). Due to their significance in regulating genetic program during development and diseases, recent efforts were directed to identify specific factors that mediate this process (arrowsmith et al ., 2012; cayuso mas et al ., 2011; kim et al ., 2012a; 2012b). In case of methylation, histone h3 lysine 4 residue (h3k4) is sequentially modified through mono-, di-, and tri - methylation status, each of which step is mediated by specific enzymes . Mlls (mll15), setdb1, and setdb2 have broad roles for h3k4 methylation, and can methylate all type of h3k4, such as non-, mono-, di - methylated h3k4 (black et al ., 2012; sims and reinberg, 2004). In contrast, smyd3 methylate mono- and di - methylated h3k4, and setd7 can only catalase the non - methylated h3k4 to transit to mono - methylated h3k4 (hamamoto et al ., 2004; wang et al ., 2001) set and mynd domain - containing proteins (smyds), involving smyd15, are high conserved protein family across plant, fungi, and animal as well as some protozoa, and have two functional protein domains, set and mynd domains (del rizzo and trievel, 2011; dillon et al ., 2005). Especially, set domain is important for histone lysine methylation activity and mynd domain can mediate the protein - protein interaction and bind to dna motifs . Smyd3 was originally reported as histone lysine methyltransferase (hmt), which methlylates the mono-(h3k4me1) and di - methylated lysine 4 residue (h3k4me2) to generate h3k4me3, of histone h3, but smyd3 also can bind to 5-ccctcc-3 motif on promoter region of nkx2.8 gene to induce its expression in cancer cells (hamamoto et al ., 2004). Recent researches showed the global decrease of histone h4 lysine 5 (h4k5) methylation in smyd3 knock - down condition and structural preference for histone h4 lysine 20 (h4k20) of smyd3, suggesting the substrate diversity of smyd3 (foreman et al . It is known that smyd3 is highly expressed in a various cancers, including liver, prostate, rectal, and breast carcinomas, and stimulates the oncogenic activities, such as cell proliferation, adhesion and migration, of tumor cells (frank et al ., 2006; hamamoto et al ., 2006; silva et al ., in addition, smyd3 is essential for myogenic differentiation through myod regulation (proserpio et al ., actually, the deficient zebrafish embryos displayed the developmental cardiac and somite abnormalities, illuminating its functions in cardiac and somite muscle formation (fujii et al ., 2011). Set domain containing protein 7 (setd7), also termed as set7/9, is another type of histone lysine methyltransferase (hmt) and only have set domain for methyltransferase activity, but not mynd domain (wang et al ., 2001). It was initially discovered as a specific methyltransferase for only non - methylated histone h3 lysine 4 (h3k4) that converted to mono - methylated histone h3 lysine 4 (h3k4me1) (wang et al ., 2001). Subsequently, recent accumulating evidence suggests that non - histone proteins, such as yap, dnmt1, e2f1, and stat3, are also the substrates for setd7 (estve et al ., 2009; oudhoff et al ., 2013; pradhan et al ., 2009; yang et al ., 2010). The mouse knock out allele of setd7 is a viable and did not show any developmental and gross defect (campaner et al . Knock - down of setd7 showed the defects in skeletal muscle formation and myofibril structures (tao et al ., 2011). Here, we examined the function of smyd3 and setd7 in heart development in zebrafish . Collectively, our results demonstrate that smyd3 and setd7 function synergistically for skeletal muscle development and non - redundantly for heart morphogenesis . Zebrafish ab strain as wild - type control for all experiments, expect the experiments using tg(cmlc2:egfp) line . Zebrafish strains were maintained as previously described (westerfield, 1993). To visualize the heart morphology in live embryos, tg(cmlc2:egfp) control, smyd3, and setd7 mos were designed by author and ordered to genetools company . Mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations . The sequences of mos used are control mo, 5-cctcttacctcagttacaatttata-3; smyd3 mo, 5-aagagctcttacctgacactgaacg-3; and setd7 mo, 5-agcttctcgtaccttccaccacttc-3. Embryos injected mos were observed or collected at desired developmental stages for further experiment . To make digoxigenin - labelled antisense ribo - probe, zebrafish smyd1a (nm_205540.1), smyd2 (nm_001013550.1), smyd3 (nm_001037400.1) and setd7 (nm_001002456.1) full orfs were cloned into pgem - t vector (promega) after amplification of 24 hpf cdna . Following primers were used for pcr reaction: smyd1a forward, 5-agcatgaccgtggagaagac-3; smyd1a reverse, 5-tgttcatgctttgatctgcac-3; smyd2 forward, 5-agca gcaggtcaatagcgatg-3; smyd2 reverse, 5-ttcctcccattatgtctgctc-3; smyd3 forward, 5-atggacggtcactg cagatga-3; smyd3 reverse, 5-ctagagcgtgttgagctc agc-3; setd7 forward, 5-aacatggacagcgatgatgac-3, and setd7 reverse, 5-caggtgttcagaggagtc-3. The reaction producing ribo - probes were carried out according to manufacture protocol (roche). Whole mount in situ hybridization were routinely performed as previously described (thisse and thisse, 2008). As brief, to experiment, fixed embryos washed and digested with proteinase k solution as desired concentration and times . Hybridization reaction was carried out at 65c, and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate (roche). Staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation . To generate the synthetic smyd3 and setd7 mrnas, we first cloned the full open reading frames (orf) of smyd3 and setd7 into pcs2 + expression vector, which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription . These plasmids were cut by noti digestion to linearize, and capping mrna were produced using m7g(5)ppp(5)g (roche, germany) and sp6 rna polymerase (roche, germany). Synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution . Injection was achieved at 12 cell stages as optimal concentrations (about 100200 pg / embryo). Zebrafish ab strain as wild - type control for all experiments, expect the experiments using tg(cmlc2:egfp) line . Zebrafish strains were maintained as previously described (westerfield, 1993). To visualize the heart morphology in live embryos, tg(cmlc2:egfp) control, smyd3, and setd7 mos were designed by author and ordered to genetools company . Mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations . The sequences of mos used are control mo, 5-cctcttacctcagttacaatttata-3; smyd3 mo, 5-aagagctcttacctgacactgaacg-3; and setd7 mo, 5-agcttctcgtaccttccaccacttc-3. Embryos injected mos were observed or collected at desired developmental stages for further experiment . To make digoxigenin - labelled antisense ribo - probe, zebrafish smyd1a (nm_205540.1), smyd2 (nm_001013550.1), smyd3 (nm_001037400.1) and setd7 (nm_001002456.1) full orfs were cloned into pgem - t vector (promega) after amplification of 24 hpf cdna . Following primers were used for pcr reaction: smyd1a forward, 5-agcatgaccgtggagaagac-3; smyd1a reverse, 5-tgttcatgctttgatctgcac-3; smyd2 forward, 5-agca gcaggtcaatagcgatg-3; smyd2 reverse, 5-ttcctcccattatgtctgctc-3; smyd3 forward, 5-atggacggtcactg cagatga-3; smyd3 reverse, 5-ctagagcgtgttgagctc agc-3; setd7 forward, 5-aacatggacagcgatgatgac-3, and setd7 reverse, 5-caggtgttcagaggagtc-3. The reaction producing ribo - probes were carried out according to manufacture protocol (roche). Whole mount in situ hybridization were routinely performed as previously described (thisse and thisse, 2008). As brief, zebrfish embryos were fixed by 4% paraformaldehyde (pfa) and stored in methanol . To experiment, fixed embryos washed and digested with proteinase k solution as desired concentration and times . Hybridization reaction was carried out at 65c, and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate (roche). Staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation . To generate the synthetic smyd3 and setd7 mrnas, we first cloned the full open reading frames (orf) of smyd3 and setd7 into pcs2 + expression vector, which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription . These plasmids were cut by noti digestion to linearize, and capping mrna were produced using m7g(5)ppp(5)g (roche, germany) and sp6 rna polymerase (roche, germany). Synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution . Injection was achieved at 12 cell stages as optimal concentrations (about 100200 pg / embryo). To delineate functions of hmts, such as smyd1a, smyd2, smyd3, and setd7, during heart development, we have first analyzed the expression pattern of these genes by whole mount in situ hybridization analyses and conventional reverse - transcriptase (rt) pcr . Transcripts of all hmts examined appear to be maternally deposited, and widely detected at early stages . At later stages, the expression becomes gradually restricted to several organs, including somite, brain and eye (figs . 1 and 2). While smyd1a and smyd2 showed higher expressions in developing somite region from 10 somite stage (ss) than smyd3 and setd7, hinting their roles in somitogenesis (figs . 1 and 2) (ses et al ., 2013), the developmental expression regions of smyd3 and setd7 were highly expressed within the heart forming region at 36 h post - fertilization (hpf) and 48 hpf, respectively (red arrow heads in fig . 2). These gene expression profiling data suggest functions of zygotic smyd3 and setd7 in heart development . To further delineate the roles of hmts in heart development, we carried out knock - down experiments of smyd3 and setd7 . First, to determine the functional requirement of smyd3 during zebrafish heart development, we knocked smyd3 down with a morpholino (mo)-based gene targeting system . For this purpose, splicing blocking mo that binds to the junction of exon2 and intron2 of smyd3 pre - matured mrna was designed (fig . 3a). To validate the mo efficacy, pcr primers were designed to detect normal and aberrant smyd3 transcripts in wild - type embryos and smyd3 morphants, and carried out conventional rt - pcr (fig . 3b). In smyd3 morphant embryo, mature transcript that amplified by f+r2 primer was not presented, while aberrant transcript that produced by f+r1 primer was displayed (fig . In addition, smyd3 expression was drastically reduced in smyd3 morphants at 22 ss, suggesting non - sense mediated decay of aberrant transcript caused by mo injection (fig . 3c). At optimal concentration (approximately 3.0 ng / embryo), gross morphology of smyd3 morphants was comparable to control embryos (fig . 3d and data not shown). However, pronounced heart edema (red arrow) was apparent in smyd3 morphants at 48 hpf, suggesting that smyd3 function may be essential for cardiac development (fig . Mos targeting setd7 was designed in a way to target the junction of exon 1 and intron 1 of setd7 pre - mature mrna (fig . Expression of setd7 was noticeably decreased by mo injection at 22 ss, suggest that setd7 mo is a functional to block the normal transcription of setd7 gene (fig . 4c). Similar to smyd3 deficiency, the knock - down embryos of setd7 at optimal concentration (1.0 ng / embryo) displayed cardiac edema (red arrow) without causing any obvious morphological defects (fig . Collectively, our results illustrate that the proper activities of h3k4 methyltransferases, specifically smyd3 and setd7, are important for zebrafish heart formation . During histone h3 lysine 4 (h3k4) methylation, setd7 can only methylate non - methylated h3k4 to produce mono - methylated h3k4 (h3k4me1), which then is further methylated by smyd3 to become h3k4me2 and h3k4me4 (hamamoto et al ., 2004; wang et al ., individual roles of smyd3 and setd7 during cardiac and skeletal muscle development in zebrafish have been reported (fujii et al ., 2011; tao et al ., 2011). Given that smyd3 and setd7 successively methylate h3k4, we can assume that these two enzymes function synergistically during zebrafish development . To test this possibility, we examined whether co - injection of smyd3 and setd7 mos at a suboptimal concentration can give rise a synergistic effect for induction of developmental defects (fig . The smyd3 and setd7 mos, by themselves at optimal concentrations (3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7), produced no discernible gross morphological defects except cardiac edema at 48 hpf (figs . 3 and 4). However, co - injection of these mos at optimal concentrations caused severe somitogenesis defects as indicated by the phenotype of shortened and curled body morphology (fig ., the severity of the abnormalities was sharpened by co - injection of increased concentrations of these mos (fig . As expected, these results suggest that smyd3 and setd7 have a synergistic function in zebrafish development . Next, we delineated the synergistic roles of smyd3 and setd7 in zebrafish heart development . For this purpose, we used the heart - specific transgenic fish tg(cmlc2:egfp) line to allow easy visualization of the heart morphology in live embryos (fig . The injections of control, smyd3, setd7, and smyd3 + setd7 mos were carried out at the optimal concentrations (3.0 ng / embryo for smyd3, 1.0 ng / embryo for setd7). Morphants without body morphological defects were selected for further analyses of heart development . At 48 hpf, heart tubes in the smyd3 and setd7 morphants displayed defects in the looping process and remained linear in shape (fig . Single knockdown of these genes caused a defect in heart morphogenesis, suggesting that each gene is indispensable for heart morphogenesis . Interestingly, double knock - down of smyd3 and setd7 presented a more severe defect than single knockdown of either of those genes (fig . However, the global expression level of clmc2 detected by whole mount in situ hybridization (kim et al ., 2014) was not altered in these smyd3, setd7, and smyd3 + setd7 morphant embryos at 48 hpf (data not shown), suggesting that specification and/or maintenance of cardiomyocytes were not compromised in those embryos . Collectively, our results show that smyd3 and setd7 likely function synergistically in zebrafish development . Moreover, our data suggest that heart development is more sensitive than somitogenesis to the decrease of smyd3 and setd7 levels . To test whether the developing heart defects in the smyd3-and setd7-deficient embryos are caused by the expressional changes of early cardiac muscle genes, we determined the early expressions of cmlc2, vmhc, and amhc (kim et al ., 2014) by whole mount in situ hybridization experiment . The expression level of these genes was not altered by the knock - down of smyd3 and/or setd7 and the gathered shape of cardiac precursor cells in these morphant embryos was also comparable to wild - type embryos (fig . This finding suggests that smyd3 and setd7 are dispensable for proper expression of early cardiac muscle genes and differentiation of cardiac precursor cells . To investigate whether enforced expression of smyd3 and setd7 causes the cardiac defect in zebrafish, synthetic mrna encoding smyd3 or setd7 the co - injection of smyd3 and setd7 mrna as optimal dose (each 100200 pg / embryo) caused similar developmental defects as in embryos lack either smyd3 or setd7, such as defective body axis formation . Moreover, these embryos showed pronounced cardiac edema, indicating the defects in heart formation (red arrow in fig . 7b). To better examine the defects caused by overexpression of these smyd3 and setd7, we analyzed the expression of known cardiac markers, including cmlc2, vmhc, and amhc . Despite the obvious cardiac edema, the expression of these genes was unaltered in embryos overexpressing smyd3 and setd7 (fig . Therefore, it appears that excessive h3k4 methyltransferase activity can lead to defects in heart and skeletal muscle development in zebrafish . Taken together, our results indicate that methylation can provide essential regulatory input during organ development in zebrafish . Since similar developmental defects were observed in embryos with either excessive or reduced level of smdy3 and/or setd7 activity, it appears that appropriate level of histone methylation is critical to ensure the formation of cardiac and skeletal muscle in zebrafish . Heart morphogenesis in vertebrate development is a highly complicated event that achieved by correct cell specification, migration, proliferation, apoptosis, and transition as well as controlled beating of heart myocytes (barnett et al ., 2012). In this process, proper gene expressions for structure of cardiac chamber at early developmental stages are critical for late heart forming event . Our data presented here indicates that proper level of histone methylation, which is mediated by a series of enzymes commonly known as hmts, including smyd3, and setd7 appears to be essential for heart development . During zebrafish development, the expression pattern of hmts indicates that transcripts encoding these proteins may be maternally deposited . In addition, considering their ubiquitous expression, these proteins may provide essential functions in development by regulating histone methylation for all types of cells (figs . 1 and 2). However, knock - down of smyd3 and setd7 in zebrafish embryos did not cause any obvious defects in early development, such as in gastrulation movement, germ layer formation, cell proliferation, or cell death . Therefore, these genes may redundantly function to regulate early vertebrate development (figs . 1 and 2). In later developmental stages, the expressions of all hmts tested in this paper were gradually restricted and often increased in specific organs, including somite, brain, eye primordial cells, fin primordial region, and heart . For instance, smyd2 becomes highly expressed within eye primodium, while smyd3 and setd7 are enriched in developing heart, suggest that each hmts might have a non - redundant and organ - specific roles during late vertebrate developmental periods (figs . 1 and 2). It was report that knocked - down smyd3 and setd7 by mo led to developmental defects in heart and/or skeletal muscle (fujii et al . Consistent with previous reports, we also find that mo injections as high concentrations (over 10 ng / embryo for smyd3 mo and 4.2 ng / embryo for setd7 mo) cause the similar somite defects . However, the optimal injection conditions (concentrations under 3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7 mo) morphant embryos showed only developmental heart abnormality without any somite defects, indicating that each organ may have different sensitivity to the fluctuation in smyd3 and setd7 level during organogenesis . Interestingly, in skeletal muscle development, setd7 directly interacts with myod to induce myogenin and mef2 expression to generate differentiated myocytes (tao et al ., 2011). Therefore, it is tempting to speculate that setd7 manipulation in zebrafish may lead to the alteration in expression of certain structural genes . However, we did not find any discernible change in the expression of cmlc2, vmhc, and amhc in our smyd3 and setd7 double - deficient or overexpressed embryos, suggesting the presence of distinct mechanisms underlying setd7-mediated differentiation of the cardiac muscle and skeletal muscle cells . Moreover, smyd3 and setd7 appear to be dispensable for the specification of cardiomyocytes, since the lack of smyd3 or setd7 did not abrogate the expression of cardiac markers (figs . 6 and 7). Here, we have shown the knock - down and overexpression phenotypes of smyd3 and setd7 in zebrafish development . These findings extend our current knowledge of the roles of hmts and increase our understanding of the functional mechanism of hmts in heart development.
How does the pattern of retinal activity produced by looking at the image of halle berry ultimately lead to the perception of the actress rather than a violin? How do the myriad different associations with halle berry (actress, specific roles) or violin (inanimate object, music) form through learning and memory? They have been approached by a variety of techniques that fall into two broad categories, watching brain activity and disrupting brain function . In this review we will discuss the development of genetic techniques that bridge the divide between these two approaches and allow the targeting of molecular changes specifically to anatomically dispersed neural representations that are activated by discrete environmental stimuli . These new tools allow the establishment of causal relationships between the activation of sparsely distributed neural ensembles and changes at the behavioral level . The use of single unit recordings in awake behaving animals provides an exquisitely precise measure of the temporal activity of neurons . This has been used to extract information about how the brain encodes information by studying the correlation between neuronal activity and the presentation of specific sensory stimuli . The best - studied example is probably in the primate visual system where a hierarchical pathway has been defined (van essen et al ., 1992). Neurons in the primary visual cortex (v1) fire in response to very general visual features such as orientation whereas following processing through the ventral visual pathway, neurons in inferior temporal cortex respond to complex object features . This visual information is then relayed to the medial temporal lobes, which integrate multimodal sensory information and play a critical role in memory . Single unit recording studies in the medial temporal lobe in humans have detected neurons with responses to highly defined categories . In the limit, units were found that responded to the presentation of a single individual in a variety of contexts (quiroga et al ., 2005). One neuron in the right anterior hippocampus responded to the actress halle berry, presented in a photograph, as a masked character (catwoman), as a drawing, or as the letter string this level of response specificity shows that the cells are not tuned to general visual features common to images of halle berry but to the concept of the specific actress . The striking degree of responsive specificity of these units strongly suggests that they participate in the neural representation of specific individuals . However, these studies are still correlative in the sense that they allow us to watch neurons that fire in a manner suggesting a role in encoding specific information, but they do not allow us to disrupt these neurons specifically to test this hypothesis . Historically much of what we know about the functional parceling of the brain has been obtained from lesion studies in experimental animals and in patients with damage to specific brain regions (squire, 2004). In learning and memory, studies of patient hm, who underwent a bilateral resection of the medial temporal lobe, have helped define this area, and in particular the hippocampal formation, as critical in the formation of long - lasting declarative memories . At a more molecular level both pharmacological and genetic manipulations have been used to test ideas about the cellular signaling mechanisms that underlie behavioral plasticity . However, each of these approaches is limited in that they act as sledgehammers, altering every neuron in a given brain region, when the electrophysiological studies suggest that it is really a very sparse group of neurons that is truly of interest in any given experimental context . One approach to circuit analysis is to describe the precise pattern of wiring within specific processing units like the hippocampus or a cortical column . For example, in the hippocampus there is the classic tri - synaptic circuit where information from the entorhinal cortex enters through the dentate gyrus, is relayed to ca3 neurons via the mossy fiber pathway and then to ca1 neurons via the schaffer collateral pathway and finally back out to the entorhinal cortex (squire, 2004). This connectivity diagram can be obtained to finer and finer levels of resolution and in principle an entire wiring diagram of a single brain at a single time point could be produced to the level of individual synaptic connections, similar to that obtained in c. elegans (white et al ., 1986). However, even if this were precisely defined down to the level of single synapses it is not likely that the mechanisms that give rise to a neural representation or memory trace would become apparent . As the example of the halle berry neuron indicates, these representations are likely to be quite sparse and embedded within a matrix of apparently identical neurons . The particular response patterns of an individual neuron are likely determined by the strength of specific synaptic connections that have been altered through experience . Moreover, even if one could explain how these specific firing patterns arise through circuit plasticity, it would be difficult to experimentally establish the contribution of a particular ensemble of neural activity to an actual representation of the environment . An alternate view is that what defines a circuit is the environmental contingencies that lead to its activation (figure 1). In the mammalian brain this is generally referred to as an ensemble code or neural representation of the particular environmental stimulus . In the case of simple systems or reflex pathways, the wiring diagram often predicts the location of the neural ensembles that encode specific environmental information . For example, in the aplysia gill withdrawal reflex a group of sensory neurons are activated by tactile stimulation and synapse directly onto motor neurons to control withdrawal behavior (kandel, 2001). Here the primary sensory neurons are defined by their enervation of the gill and their activation by tactile stimulation of the gill . The behavioral plasticity of the withdrawal reflex is controlled by synaptic plasticity within these sensory neurons . Because of the uniformity and anatomical isolation of this group of cells, it has been possible to apply techniques for both watching and manipulating neurons within the context of a defined circuit (representation) in a behaving animal . The application of these convergent approaches has proven quite powerful in defining the cellular and molecular mechanisms that underlie behavioral plasticity in this system . The goal of this review is to discuss recent attempts to develop approaches that allow similar convergent molecular and physiological access to the more dispersed neural representations of the mammalian brain . The top panel shows a simplified version of the gill and siphon withdrawal circuit in aplysia . The sensory neuron cell bodies are located adjacent to each other in a cluster and possess similar biochemistry and response properties . The bottom panel represents the hippocampal circuit of the mammalian brain . The green circles represent neurons that are activated by a specific pattern of sensory stimulation and that when activated contribute to a specific behavioral response . The hippocampal circuit, like many other circuits in the brain, responds to sensory stimulation with activation patterns that cannot be predicted from their wiring diagram . Each of these neural ensembles involves a sparse subset of neurons that have an unpredictable spatial distribution . The aplysia neurons are primary sensory neurons and their response properties can be predicted by their physical location in the ganglion . One technique that has been used for many years to watch brain activity has taken advantage of a class of immediate early genes or iegs that are expressed in response to high - level neural firing (sagar et al ., 1988). The three most commonly used iegs for this purpose are cfos, arc, and zif268 . The expression of these genes is induced by action potential firing, and the -life of the gene products are relatively short . Thus the expression pattern of iegs in brain sections from an animal provides a record of the neural activity from several hours prior to sacrifice and has been used extensively to map brain activation from a wide variety of environmental stimulation and in learning and memory relevant paradigms (guzowski et al ., 2005). One of the limitations of this approach is that it provides only a single time point record of activity patterns, making it difficult to determine how plasticity modulates activity or even how stable this pattern of gene expression is in relation to an identical stimulus . This problem was addressed using the expression of the ieg arc (guzowski et al ., 1999). By using fluorescent in situ hybridization to examine expression of arc mrna they were able to detect the pattern of arc expression at two separate time points in the same animal . They took advantage of the fact that they could detect the expression of the arc precursor rna while it was still in the nucleus as well as the mature mrna which was present in the cytoplasm and dendrites . The nuclear arc signal represented very recent and ongoing expression reflecting neural activity several minutes prior to sacrifice of the animal, while the cytoplasmic signal reflected activity that had occurred 30 min or more prior . They used this approach to examine the consistency of activation of the hippocampus when an animal was repeatedly exposed to the same environment . They found that when animals were allowed to explore the same environment, they re - expressed arc in many of the same neurons that had also expressed it on the first exposure . This is a critical result in that it demonstrated for the first time that ieg expression could be used to consistently reflect patterns of activity associated with a discrete representation and provided results that were qualitatively and quantitatively similar to results obtained with electrophysiological recordings of the hippocampus . The temporal information regarding neural activity that can be obtained using ieg expression is clearly limited relative to electrophysiological recordings . For example, while it is clear that high - level firing induces expression, it is not clear what the precise threshold is and how this might vary among different neuronal cell types . One advantage is that large brain regions can be surveyed and precise anatomical information can be obtained . A second advantage is that the promoter regulatory elements that confer neural activity dependence can, in theory, be used to drive expression of any linked heterologous transgene . 1992) using the cfos promoter to drive activity dependent expression of e. coli -galactosidase . More recently, an axonally targeted -galactosidase was expressed from the cfos promoter, providing the potential to trace the projections of specific active neuronal populations (wilson et al ., 2002). The use of these promoter elements is general and provides the potential to introduce functional effector molecules directly into activated neural ensembles to allow their molecular manipulation . The use of ieg promoters as tools for both watching and potentially manipulating functional neural circuits is limited in a number of ways . For example, the direct introduction of toxins or other molecular regulators via the cfos promoter could be complicated by developmental effects of their expression . In addition, it would be useful for many studies to allow the molecular change introduced into the activated neurons to be maintained for more prolonged periods than the short (minutes to hours) times afforded by the promoters themselves . We therefore set out in a recent study to develop a genetic system with the following features . (1) the expression of any transgene of interest should be linked to neural activity only during a specific experimenter controlled time window . (2) the transgene expressed in those active neurons should be maintained for a prolonged period, but no further labeling of active cells should occur following closure of the permissive time window . We achieved activity dependent regulation of transgene expression with these two features by combining elements of the tetracycline system for gene regulation with the cfos promoter as shown in figure 2 (reijmers et al . The first uses the cfos promoter to drive expression of the tetracycline transactivator (tta or tet - off). In mice carrying only this transgene high - level neural activity will result in the induction tta, which is a transcription factor that can be blocked by the antibiotic doxycycline (dox). In the absence of dox tta drives expression of genes linked to a teto - promoter sequence . The second transgene incorporates both a teto - linked reporter (in this case the somato - axonal marker taulacz) as well as a transcriptional feedback loop to maintain teto - linked gene expression indefinitely once it is activated . The tta (tta ) in this construct was made dox insensitive by introduction of a point mutation in the tet binding domain . In the presence of dox the teto - linked reporter is not activated even in those neurons in which the cfos - linked tta is expressed . However, if dox is withdrawn then both taulacz and tta are expressed, but only in those neurons that were active to a high enough level to induce the cfos - linked tta . Once activated, the tta * sets up a transcriptional feedback loop that can be maintained even in the presence of added dox . In this manner discrete time windows for genetic tagging of active neurons can be opened and closed through the use of dox . In the absence of dox, any neuron that has sufficient induction of cfos - linked tta to activate the feedback loop will persistently activate the taulacz reporter, as well as any other teto - linked transgene that is introduced into the mouse (aiba and nakao, 2007). This expression will be maintained even when the time window for sampling active neurons is closed by the readministration of dox . In this way a persistent record of neurons that were active during the off - dox period can be maintained . We called this the tettag mouse, which stands for tetracycline transactivator controlled genetic tagging of active neural circuits . The tettag mouse . Mice carrying two transgenes were used . The first transgene uses the cfos promoter to drive expression of the tetracycline transactivator (tta). Tta activates the teto promoter in the absence but not presence of doxycycline (dox). The second transgene uses the teto promoter to drive expression of a dox insensitive tta (tta *), which, once expressed, sets up a positive feedback loop that continuously drives expression of a -galactosidase reporter coupled to the tau protein (taulacz). Neurons activated during fear conditioning (while off dox) were tagged with long - lasting expression of taulacz (lac; red circle). Mice were put back on food with doxycycline and a retrieval test was done 3 days later, followed by analysis of the brains 1 h after retrieval for expression of lacz and zif268 . Neurons activated during learning expressed lacz and those active during retrieval expressed zif268 (zif; green circle). The number of neurons in the amygdala that expressed both lac and zif, indicating that they were activate during both learning and retrieval, was positively correlated with the strength of the fear memory that the animal displayed . We used the tettag mouse to examine the neural circuit that mediates fear memory (reijmers et al ., 2007). We asked whether neurons that are stimulated during learning in a pavlovian fear - conditioning paradigm were reactivated during retrieval of the memory as shown in figure 2 . We subjected tettag mice to a learning trial consisting of paired presentations of a tone (cs) and a foot - shock (us). This results in a long - lasting fear memory for both the tone (cued conditioning) and the conditioning box in which the animals were shocked (context conditioning). The learning trials took place during a time window in which the animals were free from dox, allowing activated neurons to be tagged with long - lasting expression of taulacz . The animals were then returned to dox to prevent further tagging of activate neurons, tested for retention of the memory in a retrieval trial, and sacrificed after 1 h for analysis using the endogenous ieg zif268 as a measure of recent neural activity . By comparing the expression of lacz (activity during learning) and zif268 (activity during memory retrieval) we could determine the degree of circuit reactivation . We found that the number of reactivated neurons in the amygdala, a region critical in fear conditioning (ledoux, 2007), correlated with the retrieval of the fear memory . We hypothesized that these reactivated neurons represent a component of the memory trace for conditioned fear . To test this idea we weakened the strength of the memory by extinction training; giving repeated cs presentations without the shock us . Animals were first fear conditioned while free from dox to tag the learning activated neurons . The extinction training then took place following re - exposure to dox (to prevent further labeling) and a memory retrieval trial was conducted 1 h prior to analysis . We found that there was a significant correlation between the strength of the remaining fear response and the degree of circuit reactivation; animals with a high fear response during retrieval showed strong reactivation of the learning circuit while those with low fear responses showed a low degree of reactivation . In addition, we found some specificity in the anatomy of the responses so that reactivation in the basal amygdala was correlated with context fear while reactivation in the lateral amygdala was associated with the strength of the cued (tone) fear memory . These results are consistent with the known role of these subdivisions of the amygdala with the two different forms of fear memory (quirk et al ., 1995; these results demonstrate that memory retrieval results in a reactivation of some of the same neurons that were active during the initial learning . We suggested that neurons activated by the us (shock) but also receiving weak cs inputs were altered during learning such that the cs alone could now activate them . In this way presentation of the cs alone after learning would recapitulate a portion of the aversive us leading to downstream fear responses . This approach represents a somewhat elaborate way of simply watching neural activity and the results are still purely correlative . However, by using a regulatable and binary genetic system, it should be relatively easy to introduce additional effector transgenes into the mice to control the activity or biochemistry of these neurons and directly test their role in memory . One recent study by josselyn and coworkers has achieved this direct manipulation of neural ensembles associated with a specific memory trace using a somewhat different approach . The use of neural activity to introduce genetic alterations into neurons offers the possibility of obtaining direct molecular control over the neurons that participate in a specific neural representation or memory trace . An alternate approach that realized this goal took advantage of the finding that certain molecular manipulations could recruit neurons to participate in control of a specific memory . In one recent study, (han et al ., 2007), it was found that over expression of the transcription factor creb in neurons resulted in their preferential recruitment into a fear memory trace . In this study, neurons in the amygdala were randomly infected with a viral vector that over expressed creb and the animals were then trained in fear conditioning . They then performed a retrieval trial and examined the expression of the ieg arc in the amygdala . They found that the creb over expressing neurons were more likely to be activated during the memory retrieval . This result suggests that these creb over expressing neurons were predisposed to participate in the memory trace . The mechanism by which these neurons are preferentially recruited is unclear but it does demonstrate that, at least in the amygdala, there is a good deal of flexibility in which neurons can be used to encode a specific memory . The system is not hard wired at the level of individual neurons but there is a sort of competition, with creb over expression favoring a neuron's recruitment into the memory trace . Josselyn and coworkers went on to take advantage of this creb priming trick to directly manipulate a specific fear memory trace (han et al ., study the viral vector that delivered creb to amygdala neurons also carried a gene that allowed for the expression of the diphtheria toxin receptor (dtr) (figure 3). The creb over expression recruited the neurons to the memory trace and expression of the dtr allowed for the selective ablation of these specific neurons with diphtheria toxin (dt). Ablation of the creb over expressing neurons disrupted the fear memory while ablation of a similar number of random neurons in the amygdala did not . The memory effect was long lasting and specific (the same animals could learn a second fear association) demonstrating that this limited group of neurons played a critical role in the specific memory encoded during the creb expression time window . This is the first example of the disruption of a specific memory within a distributed network . Disrupting a specific memory in the mouse . A viral vector expressing both creb and cre recombinase was injected into the amygdala of idtr mice leading to the expression of both cre and creb in a random subset of neurons (circles with creb / cre). The cre recombinase removed a transcriptional stop sequence and allowed for expression of dtr in these creb expressing neurons . An earlier study from the same authors (han et al ., 2007) demonstrated that the creb expressing neurons participate in the storage of the fear memory (green circles symbolize neurons that participate in the storage of the memory). After fear conditioning, mice were injected with diphtheria toxin (dt), which killed the creb expressing neurons which participated in the encoding of that memory (red circles). This caused a significant reduction in the strength of the fear memory measured during retrieval . While the ability of creb over expression to recruit neurons to participate in a specific memory is interesting in its own right, it would be useful to have an approach to manipulate neurons that were naturally activated by any general environmental stimulus . This has recently been accomplished using a technique in which neurons expressing -galactosidase can be specifically disrupted with a pharmacological agent (koya et al ., 2009). The study used rats that carry a cfos - promoter driven -galactosidase to label activated neurons . To manipulate the neurons they use a drug that is inactive in the absence of -galactosidase (daun02) but can be hydrolyzed to a compound that can reduce ca dependent action potentials (santone et al ., 1986). They examined context specific sensitization to cocaine, which is an associative paradigm where the response to a drug of abuse is potentiated when it is administered in the same environment in which it has been repeatedly taken . Animals were given repeated injections of cocaine over 1 week in context a to produce the context specific sensitization (measured as increased locomotor response to the drug). Following the training, a final sensitization trial to induce -galactosidase was given and 90 min later daun02 was injected into the nucleus accumbens to disrupt the -galactosidase expressing neurons . Previous studies had suggested that the nucleus accumbens was a critical site of plasticity mediating this behavior (mattson et al ., 2008). The injected animals showed a reduction in the context specific component of the sensitization but retained normal responses to cocaine in a novel context b when tested several days later . Like the results with the creb over expression, this study suggests that a specific associative representation (context a + cocaine) is being interfered with selectively . While the behavioral results in this study are intriguing and the linkage to cfos based expression provides a potentially general approach for manipulating discrete neural representations, there are a number of important questions that remain to be addressed with this technique . First, the electrophysiological effect of the daun02 treatment was not examined directly in the neurons but inferred from studies in cell lines . Whether the effects are mediated by suppression ca dependent action potentials or some other effect of the time course of any neural excitability or ca channel changes is also a critical parameter . The effect of the daun02 treatment was examined 3 days after the initial injection of the compound and it is unclear whether the observed effect was due to ongoing suppression of activity or to a persistent effect manifest during the initial treatment . Nevertheless, it demonstrates the general principles of this approach, which could be combined with the host of recently developed genetic regulators of neural activity . While neural representations are encoded in the specific ensemble of neurons that are activated in response to a stimulus, the plasticity that molds these patterns of activation is thought to occur at the synapse . It has been known for some time that long - term memory lasting 24 h requires new gene expression initiated at the time of learning, while short - term memory lasting a few hours lacks this requirement (davis and squire, 1984). Since the short and long - term memories for the same event presumably involve the same pattern of synaptic changes, it raises the question of how the required gene products exert their effects selectively on the appropriate synapses . A potential answer to this question was suggested by frey and morris in studies of long - term potentiation (ltp), a form of synaptic plasticity thought by many to underlie memory (frey and morris, 1997, 1998). They found that synaptic activity could produce a sort of molecular tag at a synapse that would allow it to utilize newly expressed gene products to maintain ltp for long periods . We recently used the cfos based genetic approach to demonstrate a similar mechanism in behavioral learning and memory . A number of studies have implicated the regulated trafficking of the glutamate receptor glur1 to synapses as an important mechanism in both ltp and fear learning (kessels and malinow, 2009). In addition to allowing the genetic tagging of activated neurons, the ieg promoters like cfos show a very rapid onset and offset of expression, making them useful for cellular trafficking and turnover studies . We took advantage of this property to examine the trafficking of glur1 following learning in the fear - conditioning paradigm (matsuo et al ., 2008). Mice carrying both a cfos - tta and teto - gfpglur1 fusion transgene were used in this study (figure 4). In the absence of dox neural activity will induce a pulse of expression of the gfp tagged glur1 and the distribution to synapses of this newly synthesized receptor can be followed histologically . Animals were fear conditioned in the absence of dox to both induce a contextual fear memory and to induce synthesis of the gfp tagged receptor in activated neurons . The distribution of the receptor to dendritic spines (the site of most excitatory synapses) in the hippocampus was examined 24 h after the conditioning . We found that the receptor was not evenly distributed but present in only about 50% of spines, even in controls . In the fear conditioned animals we found a similar distribution except that there was an increase in trafficking to one morphological type of spine, the mushroom spines . This preferential trafficking only happened when the conditioned stimulus (cs: novel box) and the unconditioned stimulus (us: foot shock) were paired, but not when cs or us were presented separately . The newly synthesized glur1 requires 2 h to begin to reach the dendritic spines, yet is somehow preferentially recruited to a specialized class of spine based on the associative conditioning that occurred 2 h prior to its arrival . This is indicative of a synaptic tagging event acting in behavioral memory similar to that described for ltp (frey and morris, 1997). Learning regulated targeting of glutamate receptors . The first transgene is identical to the one described in figure 2 and uses the cfos promoter to drive expression of a tetracycline transactivator (tta). The second transgene was a teto - promoter gfp tagged glutamate receptor subunit (glur1-gfp). Animals were fear conditioned in the absence of dox to produce both a fear memory and a pulse of gfp - glur1 expression in active neural ensembles in the hippocampus . The distribution of gfp - glur1 in dendritic spines was analyzed 24 h following the conditioning using dii to label all spines on a given neuron . Fear conditioning led to an increase in trafficking of the receptor specifically to mushroom type spines . This experiment demonstrates how genetic tools can be used to image a specific molecular event selectively within an activated neural circuit . The ability to genetically manipulate activated neuronal ensembles or neurons participating in a sparsely encoded memory trace offers a number of advantages that are only beginning to be realized . A parallel line of technological development has focused on generating genetic tools for manipulating neuronal activity . The light regulated channelrhodopsin chr, developed by deisseroth and colleagues (zhang et al ., 2006), allows for the very precise light regulated control of action potential firing in neurons expressing the channel . There is an expanding tool box of genetic effectors like chr that are light or ligand controlled and can be used to either stimulate or suppress neural activity (luo et al ., 2008). In addition, there are a number of similar effectors that can be used to regulate second messenger signaling pathways when expressed in heterologous cells (isiegas et al ., 2008; the combination of these new tools with activity based genetic delivery and multi time point brain activity mapping at cellular resolution will open up a variety of new questions to experimental analysis . To return to the initial discussion of neural representations, the fusion of these approaches should allow one to address the question of what neural firing patterns mean to the animal . To take the example of the halle berry neurons (actually the equivalent in genetically accessible animal models) one could ask what would be the consequences of silencing this specific group of neurons . What fraction of neurons in the representation need to be silenced in order to impair recognition? How does the silencing of a specific group of neurons in one brain region affect the activation patterns in downstream areas? An alternate approach is to ask whether a representation can be built by experimenter driven stimulation of the appropriate neurons . In one recent study, svoboda and colleagues delivered the chr2 molecule to random populations of neurons in the somatosensory cortex . They found that stimulating as few as 300 neurons could be detected by the animals and used to alter behavior in a conditioning task (huber et al ., 2008). However, the coordinated activation of those neurons presumably does not form any natural representation . Suppose that instead of a random group of neurons one could activate a subset of neurons that responded to a natural stimulus, say the tone cs in a fear - conditioning task . The artificial stimulation of those neurons paired with a foot shock would presumably lead to a conditioned fear of the extrinsic stimulation cs . If so would all the features of this sensory representation be maintained, for example frequency selectivity? With what fidelity does artificial stimulation of the tone representation in one brain region recapitulate the brain activity patterns produced by the natural tone itself? This type of approach should now be achievable and allow direct functional investigation of the structure of neural representations . Finally, the ability to genetically alter activated neural ensembles provides an entre into a more specific biochemistry of the brain . In the glur1 trafficking studies discussed above, the gfp tagged receptor provides not only a signal to watch molecular movements, but also a tag for specific biochemical analysis . For example, one could ask how the synapses from activated neurons that received new receptor differ biochemically from those that did not by using the antibodies to the gfp tag to affinity purify positive from negative material . A similar approach could be used to tag specific cellular compartments or molecular complexes so that biochemical studies can be limited to just the activated neurons, sparsely embedded in a matrix of inactive neurons and glia . It should also be possible to improve the specificity of the genetic modifications using a variety of genetic tricks such that only neurons active in one brain region or active at time point 1 but not time point 2 would be tagged . The increased specificity along with the new genetic tools and biochemical tagging should provide a new level of circuit analysis in the brain and break down the barrier between watching neural firing and manipulating neural function . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Angiogenesis, the formation of new blood vessels by sprouting of pre - existing ones, is a main mechanism of vascularization during embryonic development, growth, formation of the corpus luteum and endometrium, regeneration and wound healing . However, deregulated, abnormal angiogenesis is involved in many pathological processes [1, 2]. The complex sequence of events involved in angiogenesis is related to changes in endothelial cell biosignalling . The relationships of angiogenesis with cancer have special relevance, since angiogenesis has been described as one of the hallmarks of cancer, playing an essential role in tumour growth, invasion, and metastasis . Since tumour blood vessels show many differences from normal vessels and are not genetically unstable, they are potential targets for therapy of all types of cancer . Due to the pivotal role played by endothelial cells in tumour angiogenesis, most previous efforts were devoted to the development of agents that could block their activation by an angiogenic signal (mainly vegf), or to inhibit one or several specific functions of activated endothelial cells (proliferation, adhesion to extracellular matrix, proteolytic activities, migration, invasion or differentiation). National cancer institute database showed that in august 1999 a total of 20 angiogenesis inhibitors were being tested in clinical trials . Remarkably, most of them were monotherapies with the antiangiogenic agent, and those compounds that had then reached the phase iii, including several inhibitors of matrix metalloproteinases, were discontinued due to their lack of activity or the appearance of undesirable toxicities . In spite of the great number of angiogenesis inhibitors described so far (estimated to be> 300 drug candidates in 2001), and the interesting results obtained in experimental models, even showing complete tumour regressions in pre - clinical studies, modest or even negative results emerged from the first generation of compounds entered in clinical trials . Nevertheless, there is no reason for premature pessimism, as revealed by current ongoing trials and the clinical developmental status of anti - angiogenic drugs . A critical analysis of the disappointing results obtained in previous clinical trials points to different reasons for this failure . These include flaws in the methods used to select these inhibitors and in the design of the clinical trials to test their effects, as well as an oversimplified view of tumour vasculature pathophysiology . Angiogenesis inhibitors are initially selected by means of in vitro assays that make use of endothelial cells from different sources . The results obtained in this primary screening can be dependent on the type of endothelial cell . Afterwards, the antiangiogenic activity of the selected compounds is usually tested with several in vivo assays (for a review, see). Although useful, these have limitations . Some of them do not take into account the tumour microenvi ronment (this is the case of vascularization assays in the chicken chorioallantoic membrane and in the mouse cornea). Other assays make use of rapidly growing tumours and/or animals that do not fit into the clinical reality . Most of the tumour systems used with laboratory animals show angiogenic responses much higher than those induced by human tumours . Furthermore, the effectiveness of an angiogenesis inhibitor can be hampered by the intrinsic heterogeneity of human tumour angiogenesis . Animal and preliminary clinical trials have revealed that different tumours respond very differently to antiangiogenic therapy . In fact, the responses have been extremely heterogeneous, most probably due to the randomized design of the trials . A clinical challenge in antiangiogenesis is the finding of biological markers that help to identify subsets of patients more likely to respond to a given antiangiogenic therapy, as well as to determine optimal dosing of therapy, to detect early clinical benefit or emerging resistances and to decide whether to change therapy in second - line treatments ., microvessel density has been proven to be a useful prognostic indicator but, at the same time, does not seem to be a good direct indicator of antiangiogenic treatment efficacy . Surrogate biomarkers could include those related to the various steps of the angiogenic process, including variations in endothelial - cell survival, alterations in the endothelial - cell signaling, and variations in the number of circulating endothelial progenitor cells [3, 13, 15, 16]. Another possibilityis the fractal analysis of the vascular network in tumour biopsies . However, these approaches are far from an ideal biomarker for clinical practice . Although some authors consider taking biopsies repeatedly to be feasible, most physicians consider this to be very cumbersome for patients . Since tumour angiogenesis produces interstitial hypertension in tumours, the determination of interstitial pressure of tumours can be considered an alternative surrogate biomarker . Easier to determine and less invasive approaches include the measurements of circulating levels of several angiogenic factors, but so far no growth factor has been validated for predicting response to antiangiogenic therapy, as well as high resolution image analysis that requires expensive instrumentation that could not be available in all institutions . On the other hand, the clinical end - points for dose - defining trials (phase i) and efficacy trials (phase ii) should be reconsidered . The expected good tolerability and low toxicity of well selected antiangiogenic compounds give little relevance to the determination of maximum tolerated doses (mtd) and dose - limiting toxicity (dlt), which could be replaced by the determination of optimal biological dose (obd) in phase i trials . More and better - designed pharmacokinetic studies are required not only to determine the obd, but also to determine the optimal schedule of drug administration . Some years ago, trials with this antiangiogenic compound were discontinued due to low responses and toxicity . However, an improvement in the treatment regimen yielded enhanced response with decreased toxicity in phase i and ii trials . Probably, a readjustment of doses and/or schedule could contribute to diminish or even abolish some of the side effects produced by other previously tested antiangiogenic compounds . In phase ii trials, objective responses (i.e. The degree of tumour regression) alternative parameters such as disease stabilization, progression - free survival and time to progression should be used . However, these parameters are more difficult to be evaluated properly and they require larger patient samples and more prolonged treatments . Validation and standardization of monitoring techniques for antiangiogenic therapy are urgently required . The fact that tumour vasculature has been understood in an oversimplified fashion is another explanation for the poor results obtained in the first generation clinical trials . It is now known that different tumour types may acquire their blood supply by different mechanisms . Tumour vasculature is not necessarily derived from endothelial cell sprouting;instead cancer tissue can acquire its vasculature by a number of alternative mechanisms that could be the basis for developing effective clinical modalities using antivascular therapy of cancer . The recruitment of circulating endothelial progenitor cells, mainly from bone marrow origin, can contribute to the tumoural neovascularization by vasculogenesis, a process that was initially thought to be limited to embryonic development . In fact, as recently reviewed, there is accumulating evidence that progenitor cells as well as other stromal cells are actively recruited into tumours and that this recruitment is essential for the proangiogenic environment of tumours . Another possibility is the co - option of vessels during early growth of tumours in the absence of angiogenesis [11, 28]. On the other hand, vascular mimicry, the generation of microvascular channels by genetically deregulated and aggressive tumour cells, is an alternative way to provide blood supply to tumours and is independent of angiogenesis . The potential of mono - cytes / macrophages to contribute to neovascularization has recently come into focus . Some experimental evidences indicate that infiltrating mononuclear cells may incorporate in the lumen microvessels and that peritoneal macrophages may form capillary - like lumens and branching patterns in vitro[31, 32]. Pathological angiogenesis is characterized by structurally and functionally abnormal vessels and lymphatic vessels [1, 2]. These abnormalities result from an imbalance between levels of pro- and antian - giogenic molecules . As a result, this, in turn, compromises the delivery and effectiveness of conventional therapies, as well as molecular targeted therapies . Finally, in this brief analysis of the rationale behind the failure of the first generation of antiangiogenic agents, concern should be given to the way in which scientists communicate their findings to society . Angiogenesis research is an especially competitive area in which the promising preclinical results have been very often prematurely amplified by mass - media releases . The high prevalence of cancer and the extremely high sensitivity of society towards this primary medical problem facilitate that great expectations could lead to deep disappointment . A second generation of antiangiogenic trials is beginning to show highly significant potential [8, 35]. The first study showing phase iii data validating an antiangiogenesis strategy for treating human cancers was obtained with bevacizumab, a humanized recombinant monoclonal antibody that neutralizes the biologically active forms of vegf that interact with vegf receptors 1 and 2 . In a communication that received much attention in the 2003 asco meeting, the authors reported that the beva - cizumab / ifl (irinotecan / fluorouracil / leucovorin) combination led to significantly prolonged survival and had a better ability to shrink tumours than standard ifl alone, without statistically significant increases in adverse events in patients with metastatic colorectal cancer . The results were based on 412 patients in the ifl / placebo arm and 403 patients in the ifl / beva - cizumab arm . The presence of bevacizumab in the treatment produced remarkable and statistically very significant increases in all the four determined survival and response parameters: median survival, progression - free survival, objective response, and duration of response . These impressive results led the fda to approve the use of bevacizumab in patients with metastatic colorectal cancer and they have been finally published in the form of a research article in the new england journal of medicine . However, as stated in the accompanying editorial, although it is tempting to attribute the effect of bevacizumab to a direct antiangiogenic mechanism, the validity of this assumption is presently uncertain . Once more, mass - media releases have led to unrealistically high expectations . As commented in the aforementioned editorial, patients need to be informed that beva - cizumab does not cure metastatic colorectal cancer and that there is no evidence as yet that the antibody has antitumour activity when administered as a single agent for this disease. Although hurwitz et al . Did not measure surrogate markers of antiangiogenesis, they mention that bevacizumab may have altered tumour vasculature and decreased elevated interstitial pressures in tumours, thereby enhancing the intracellular delivery of chemotherapy agents . Recently, bevacizumab plus folfox4 (oxaliplatin/5-fu / leucovorin) treatment was approved for second - line metastatic colorectal cancer . In october 2006, bevacizumab in combination with paclitaxel and carboplatin treatment was approved for the first - line treatment of patients with non - small cell lung cancer . Currently, more than 100 clinical trials with bevacizumab are ongoing, including phase iii trials in kidney, breast, prostate and ovarian cancer, among others . In fact, successful clinical trials of multitargeted compounds have yielded two significant fda approvals . In december 2005, sorafenib (bat 43 - 9006), an inhibitor of the faf / mek / erk and the vegfr and pdgfr signaling pathways, received fda approval for the treatment of renal cell carcinoma . Sunitinib (su11248), an oral inhibitor of vegfr2, pdgfr, flt-3 and c - kit, received fda approval in january 2006 for patients with gastrointestinal stromal tumours (gist) and advanced kidney cancer, being the first time the agency had approved a new oncology product for two indications simultaneously [42, 43]. It seems that, after a period of flawing interest, antiangiogenic compounds have regained their place in the centre of anticancer treatment trials, as shown by the eastern cooperative oncology group portfolio of clinical trials . From the results obtained so far in clinical trials, it can be concluded that the future clinical success of angiogenesis inhibitors could be related to their use in combination with chemotherapy or radiotherapy . Since abnormal angiogenic vessels compromise the delivery of drugs targeting tumour cells, the normalization of tumour vasculature with antiangiogenic therapy has emerged as a new paradigm for combination therapy [19, 45]. Synergic effects can be expected, since judiciously applied antiangiogenic therapy can increase the penetrability of chemotherapeutic agents, as well as the radiosensibility of tumour cells . A detailed analysis of how antiangiogenic compounds reduce vessel density shows that these drugs reduce vascular permeability, destroy immature vessels and increase the recruitment of pericytes to stabilize other vessels . This transient stabilization has been termed the normalization window, defined as a period of time where tumour blood flow and oxygenation increases, thus providing an opportunity to better deliver chemotherapeutic drugs and radiation therapy . As previously mentioned, the heterogeneity of blood vessel growth, the fact that angiogenesis differs among tumour types is a basis for the observed differences in response to antiangiogenic therapy in both animal and clinical trials . Therefore, a multidrug approach might be more successful than monotherapy . The combined used of several antiangiogenic compounds targeting different steps of angiogenesis should be explored . As stated before, reliable biomarkers are strongly needed to validate the efficacy of antiangiogenic therapy, to identify responsive patients and optimal doses, to predict efficacy of regimens that include anti - angiogenic agents, and to detect and prevent tumour escape . The lack of reliability of measurements of circulating levels of angiogenic factors has made the search for new biomarkers to shift away from measuring their levels to measuring their effects, such as the recruitment of endothelial progenitor cells from the bone marrow to the tumour where they contribute to neovascularization . Preclinical studies have shown that circulating endothelial cells, which are probably derived from blood vessel wall turnover, and circulating endothelial progenitors kinetics correlate well with several standard laboratory assays, that cannot be used in humans . The initial suggestion that variation in the levels of circulating endothelial progenitor cells could be a useful surrogate marker to monitor angiogenesis has been confirmed and extended in an outstanding report published in cancer cell . This report provides evidence that the levels of circulating endothelial progenitor cells are genetically predetermined and regulated by regulators of angiogenesis, including vegf, tie-2 and thrombospondin-1 . Moreover, antiangiogenic therapy can be optimized by monitoring the levels of both circulating endothelial cells and circulating endothelial progenitor cells [13, 25]. Therefore, the kinetics of these cells in peripheral blood is suggested to be useful surrogate markers of pathological angiogenesis with potential application for the monitoring of antian - giogenic therapy response . There are clear signs that during the last year antian - giogenesis research has entered a new age . Table 1 tries to summarize the trials and errors in past failures and possible solutions to them . Antiangiogenic cancer therapy: past and future; clinical trial lessons the development of new and better models for the in vivo assay of potential inhibitors of human angio - genesis should be considered a priority in this field of research . There is increasing concern that by using approaches based on traditional end - points, potentially interesting angiogenic modulators might be rejected prematurely . Consequently, the extensive use of correlative studies in the early phases of drug development to establish surrogate biomarkers for use in efficacy trials is strongly recommended . Methods of imaging will be helpful to assess the efficacy of treatment [12, 13, 20]. A careful selection of the clinical setting for the investigation (for example, tumour type and stage of disease) and innovative statistical designs to optimize the selection of patients must be carried out before expensive, definitive phase iii clinical trials . An example is the randomized discontinuation trial design (rdtd), aimed to select a subset of enrolled patients who are more homogeneous with respect to important prognostic factors than the group of patients that would otherwise be randomized in the trial . Frequent administration of chemotherapy at low doses, ranging from one - tenth to one - third of the mtd, significantly increases the antiangiogenic effect . Metronomic scheduling has shown impressive antitumour activity in animal models and is now being tested either alone or in combination with other antiangiogenic agents in clinical trials [35, 47, 48]. Furthermore, this metronomic approach is also used with radiation therapy, when administered at lower than normal doses, known as hyper - fractionated radiation. The concept of vascular targeting is related to antiangiogenesis but involves a different approach . Juliana denekamp outlined the concepts behind vascular targeting for cancer treatment in the early 1980s, showing that physical occlusion of the blood supply to tumours in rodents led to tumour regressions . Vascular targeting agents exert their primary action on the pre - existing blood vessels of solid tumours . Vascular targeting therapies would share the advantages of antiangiogenic therapies and could offer some additional advantages . First, blood flow is a defined surrogate marker of biological activity that can be measured in the clinic . Second, temporary effects on vascular functioning may be sufficient . And third, unlike angiogenesis inhibitors, vascular targeting agents should require only intermittent administration to synergize with conventional treatments rather than chronic administration . The clinical studies completed to date with vascular targeting agents are encouraging . Progression into combination studies has begun . Recently, concerns have been raised on the possibility of resistance to antiangiogenic therapy [13, 21]. In fact, an effective antiangiogenic therapy could select for resistant and aggressive cancer cells during therapy - induced tumour regression . Ideally, the most effective therapy would suppress all cancer cells, avoiding relapse . On the other hand, hypoxia is common in tumours, despite the increase in their vascularization, because of a poor perfusion caused by aberrant vessels . Strategies that target hypoxic cells may therefore synergize with antiangiogenic treatments . The contribution of inflammatory cells to tumour angiogenesis the inhibitors of this pathway exhibit high tolerability and they can be administered chronically . Their performance in clinical studies is currently being tested . However, the recent problems with cycloxygenase inhibitors in cancer prevention treatment raise serious concerns for their use [55, 56]. Inhibitors of angiogenesis could slow the progression of premalignant lesions and reduce the risk of developing invasive tumours . They have the potential to be used in primary, secondary or tertiary cancer prevention settings . In fact, monotherapies with antiangiogenic compounds could be useful as adjuvant treatments in situations of minimal residual disease following either cytore - ductive surgery or cytotoxic treatment . However, in spite of the fact that past clinical studies have shown that many angiogenesis inhibitors can be given safely to patients, more long - term toxicity studies are needed . How much antiangiogenic therapy will be incorporated in the future to the treatment of cancer patients depends on further advances in the understanding of the molecular mechanisms involved in tumour angio - genesis, the development of standardized methods to assess surrogate predictive markers of response, and the capability of performing a new generation of appropriately designed clinical studies . A convergence of the efforts carried out in basic, applied and clinical research would contribute to achieve these goals . This knowledge will be applied not only to cancer treatment but also to other diseases characterized by abnormal vasculature such as hemangiomas, diabetic retinopathy, macular degeneration and psoriasis, among others for which antiangiogenic approaches have already shown benefits [1, 2].
Regulation of gene expression is one of the most enigmatic facets of molecular genetics that results in intricate appearance of a biological entity . Scientists have been attempting to elucidate the regulatory mechanisms of gene expression for long and the radical discovery of regulatory function of endogenous small noncoding rnas is overwhelming the scientific community with their ever increasing potentials . Small noncoding rnas of 1840 nucleotides (nt) in size have been proved to play a vital role in a remarkably wide range of biological processes, including cell proliferation, developmental timing and patterning, chromatin modification, genome rearrangement, and stress response in plants and animals . Small rnas regulate a variety of biological processes in plants by interfering with messenger rna (mrna) translation, directing mrna cleavage or promoting the formation of compact, transcriptionally inactive chromatin . Several distinct classes of small rnas have been reported so far including micrornas (mirnas), small interfering rnas (sirnas), repeat - associated small interfering rnas (ra - sirnas), piwi interacting rnas (pirnas), natural antisense transcript derived small interfering rnas (nat - sirnas), transacting small interfering rnas (ta - sirnas), heterochromatic small interfering rnas (hc - sirnas), secondary transitive small interfering rnas, primary small interfering rnas, competing endogenous rnas (cernas), and long small interfering rnas [1113]. It has only been a few years since it was appreciated that micrornas provide an unanticipated level of gene regulation in both plants and metazoans . Mirnas are well differentiated due to some of their particular characteristics; they are derived from distinct genomic loci and processed from transcripts that can form local rna hairpin structures, and usually mirna sequences are nearly always conserved in related organisms [13, 14]. Most mirnas are transcribed by rna polymerase ii which folds into a stable, usually imperfect, hairpin structure; pri - mirna transcript is cleaved to pre - mirna by rnaseiii - type dicer - like 1 (dcl1) protein to produce a distinctive ~21 nt, double - stranded rna . This duplex is exported into the cytoplasm by hasty and methylated at the 3 end by hen1 . A cytoplasmic helicase unwinds the translocated duplex into a single - stranded mature mirna, which is finally incorporated into rna - induced silencing complex (risc) [5, 17, 18]. A mature mirna sequence can range from 19 to 24 nucleotides (nt) in length and act as a regulatory molecule in posttranscriptional gene silencing by base pairing with target mrnas . Within the risc complex, mirnas function in the direct cleavage of 3 untranslated region of protein - coding genes or translational repression depending on its perfect or imperfect match with the targets . The same mature mirna can also be present as several length variants; these populations of mirna variants are called isomirnas, which are isoforms of micrornas caused by an imprecise or alternative cleavage of dicer during pre - mirna processing . Several mirnas have been identified in plants, and they have been characterized in a wide variety of metabolic and biological processes with important functions . The first plant mirnas were described in arabidopsis thaliana; currently the latest mirbase release (v20, june 2013) contains 24,521 microrna loci from 206 species, processed to produce 30,424 mature microrna products . Earlier, mirnas have been identified through either bioinformatics analysis or sequencing; various methods have been used to identify mirnas in rice, wheat, tomato, and maize . Besides the mirnas that are highly conserved in different species, there are species - specific mirnas originating from recently evolved mirna genes [28, 29]. The expression of these species - specific mirnas is often low and can therefore be difficult to detect by traditional methods . In recent times, high - throughput sequencing platforms are showing significant promise for small rna discovery and genome - wide transcriptome analysis at single - base pair resolution [23, 31]. Sequencing techniques such as the solexa platform, solid, and 454 technology as well as other massively parallel sequencing strategies have been successfully applied in order to identify mirnas in many plant species, such as rice, alfalfa, grape, tomato, orange, soybean, peanut, poplar, and black gram . In comparison with microarray, deep sequencing has several advantages, the major one being its application in comprehensive identification and profiling of small previously unknown rna populations . Nevertheless, analyses of these data are not perfect, especially in the absence of native genome sequence . Jute (corchorus olitorius and corchorus capsularis) is a bast fibre, like flax and hemp . Cultivation of this environmentally friendly as well as the most affordable fibre producing plant is concentrated around the ganges delta region of bangladesh and india where the warm, wet climate during the monsoon season provides ideal growing conditions . In terms of usage, production, and global requirement, jute is second only to cotton . Jute plants are easy to grow, have a high yield per acre, and, unlike cotton, have little need for pesticides and fertilizers . The leaves and roots left after harvest enrich the soil with micronutrients, maintaining soil fertility . When used as a geotextile, it puts nutrients back in the soil when it decomposes . This rain - fed crop during its growth helps to clean the air by assimilating three times more co2 than an average tree, converting the co2 into oxygen . Despite its great agronomic importance, research on jute at the molecular level so far only 1,210 sequences are found in the genbank with no deposits of any mirna sequences in mirbase database . Within this context, the current study has employed the deep sequencing strategy in an attempt to effectively identify conserved and novel jute mirnas . Quantitative real - time pcr (qrt - pcr) has been performed to determine the expression of these mirnas . For mapping the identified mirnas, genome of vitis vinifera seeds of farmer popular o-9897 variety of tossa jute (corchorus olitorius) were collected from bangladesh jute research institute (bjri). They were surface sterilized with 70% ethanol, subsequently washed in distilled water, and allowed to germinate on sterile petri dishes containing 3 mm moist filter paper (whatman) at 30 1c and 65% relative humidity . Seeds were allowed to grow for 4 days under the specified conditions . On the fourth day of germination, seedlings were collected and immediately snap - frozen in liquid nitrogen and stored at 80c for subsequent use . Rna was isolated from collected seedlings using trizol reagent (invitrogen, usa) by following the manufacturer's instructions . Later, rna samples were sent to beijing genome institute (bgi, shenzhen, china) for deep sequencing of small rna by illumina hiseq high - throughput sequencing platform . In short, after ligation with 5 and 3 adaptors, the short rnas so obtained were reverse - transcribed to cdna according to the illumina protocol . The resulting small rna library was then sequenced following sbs method (sequencing by synthesis) by illumina hiseq high - throughput sequencing . Raw data obtained from illumina hiseq high - throughput sequencing was at first filtered by removing contaminants which include low quality reads, reads with 5 primer contaminants, reads without 3 primer, reads without the insert tag, reads with poly a, and reads shorter than 18 nt . After cleaning, clean reads fully matching other rnas, including mrna, rrna, trna, snrna, snorna, and repeat rna, were excluded by using blastn - short alignment (blast2.2.26 +, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.26/) and aligning against sanger rna family database (rfam 11.0, ftp://ftp.sanger.ac.uk/pub/databases/rfam). The remaining unique sequences were further aligned against mirbase - v20 allowing up to 3 mismatches to identify known mirnas present in c. olitorius . Mature mirnas present within a genome encoding identical or nearly identical sequences were then grouped together into a family . Prediction of novel mirna was done using prediction software, mireap (http://sourceforge.net/projects/mireap/), developed by bgi by taking into consideration secondary structure, cleavage position of dicer protein, and minimum free energy of the unannotated small rna tags . Strategic conditions for selecting unique mirna are as follows: (i) the tags which are to be used to predict novel mirna should be from unannotated tags which can match reference genome (vitis vinifera), from the tags which align to introns as well as antisense exons; (ii) genes, whose sequences satisfy the above standards and their secondary structures which allow hairpin mirnas to fold within them and the presence of mature mirnas in one arm of the hairpin precursors, are considered as candidate genes for mirna; (iii) the possible candidate mature mirna strand contains 2-nucleotide 3 overhang; (iv) hairpin precursors of the candidate mirnas are devoid of large internal loops or bulges; (v) secondary structures of the hairpins are stable, with the minimum folding energy (mfe) lower than or equal to 20 kcal / mol . The rules used for target prediction in plants are based on those suggested by allen et al . And by schwab et al . These are (i) presence of maximum 4 mismatches between small rna and target (g - u bases count as 0.5 mismatches), (ii) not more than 2 contiguous mismatches in the mirna / target duplex, (iii) no end - to - end mismatches at the 5 of mirna from 2 to 12 positions of the mirna / target duplex, (iv) no mismatches in positions 10 - 11 of mirna / target duplex, (v) a maximum of 2.5 mismatches in positions 112 of the mirna / target duplex from 5 region of mirna, and (vi) minimum free energy (mfe) of the mirna / target duplex which should be 74% of the mfe of the mirna bound to its perfect complement . The targets of mirnas were further validated by a well - recognized mirna - target prediction tool: psrna target . In addition to potential target prediction for known and novel mirnas, pathways which include the corresponding target genes as well as the biological function of such genes are taken into consideration by using the grape genome as a reference . Gene ontology (go) is an international standard classification system for gene function, which provides a set of controlled vocabulary to comprehensively describe the property of genes and gene products . There are 3 ontologies in go: biological process, cellular component, and molecular function, containing lists of biological functions that illustrate each gene and its product (http://www.geneontology.org/). Each category defines precise participation of a given gene within an organism . As for pathway identification, kegg (http://www.genome.jp/kegg/) kegg analyses reveal the main pathways with which the target gene candidates are involved . To verify the identified known and potential novel mirna candidates in jute, stem - loop reverse transcription - pcr was performed . Stem - loop primers were designed according to the method described by chen et al . . This primer binds to specific mirna at the 3 region owing to the precision conferred by the primer with the exact reverse complement of six nucleotides from the 3 end of each particular mirna sequence, which is reverse - transcribed by the rt enzyme . Two thousand nanograms of total rna were used to perform the rt reaction with superscript iii first strand synthesis system (invitrogen, usa) according to the protocol of varkonyi - gasic et al ., which has been further standardized for jute . For a reaction volume of 20 l, 0.5 l of 10 mm dntps was at first taken together with an appropriate amount of rna and an adjusted amount of depc - treated h2o followed by heating the mixture for five minutes at 65c and then immediately transferring the same on ice . While keeping on ice for approximately 2 minutes, 4 l of 5x fs buffer, 2 l of 0.1 m dtt, 1.2 l of 1 m stem - loop primer, 0.25 l of m - mlv superscript iii rt (200 u/l), and 0.1 l rnaseout (40 u/l) were added to the reaction mix . The rt reaction was carried out in a thermal cycler (mastercycler, eppendorf, germany), followed by a pulse rt cycle starting from incubation at 16c for 30 minutes, then 60 cycles of 30 sec . At 30, 30 sec . At 42c, and 1 sec at 50c . This step was followed by another incubation step of 5 mins at 85c to inactivate the rt enzyme . End point pcr was then conducted with mirna specific forward primer and a universal reverse primer to check the presence of the specific mirnas . Pcr products were electrophoresed on 3% agarose gel in 1x tae and stained with ethidium bromide before visualization under a transilluminator . We further conducted quantitative real - time pcr for confirming the expression of some selected known and novel mirnas using a 32-well plate roche lightcycler nano system and the roche sybr green master i (roche diagnostics, germany). Briefly, equal amount of cdna was taken in a reaction volume of 7.5 l in triplicate with 0.1875 l of each primer (forward and universal reverse) and 3.75 l of sybr green master i. thermo cycling conditions were set at an initial polymerase activation step for 600 seconds at 95c, followed by 45 cycles of 5 sec at 95c for template denaturation, 10 sec at 60c for annealing, and 1 sec at 72c for extension and fluorescence measurement . Later, a dissociation protocol with a gradient from 50c to 95c was used for each primer pair to verify the specificity of the rt - qpcr reaction and the absence of primer dimers . In order to identify micrornas in jute, rna was isolated from the total tissue of four - day seedlings and subjected to illumina hiseq high - throughput sequencing by synthesis (sbs) technology . Among a total of 16912862 raw reads, the details of tag cleaning are summarized in table 1, which shows that a total of 16644324 clean reads were obtained by removing 3 adapter null, insert null, 5 adapter contaminants, sequences smaller than 18 nt, and poly a. this comprises about 99% of the high quality reads . Distribution of these clean reads that contain a pool of small rnas ranging from 18 to 30 nucleotides is shown in figure 1 . However, the sizes of small rnas were not found to be uniform; majority (92.69%) of the srnas are 2024 nt in size, with 21 nt being the most abundant (42.19%), followed by 24 nt (22.95%) and 20 nt (14.25%), respectively . These sequences were then aligned to rfam 11.0 and genbank database (blastn) to identify common srnas other than mirnas, as well as to remove mrnas (see supplementary file-1 in supplementary material available online at http://dx.doi.org/10.1155/2015/125048). The remaining sequences were matched against mirbase-20 database for the prediction of mirnas, revealing 33433 unique and 8994892 redundant reads, which were finally used to identify known mirnas . The novel mirnas in jute were identified from unannotated tags by using mireap, software developed by bgi (described in section 2). In the absence of the complete genome sequence and with practically no information on mirna of jute in mirbase, clean reads were aligned to the mirna precursor / mature mirna of all plants in mirbase allowing up to three mismatches or free gaps to identify known mirnas . The expression of mirna is generated by summing the count of tags which can align to the temporary mirna database, generated by choosing the most expressive mirna of each mature mirna family . A total of 227 known mirnas were identified in this study, of which 164 belong to 23 conserved and 63 to 58 nonconserved families . Conservancy of mirna families found in jute showed high homology with their respective homologs in other model plants (figure 2). However, the number of family members of conserved mirnas was highly variable, with mir156 being the largest family, consisting of 26 members, whereas mir403, mir394, mir827, mir477, and mir2111 were the smallest among the families, comprising only one member . Mir166 and mir169 were the second largest with each having 18 members and mir171 was the third largest family with 14 members (figure 3). Most of the conserved families contain both 5p and 3p mature mirna sequences, attaching a high confidence to the data set . Highly variable reads numbers were also found among the families, even in members of the same family, indicating different expression levels of these mirnas . Among them, col - mir157a had the highest level of expression, having 5531609 counts, and the other mirnas like col - mir156a, col - mir166a, and col - mir167h also had relatively high reads numbers, counting more than 150000 . Several conserved mirnas (like mir171, mir398, and mir159) and, as expected, most of the nonconserved mirnas had relatively low copy numbers . Interestingly, a mirna named col - mir3954 from an undefined family had very high level of expression, having 868222 reads, third highest of all mirnas found in jute . High expression frequency of mirnas derived from the 3 arm of some pre - mirnas, like col - mir166h-3p, col - mir166g-3p, col - mir166j-3p, col - mir165a-3p, col - mir396b-3p, col - mir396e-3p, and so forth, compared to their corresponding 5 arm - mirnas, supports the observations of functional activity of both arms of pre - mirna hairpins [55, 56]. The mirna hairpins are mostly located in intergenic regions, introns, or reverse repeat sequence of coding sequences . Thus tags belonging to these regions characteristic hairpin structure of mirna precursor was used to predict novel mirna with prediction software mireap (http://sourceforge.net/projects/mireap/) by exploring the secondary structure, the dicer cleavage site, and the minimum free energy of the unannotated small rna tags which could be mapped to the reference vitis vinifera genome . Predicted secondary structures were further validated by mfold (supplementary file-3) and novel mirnas were identified based on the selection criteria described in section 2 . 17 potential novel mirnas have been identified in this study of which 9 mirnas were derived from 3 arm of the pre - mirna sequences and 8 from the 5 arm (table 3). Average length of the pre - mirnas sequences ranged from 78 to 349 nt, similar to those found in maize and rice; minimum folding energy (mfe) for jute mirnas was observed to be within a range from 21 to 105.3 kcal / mol, similar to the range observed in cucumber (supplementary file-4). Expression of novel mirna was determined by summing the count of such mirnas which have no more than 3 mismatches on either the 5 or 3 ends and with no mismatch in the middle . Novel mirnas usually have lower levels of expression than the conserved mirnas, as evident from findings of several plant species like soybean, brassica napus, maize, arabidopsis, and wheat [59, 6164]. For a precise elucidation of the role of mirnas, target identification and determination of their biological functions it is now evident that cleavage or translational repression site of most known plant mirnas is located in the cds (coding sequence) region of their target mrna with perfect or nearly perfect sequence complementarity, making it feasible to identify plant mirna targets [4, 21, 66]. In this study, target genes of mirnas were identified by blastn against the genome sequence of vitis vinifera, following methods described by allen et al . And schwab et al . [9, 45]. Among a total of 79 identified mirna (both conserved and nonconserved) families, 116 potential target genes were predicted for 39 families (supplementary file-5). A total of 46 genes from this prediction overlapped with targets identified by psrna target which found 99 target genes for 19 mirna families (supplementary file-9). Highest number (16) of targets was identified for mir397 family, all of which are laccase, an enzyme, involved in plant cell wall lignification . Most of the other families targeted only a single gene . For the novel jute mirnas, a total of 11 targets were predicted for 4 among the 17 identified mirnas (table 4, details in supplementary file-6), with a maximum number of target genes (6) recognized for col - mirn7 . Most of col - mirn7 targets are nb - arc domain containing protein, which is a resistance (r) protein, involved in pathogen recognition and subsequent activation of innate immune responses . To better understand the functions of mirnas, target genes were analyzed by gene ontology (go) level 3 to divulge the regulatory network of mirnas and target genes . Such analysis demonstrates that for jute 133 predicted target genes (both for known and novel mirnas) can be classified into 20 having biological, 5 cellular, and 5 molecular functions . Same gene was found to be involved in multiple processes with the reverse being also true (figure 4 and supplementary file-7). As illustrated by kegg pathway analysis (supplementary file-8), the predicted target genes of jute mirnas were found to be involved in 42 different pathways . Some of the mirnas identified through deep sequencing were verified by the standard stem - loop rt - pcr method followed by end point pcr and qrt - pcr . The stem - loop primers were designed with a 3 specificity for a particular mirna which hybridizes to the same and is reverse - transcribed by the rt enzyme . These primers increase the sensitivity of the reactions such that this method can significantly distinguish two mirnas with only one single nucleotide change . The rt product is then subjected to end point and qrt - pcr . Forward primers were precisely designed from the first 15 bases of each mirna with 5 extension of random gc rich sequence to increase the melting temperature as mentioned by varkonyi - gasic et al . In 2007 while the reverse primer is a universal sequence that is designed from the 5 region of the stem - loop rt primer . A set of 11 randomly selected conserved mirnas as well as 9 novel mirnas were used for verification . In this study, the stem - loop primer used was 50 bp long together with 5 (forward primer) and 3 extensions, and the end point pcr product size ranged from 60 to 70 bp . Amplification of the product gave a sharp band for each of the selected known and novel mirnas (shown in figure 5). Cdnas were further amplified by qrt - pcr in technical triplicates from which log 2 values of cq were calculated for each of the mirnas and average of these values was compared with the log 2 value of read counts obtained from deep sequencing . Most of the qrt - pcr results acceded with the sequencing data; however, in some cases, discrepancy was observed (figure 6). Widespread discovery of mirnas and their critical role in gene regulation has made it ever important to recognize them in different species . While a large amount of mirnas are reported and deposited in databases from different plants, mirna associated research in jute is still to be instigated . Without the genome sequence of jute at hand, identification of mirna and their targets in jute by deep sequencing of small rnas use of closely related species' genomes as proxy references can facilitate mirna identification in nonmodel species like jute for which genome sequence is not available . We have used the grape genome as the background because of sequence similarity between these two species . . Analyses of size distribution patterns of the reads show that the most abundant srnas in jute are 21 nt in size which is about 42.19%, consistent with recent identification of srnas in different plant [34, 62, 64]. Sequencing frequencies for mirnas in a library can be used as an index for estimating the relative abundance of mirnas . Numerous small rna sequences, engendered from illumina hiseq high - throughput sequencing platform, show the presence of different mirna families and are even able to differentiate between distinct members of a given family . Mir156 family which is highly conserved across the species was found to be the largest family in jute seedlings with the highest expression of col - mir157a followed by col - mir156a . Two other members of the same family, namely, col - mir156c and col - mir156k, also show significant levels of expression . During shoot development, mir156 regulates the transition of plants from juvenile to adult phase by targeting spl genes . In arabidopsis, mir156 is strongly expressed during seedling development and shows weak expression in mature tissues . This could explain the relative abundance of the members of mir156 family since rna used in sequencing was extracted from jute seedlings . Deep sequencing technology allows distinguishing and measuring mirna sequences with only a few nucleotide changes . For example, the abundance of mir156 family varied from 1 read (col - mir156p) to 5531609 reads (col - mir157a). This was also the case for some other mirna families, such as col - mir166 (from 3 to 215636 reads) and col - mir167 (from 12 to 154973 reads). Presence of a prevailing member in a mirna family may indicate the dominant role of this member during the growth phase at which the samples were collected . It is also to be noted that most of the conserved mirna families consist of more than one member, whereas nonconserved mirnas identified in this study are mostly represented by a single mir (mirna) gene . It has been hypothesized that mir genes originate by gene duplication events followed by random mutation processes to evolve in multiples of imperfectly paired hairpins [77, 78]. Consequently, ancient evolutionarily conserved mirnas are represented by multiple mir genes whereas nonconserved mirnas (believed to be evolutionarily recent) generally originate from a single locus . It is plausible that the conserved mirnas are responsible for control of basic cellular and developmental pathways common to most eukaryotes whereas nonconserved mirnas are involved in regulation of species - specific pathways and functions . Species - specific mirnas are believed to have recently evolved and, in general, expressed at levels lower than those of strictly conserved mirnas [34, 77]. Data acquired from sequencing frequencies of conserved and nonconserved mirnas fits well with this extrapolation, where the nonconserved and species - specific mirnas show residual accumulation in the tested tissue . However, one mir-3954, a single member of an undefined family, appears to be expressed in significantly high levels . Its only homolog deposited in mirbase v20 is in c. sinensis, showing high frequency of reads . 17 new jute specific mirnas identified in this study show a size anticipated for srnas derived from dcl1 processing, although sequence variants that possess shortened or extended 5 or 3 ends were also found . Ten among the seventeen new col - mirnas are 21 nt in size, consistent with canonical dcl1 products . However length variation was also found . Two, col - mir2 and col - mir9, are 20 nt in size; three, col - mir5, col - mir6, and col - mir14, are 22 nt long . Col - mir13 was found to be 23 nt in size which can probably be explained by the fact that diverse mirna families are also independently processed by dcl3 to generate a new class of bona fide (2325 nt) mirnas with no canonical size, called long mirnas . A total of 20 mirnas of both conserved and species - specific origin were corroborated by stem - loop rt - pcr and their expression pattern was assessed by qpcr to validate the data obtained from deep sequencing . Discrepancies in the expression pattern of some mirnas found by deep sequencing and qpcr can be attributed to practical differences between the sensitivity and specificity of these two techniques . The sensitivity and large dynamic range of next generation sequencing (ngs), along with its consistent prediction of fold changes when compared with gold - standard qpcr, support its use for discovery - oriented and exploratory mirna profiling experiments [84, 85]. To evaluate and outline a putative function for a mirna in plants, we have predicted target genes for known and potential new mirnas identified in this study using the genome of vitis vinifera as a reference . Most of the target genes for conserved mirna families predicted in jute have already been confirmed in model plants, as target genes are commonly conserved [78, 80]. Mir156/157-squamosa promoter - binding protein, mir166-homeodomain leucine zipper protein iii (hd - zip iii), mir167-auxin response factor (arf), mir164-nac domain protein, mir172-transcription factor apetala2, mir159-myb transcription factor, mir171-gras family transcription factor, mir394-f - box family protein, and mir395-atp sulfurylase well characterized mirna - target pairs in other plants have been found in jute . However, a number of widely studied mirna - target pairs, such as mir398-copper superoxide dismutase, mir399-e2 ubiquitin conjugating protein, and mir162-dicer - like 1(dcl1), were not found in this study . This could possibly be due to the fact that the jute genome sequence is not available to be used as a reference . However, conserved mirnas with their nonconserved targets, including mir167-peroxidase29, mir396-eukaryotic translation initiation factor 2c, mir168-nac domain containing protein, mir164-growth regulating factor 1, mir390-ap domain containing transcription factor, mir160-myb transcription factor, and mir393-gtp - binding protein alpha subunit, were also found to be present in jute, allowing presumption of nonconserved targets for conserved mirnas . Highest number of target genes were identified for mir397, which is laccase, a well - studied enzyme, encoded by multigene families in poplar, arabidopsis, rice, and liriodendron tulipifera, reported to be involved in lignin biosynthesis of plants [99101]. Toughness of this biopolymer also poses a major obstacle to pulping, forage digestibility, and biofuel production . It has been reported that transgenic p. trichocarpa plants overexpressing ptr - mir397a result in a reduction of klason lignin content, supporting the idea that use of mir397 would be an attractive means for reducing lignin - related problems . Future experiments including in - depth studies of mir397-laccase pair may help in producing quality products from jute . This set of experimentations for identification of mirnas and their potential targets can initiate further study on understanding the mechanisms of regulation of jute mirna.
The predominant method of entry in gynecologic surgery worldwide remains a closed technique, with or without pneumoperitoneum . This approach unfortunately has been demonstrated in multiple studies to have the potential for visceral and vascular injury due to the blind insertion of veress needles or trocars . Complications reported in the literature from the closed entry technique range from 0.05% to 0.67%, and include vascular injury, enterotomy, urinary tract injury, subcutaneous emphysema, and gas embolism . These complications arise due to the normal anatomic relationship of the periumbilical site to the underlying great vessels and viscera and are particularly problematic in patients with adhesions or at extremes of body weight . Additionally, delay in diagnosis of bowel injuries frequently sited with this closed technique accounts for significant morbidity and mortality . This technique consists of creating a small umbilical incision under direct visualization to enter the abdominal cavity followed by the introduction of a blunt trocar . Hasson proposed its potential benefits to be the avoidance of blind insertion of the veress needle and bladed trocar, prevention of preperitoneal insufflation and gas embolism, guaranteed pneumoperitoneum, and a more anatomical repair of the abdominal wall . Since that time, hasson and others have corroborated these proposed benefits with data obtained from large case series . This large chart review was undertaken to examine our experience with open laparoscopy and to determine whether preoperative characteristics can predict open laparoscopic entry complication . We completed a retrospective chart review of all patients who underwent laparoscopy via an open technique at the mayo clinic arizona in the department of gynecology (8 surgeons) from january 1, 1998 through december 31, 2006 . Data were extracted from a computer - generated search, and each electronic medical record was individually reviewed to ensure inclusion criteria and obtain end points . All charts were reviewed from the point of surgery through the remainder of their care at our institution . Intraoperative complications (bowel and vascular injury, failure to enter), postoperative complications (hernia, hematoma, cellulitis, abscess, cosmetic issues), body mass index (bmi), number of previous abdominal surgeries, and length of follow - up were extracted from charts . Statistical analysis was performed using jmp 6.0 for windows (sas institute, cary, nc). The open laparoscopic technique used in this series is similar to that originally described by hasson and is as follows: the umbilicus is held and everted with 2 allis forceps . A vertical skin incision, 10-mm to 15-mm long, is created at the deepest portion of the umbilicus . The underlying fascia is grasped with 2 kocher forceps, elevated, and incised in a vertical midline fashion . If the peritoneal cavity is still intact, it is grasped with kocher or allis forceps and entered with metzenbaum scissors . Finally, the laparoscope is introduced and the organs below the entry site are inspected as is the remainder of the abdominal cavity . Closure of the umbilical entry site is performed with direct visualization and identification of the fascial layer . A 0-polyglactin suture on a #2 urologic needle is used for the fascial layer, and a 40 polyglactin suture is used for skin approximation . An occlusive dressing is removed from the site in the ensuing 24 hours to 36 hours . Mean patient bmi was 26.5 (range, 14 to 57), mean previous abdominal surgery was 1.3 (range, 0 to 18), and mean follow - up was 340 days (range, 0 to 3028). At the time of entry with the open technique, we experienced 2 (0.1%) instances of enterotomy and 3 (0.1%) cases of failed entry . Of the enterotomies, in one patient dense adhesions from a previous debulking laparotomy (requiring over 3 hours of laparoscopic adhesiolysis) were present with jejunum adherent immediately under the umbilicus . The second patient had the transverse colon adherent to the umbilicus; she had had 4 previous laparotomies . Of the failed entries, the first patient had dense umbilical adhesions from 2 prior laparotomies, and the procedure was aborted . In the second patient, surgeons were unable to reach the peritoneal cavity due to the patient's pannus (bmi 43), and a left upper quadrant entry was chosen . The third patient had dense periumbilical adhesions from a previous colectomy, so the procedure was converted to a minilaparotomy . Patients with umbilical infection were subclassified based on whether the diagnosis was made over the phone due to patient complaints of erythema or drainage, by examination and diagnosis of cellulitis, or an umbilical abscess requiring evacuation . Patients with subsequent umbilical hernias were subclassified into symptomatic (noted by a physician or patient) or asymptomatic (discovered serendipitously at a subsequent surgery) (table 1). Fisher's exact test was used to determine the association between each of the recorded complications and the number of previous abdominal surgeries and obesity (bmi>30) (table 2). A significant association was noted between umbilical infection and previous abdominal surgery (p=0.049), and between umbilical hernia and obesity (p=0.024). This chart review confirms previous findings of case series, literature reviews, and meta - analyses of the complications associated with open laparoscopic entry . A large dutch review of 12,444 cases of open laparoscopy culled from 6 previously published case series found a 0.048% rate of enterotomy and no cases of vascular injury or gas embolism . These rates contrast with an enterotomy rate of 0.083% and vascular injury rate of 0.075% from 489,335 cases of closed laparoscopic entries in this same review . Similarly, an australian meta - analysis examined 22,465 cases of one published open laparoscopy series and 760,890 patients from 22 published closed laparoscopy series . They noted an enterotomy rate of 0.049% (11 patients) in open cases and 0.067% (515 patients) in closed cases (nonsignificant difference). No cases occurred of vascular injury in open cases, while the rate of vascular injury in closed cases was 0.044% (336 patients), which was statistically significant (p=0.003). Similar rates of umbilical infection and hernia occurred with both techniques . To date, there are no reports of fatal vascular injuries and only 2 instances of nonfatal major vascular injuries in the literature associated with the use of the open technique . The first case occurred during the skin incision, when the scalpel directly entered the aorta (the hasson trocar had not been used). The second case involved a damaged metal hasson cannula with a protruding spike that caused an aortic laceration . The lack of fatal vascular injuries noted in this and other large series is of utmost significance . Data from litigious allegations related to 135 laparoscopic procedures over a 19-year period revealed a disproportionate percentage of the cases involving vascular injuries . Additionally, these injuries are associated with a significant mortality risk, with 5 of 9 vascular injuries at closed laparoscopic entry reported to the medical defense union resulting in death . Moreover, the morbidity incurred with major vascular injury includes transfusions, prolonged hospitalization, loss of limb, or other long - term sequelae . Visceral injuries, in particular small bowel and colon, are also life - threatening, and because they are commonly missed during a closed entry, and injury may later be recognized only when symptoms of peritonitis develop . Although intestinal injuries occurred in this case series, a major advantage of the open technique is the immediate recognition and repair of the enterotomy . Neither of the 2 patients in this series suffered any long - term complications related to enterotomy . Previous abdominal surgery and obesity are therefore not contraindications to an open laparoscopic entry, and in fact the open approach may offer some advantage over the closed technique in these patients . Several series have reported lower hernia formation with the open technique, perhaps because of the ability to easily identify the fascial layer during closure . The fact that none of the hernias occurred prior to 6 weeks postoperatively also suggests that a good primary reappoximation was achieved at surgery . Although this is a retrospective review of patients presenting to a referral center with somewhat limited follow - up, the large number of procedures performed via a consistent technique enhances the strength of this study . Additionally, each case was hand reviewed for complications rather than using icd-9 codes to assess for rate of complications . Finally, when available, patients charts for the entire time they received care at our institution, in some cases up to 8 years, were reviewed . The use of an open laparoscopic entry is advocated because it is a safe, simple means of accessing the peritoneal cavity . This case series confirms previous reports of the low risk of enterotomy, absence of fatal vascular injury, and comparable rates of umbilical infection / hernia associated with an open entry technique . The rapid recognition of enterotomy with this entry technique, and the utility of this technique in obese patients or those with previous abdominal procedures are additional advantages.
Pulmonary hypertension (ph) is predominantly defined by a mean pulmonary artery pressure at rest greater than or equal to 25 mm hg . It is an enigmatic vascular disease and the pathogenesis of ph is multifactorial of origin and, hence, is categorized as idiopathic type [14]. As ph develops in a wide variety of clinical circumstances and is associated with diverse histological manifestations, a classification system is developed [5, 6]. The dana point expert group has published a consensus of ph classification based on pathology, survival, natural history / epidemiology, etiology, and response to the treatment . Among them, pah specifies that the disease primarily restricted to the pulmonary arterioles, a typical characteristic which shows an elevated pulmonary arterial pressure [2, 3]. The pathological consequence of pah is the structural remodeling of pulmonary arteries (pa), where increased proliferation of pulmonary artery smooth muscle cells (pasmc) and dysfunction of pulmonary artery endothelial cells (paec) occur in the vascular bed [79]. The morphological changes consist of hypertrophy of the tunica media, multicellular vascular lesions which obstruct and obliterate pulmonary arterioles leading to intimal thickening . The obstructed vessels limit the blood flow via pa and increase right ventricular afterload leading to right ventricular hypertrophy (rvh) and rv dysfunction [1012]. At molecular level, it is believed that the remodeling events in ph demand the participation of all cell - types present in the pulmonary arteries and that influence the pathological manifestation in the pulmonary vessel wall . The contributing factors that influence the remodeling process are hypoxic state, inflammation, vessel injury, and oxidative stress in the pulmonary vessels . As all forms of ph have in common an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacyclin, or endothelin- (et-) 1, to establish the correct balance between vasoconstriction and vasodilatation [1316]. Currently, the management for ph is aimed at optimizing cardiopulmonary interactions by targeting prostacyclin, endothelin, and nitric oxide signaling pathways . The most commonly used treatment regimen of ph is the use of prostacyclin analogues (alprostadil, epoprostenol, treprostinil, and iloprost), endothelin receptor antagonists (bosentan, ambrisentan), and inhaled no . In addition, phosphodiesterases (pdes) inhibitors; pde-3 inhibitors (e.g., milrinone and enoximone), and pde-5 inhibitors (e.g., sildenafil and tadalafil) are used to treat ph . They were used as an alternative therapeutic strategy which targets downstream components of the no signaling pathway by inhibiting pde-5, the enzyme that catalyzes the conversion of cgmp to gmp . Despite the advancement of modern surgery or ph - specific therapy, although ph (or pah) is well - studied encompassing both cardiac and vascular boundaries, the precise cellular and molecular mechanism of initiation and progression of ph are not completely understood and are still being explored . There is no cure of this disease and current therapies are limited to reverse the vascular remodeling . Evolving evidence indicates that dysregulation of micrornas (mirna or mir) contributes to ph pathogenesis [1924]. Indeed, an emerging body of evidence demonstrates that a fine balance in mirna levels seems to be a fundamental to maintaining homeostasis in the pulmonary vasculature and an imbalance with mirna level playing a critical role in the pathogenesis of ph by regulating a set of targeted genes . This review will collate vascular remodeling during ph, mirna biogenesis, recent advances on mirna modulation in ph, therapeutic opportunity, and conclusion . The mirna(s) are composed of a vast family of short, noncoding rnas (~22 nucleotide long). Mirnas are found from a single cell organism to plant and higher animals and even viruses [3739]. In humans, the biogenesis (canonical) pathway of mammalian mirnas is a two - step enzymatic process initiated in the nucleus and then transported to cytoplasm (figure 1). The transcription of mirnas generally processed by rna polymerase ii (less frequently by rna polymerase iii) and are typically capped and polyadenylated [41, 42]. As a result, a long primary mirna transcripts (pri - mirna) containing a stem - loop structure is developed which is recognized by a large protein complex, called the microprocessor, the main components of which are the rnase iii drosha and digeorge syndrome critical region 8 (dgcr8) [4348] (figure 1). For most pri - mirna, drosha is contributing the cleavage process called cropping with the assistance of its binding partner protein, dgcr8 [41, 48, 49]. The resultant product is a ~60 nucleotides long, hairpin - structured, called precursor mirna (pre - mirna). Dgcr8 directly interacts with the pri - mirna stem and flanking segments, a crucial measurement for one end of the mature mirna . In addition to the canonical pathway of mirna biogenesis described above, an alternative pathway also exists that are independent of drosha . In alternative pathways, the mirna can be released as mitrons (pre - mirna like introns) from pri - mirna and proceed for mirna processing unit without assistance of microprocessor [50, 51]. The pre - mirnas are actively transported from the nucleus into the cytoplasm by exportin-5 (exp-5) (figure 1). In the cytoplasm, another rnase iii enzyme, called dicer, catalyzing the process with the assistance of partner molecules argonaute (ago2), hiv-1 transactivation response rna - binding protein (trbp) and/or protein activator of pkr kinase (pact), producing a short dsrna duplex, mirna / mirna of approximately 22-nucleotide length [44, 5356]. The mirna / mirna duplexes are then incorporated into a ribonucleoprotein (rnp) complex called mirisc (rna - induced silencing complex)that plays a critical role in the mirna - mediated mechanism of gene regulation . During assembly process of the mirisc, the mirna / mirna duplex is loaded into the ago, and the strands (called the passenger strand) are released and degraded . Eventually, the bound mirna strand (called the guide strand) dictates mirisc to interact with partially complementary sequences in target transcripts (localized within the 3utr) and primarily triggers mrna deadenylation and degradation . Micrornas (mirnas) are small, endogenously expressed noncoding rnas that regulate gene expression at posttranscriptional level, via degradation or translational inhibition of their target mrnas [37, 59]. Mirnas are ~22 nucleotides in length which bind to the 3 untranslated region of specific target genes and thereby suppress / inhibit the translation of target genes [60, 61]. Mirnas are key regulators of a wide range of cellular processes and play a pivotal role in vascular inflammation and cardiovascular pathologies inclusive of ph . From extensive studies from the past few years, it has become apparent that mirnas are expressed in a cell- and tissue - specific manner and are critically involved in various biological processes . Emerging evidence indicates that mirnas contribute an important role in the maintenance of pulmonary vascular homeostasis and in the pathogenesis of ph . In the following part, a discussion of the roles of mirnas in ph - related signaling pathways is provided . Mir-21 is a ubiquitously expressed mirna that is traditionally considered to be an oncogenic mirna (oncomir). The two - channel microarray was performed to quantify mirna expression in whole lung extracts during the development of ph or pah caused by chronic hypoxia or monocrotaline in rats . It was suggested that mir-21 expression was downregulated in mct - induced ph model, but not in chronic hypoxia rats . Mir-21 showed a similar expression level in both normoxic hypoxic cells, whereas tgf-1, an important regulator of pulmonary vascular remodeling in ph, reduced expression of mir-21 . This suggests that although hypoxia- and monocrotaline - induced ph shares some common cellular processes driving the characteristic vascular remodeling, the different pathobiology induced by hypoxia and monocrotaline may lead to the different regulation of mir-21 expression . Furthermore, the downregulation of mir-21 was confirmed in human lung tissue and serum from patients with idiopathic ph . Since bmps induce smooth muscle cell differentiation through upregulating the expression of mir-21, the downregulation of mir-21 in this setting may relate to the reduced bmp signaling and contribute to the alteration of smooth muscle cell phenotype in ph . However, yang and colleagues reported that mir-21 expression was increased in distal small pulmonary arteries of hypoxia - exposed pah mice and levels of bmpr2, wwp1, satb1, and yod1, the putative mir-21 targets, were decreased in the same tissue . Transfection of mir-21 mimics also led to the reduced expression of bmpr2, satb1, and yod1 in pasmcs . The crucial role of mir-21 in vascular pathology has been evidenced by the results that the blockade of mir-21 impeded the development of intimal hyperplasia after acute vascular injury and bleomycin - induced pulmonary fibrosis [28, 64]. Yang et al . Also found that inhibition of mir-21 alleviated chronic hypoxia - induced ph and attenuated pulmonary vascular remodeling . In human pasmcs, overexpression of mir-21 promoted, whereas sequestration of mir-21 abrogated cell proliferation and the expression of cell proliferation - associated proteins . The mir-21 null mice showed an exaggerated ph response to hypoxia, suggesting a major role of mir-21 in the pathogenesis of chronic hypoxia - induced pulmonary vascular remodeling and ph . Although an association between mir-21 and ph (pah) is identified, the function of mir-21 in the development of ph is inconsistent in many experimental models . Therefore, further investigations are required to clarify the role of mir-21 in the pathogenesis of ph . The mir-204 appears to be the first mirna that showed a mechanistic link between pulmonary arterial remodeling and cellular function . Mir-204 was reported to be decreased in rodent lungs with hypoxia- and mct - induced pah and lung specimens from patients with pah . The reports suggested that stat3 activation contributed a crucial role in regulating mir-204 in pasmc . It was further demonstrated that mir-204 inhibition increased expression of shp2, triggered the activation of src kinase, stat3, and nuclear factor of activated t cells (nfat), and thereby reduced apoptosis and was promoted proliferation of pasmcs . Finally, delivery of synthetic mir-204 mimic to the lungs lowered pulmonary artery pressure, reduced medial wall thickness, normalized levels of mir-204, shp2, and stat3, and alleviated the disease severity . This study may indicate the safe and effective use of mimic delivery to the pulmonary vasculature for future therapeutic purpose . Interestingly, wei et al . Have identified reduced level of mir-204 in the buffy coat of human subjects may correlate with pah severity and might serve as a circulatory biomarker for ph . Expression of mir-143/145 was driven by tgf- and bmp4 and induced contractile gene expression through downregulating klf4 and myocardin [31, 68, 69]. The elevated levels of mir-145 were observed in primary pasmcs cultured from patients with bmpr2 mutations and also in the lungs of bmpr2-deficient mice, suggesting a role of bmpr2 signaling in modulating mir-145 expression . Caruso and colleagues also found that mir-145 was downregulated in patient samples obtained from idiopathic and congenital ph but upregulated in plexiform lesions . The role of mir-145 in pah was further explored and elevated expression of mir-145 was shown in the wild type mice exposed to hypoxia . Mir-145 deficiency and a locked nucleic acid anti - mir-145 resulting in significant protection from hypoxia - induced ph may represent a potential therapeutic target . The mir-17 - 92 cluster encodes seven related mirnas, which result from the transcription of a single pre - mirna and is further processed and cleaved to the mature mirnas . Mir-17 - 92 cluster was retrieved as potential modulators of bmpr2 signaling by performing a computational algorithm on the bmpr2 gene . Overexpression of mir-17 - 92 resulted in a marked reduction of bmpr2 protein level, and bmpr2 was proved to be directly targeted by mir-17 and mir-20a by using a bmpr2 reporter in hek293 cells . Stat3 was found to be involved in il-6 signaling - mediated upregulation of mir-17 - 92 cluster and the subsequent downregulation of bmpr2, since a highly conserved stat3-binding site exists in the promoter region of mir-17 - 92 gene . The role of a cholesterol - modified antagomir to mir-20a in hypoxia - induced ph was explored by brock et al . Mir-20a restored functional bmpr2 signaling in human pasmcs and intraperitoneal administration of anti - mir-20a increased bmpr2 levels and alleviated vascular remodeling in lung tissue of hypoxic mice . Antagomirs to mir-17 and mir-92a reduced muscularization of pulmonary arteries in the hypoxic mouse and monocrotaline rat models of ph, but only anti - mir-17 decreased rvsp and parameters of right heart dysfunction . Bertero and colleagues identified mir-130/301 family as a master regulator of cellular proliferation in ph by constructing in silico a network of genes and interactions based on curated seed genes with known importance in ph . Mir-130/301 expression was found to be increased in lungs of mice suffering from ph induced by su5416 administration with chronic hypoxia, in lungs of rats with mct - induced ph and in lungs of juvenile sheep with shunt - induced ph . In both human paecs and pasmcs, multiple ph inducers, including hypoxia, il-1, and il-6, increased mir-130/301 expression and hypoxia was found to upregulate mir-130/301 via a dependence on hif-2 and pou5f1/oct4 . It was further validated that mir-130/301 modulated apelin - mir-424/503-fgf2 signaling in paecs and mir-130/301-ppar- axis controlled proliferation of pasmcs by increasing stat3 expression and activity and repressing mir-204 expression . In hypoxia - induced ph mouse model, induction of mir-130/301 was promoted while mir-130/301 inhibition prevented ph pathogenesis . Ph is broadly considered to be a vascular disease as vasoconstriction in the pulmonary artery is a prime cause for the development of ph . The hyperproliferation of pulmonary vascular cells (mainly vsmc) and subsequent neointima formation in the small pas are hallmark in ph . On the other hand, pulmonary arterial endothelial cells (paecs) further contribute a critical role in vascular homeostatic balance in the pulmonary vascular bed by orchestrating the vessel tone, leucocytes trafficking, and so forth [74, 75]. Proliferation of fibroblasts is reported in hypoxia - induced ph that plays a role in adventitial thickening [77, 78]. Under pathological situation like ph (pah), the endothelium becomes dysfunctional which allows the penetration of infiltrating molecules, increased leucocytes adhesion, getting resistant to apoptosis that result in severe remodeling in the pulmonary vessels . These infiltrating molecules invade the barrier of neighboring cells like smooth muscle cells and activating the resident of adventitious fibroblasts . The remodeling process is basically an interaction between these cell - types present in the pulmonary arterial layers that resulting in a marked histological change in the pulmonary vasculature . An emerging body of evidences suggests that the mirnas play a pivotal role in the vascular cell integrity and plasticity during the progression of ph . Here, although identification of mirnas in vascular diseases like ph is relatively new, but the earliest association connecting vsmc remodeling with mirnas was reported in 2007 . Described the mirna signature in the vascular wall of balloon - injured carotid artery rat model . They showed that mir-21 was upregulated and the cellular effects were targeted by pten and bcl2 for neointimal lesion formation . The initial studies of mir-21 in vascular remodeling were followed by series of milestone works that include mir-143/145 cluster [24, 31, 7983]. Two of the key mirnas involved in regulation of the phenotype of smcs are mir-143 and mir-145 . Mir-145 is transcribed bicistronically along with mir-143 from human chromosome 5 . With relevance to pah, downregulation of mir-145 downregulation of target genes klf4 and klf5 by activation of mir-143/mir-145 upregulates smc - specific genes such as sma, calponin, and sm22-, triggering differentiation and lowering the proliferation rates in vascular smcs [31, 82]. Another mirna, mir-221/222 that triggers pdgf signaling in vsmc is thought to contribute in neointimal proliferation [35, 84, 85]. It is demonstrated that inhibition of mir-221 prevented pdgf induced proliferation, while forced expression of mir-221 increased proliferation and reduced the expression of vsmc marker [35, 84]. In addition to the above mirnas, other mirnas are reported to regulate smc gene expression in hypoxia - induced ph . It is demonstrated that mir-210 that acts as a hypoxia - inducible mirna both in vitro and in vivo, inhibits pasmc apoptosis in hypoxia by specifically repressing e2f3 . A cell - based high throughput screening of a human mirna library with the nfat luciferase reporter system identified mir-124 as a new candidate in ph . It showed decreased nfat reporter activity and, decreased dephosphorylation and the nuclear translocation of nfat . An elegant study by courboulin et al . Demonstrated that mir-204 downregulation correlates with ph severity and responsible for proliferative and antiapoptotic phenotypes of ph - pasmcs targeting stat3 and nfatc . Interestingly, a recent study showed that a serine / threonine kinase mst1, a modulator of cell death, appears to be a target of mir-138 . The authors suggest that mir-138 may be a negative regulator of pasmc apoptosis in hypoxic mediated pah . A panel of mirnas which are shown to modulate smc fate and modulation are listed in table 1 [2636]. Therapeutic target which defines the treatment of a disease by means of a well - defined biological molecule . In this tiny rna molecule, have become an important gene modulator of various biochemical, physiological, and cellular functions . As we observed that either deficiency or abundance of a specific mirna or cluster of mirnas contributed a pathological state of many cardiovascular diseases including ph, it is reasonable to accept those mirnas as therapeutic target for diagnosis and therapeutic intervention . This is because we can utilize ability of a single mirna to control the expression of multiple (hundreds) proteins suggest that changes of a single mirna may influence several signaling pathways associated with pathological disease state . While each target is regulated subtly, the additive effect of coordinated regulation of a large suite of transcripts is believed to result in strong phenotypic outputs . Several animal models in diverse disease pathology showed usefulness of mirnas as target molecules for therapeutic benefit; this review will focus on vascular remodeling associated with ph . At therapeutic strategic stand - point, the aberrant mirna expression can be modulated or restored to normal by two main approaches: an anti - mirna (anti - mir) and mirna mimic . The former can be applicable to those mirnas whose expressions were increased in disease pathology and, therefore, silencing or inhibiting will be beneficial . To modulate mirna expression, the current strategy utilized chemically modified, cholesterol - conjugated single - stranded rna analogues complementary to the mature mirnas for antagomir or mimics, respectively . An elegant work demonstrated by liu et al . In a balloon - injury model represents a successful knocking down of mir-221/222 that suppressed vsmc proliferation and neointimal lesion formation . Recently, brock et al . Have shown that treatment with antagomir-20a restores the levels of bmpr2 in pulmonary arteries and prevents vascular remodeling in hypoxia - induced pah . For downregulated mirnas, a mimic approach is generally undertaken which basically rescued the underexpressed mirna in the tissue under pathological situation . This strategy is sometimes referred to as mirna replacement as it reintroduced the similar depleted mirna those were downregulated during diseases progression . Therefore, by introducing the mirna mimic, the cells can restore the function as the mirna mimic is expected to target the same set of mrnas that is also regulated by the endogenous mirna . In a very comprehensive study by courboulin et al ., they demonstrated that reestablishing of mir-204 level by delivery of synthetic mir-204 into the lungs of experimentally - induced pah animals significantly reduced the disease pathology . Although the mirna - based intervention is attractive and seems to be feasible, however, several challenges are noted . First, the critical mirnas responsible for ph must be confirmed definitively in well - accepted animal models along with explanted human pah samples, biopsies, and so forth, second, the cellular, molecular and physiological function of these mirnas should be performed in diseases progression and prevention; third, the delivery route should be precisely targeted or restricted to lung vascular cells, for example, paec, pasmc, or fibroblasts . Finally, the dose of antagomir or mimic delivery to the vascular bed should be carefully monitored further to avoid off - target effects . Therefore, our future direction should be focus of vascular cell - specific delivery of mirna mimic or inhibitors . Additionally, a regulated release of mirnas by conjugating nanoparticle will be considered for long - term use . Finally, mirna - mediated therapeutics can be achieved by inhalation which may reduce the potential off - target issues to the other organs . Biomarker generally refers to measurable substance in biological state . At clinical stand - point, it is extremely important as it predicts the medical outcome of a disease and its progression . Over the past few years, it has been demonstrated that mirnas are found in the blood, plasma, urine, platelets, and saliva in a surprisingly stable form [9197]. The stability of mirna in the extracellular environment offers a great opportunity to consider as biomarker at clinical settings . Using mirna as a biomarker offers many advantages like early prediction of diseases, differential expression during disease progression / pathologies, high degree of specificity, and sensitivity and importantly having longer half - life in the system . Accumulating evidence suggested that the stability of mirna in extracellular environment illustrated by packed with lipid vesicles, wrapped in protein or lipoprotein complexes [98100]. In the context of cardiac diseases that include myocardial infarction, hypertrophy and heart failure; several mirnas are identified and predicted to be considered as biomarker [101104]. The mirnas are circulating freely in the mammalian blood and can be predicted as biomarker for early diagnosis of cardiovascular diseases in humans [102, 105108]. Several evidences indicate that mirnas are secreted as micro vesicles or exosome and apoptotic bodies that may be responsible for release the mirnas into the circulation [109112] and are extremely stable in the blood or serum . Rhodes and colleagues measured plasma levels of mirnas in eight patients newly diagnosed with pah and eight healthy controls by use of microarray analysis . Fifty - eight mirnas showed differences in plasma concentration between the two groups and mir-150 was largest downregulated in pah . Reduced expression of mir-150 predicted 2-year survival and correlated with disease severity . The mir-150 level was also found to be significantly reduced in circulating microvesicles and lymphocytes from patients with pah . Courboulin et al . Reported that downregulation of mir-204 in buffy - coat cells correlated with pah severity and might serve as a circulatory biomarker of pah . On the contrary, increasing higher plasma levels of mir-130/301 family members were observed in patients with increasing hemodynamic severity of ph . Our previous study detected the pattern of mirnas in buffy - coat samples from mild - to - severe human ph subjects compared with the control subjects by mirna array . Our study revealed that moderate to severe ph in human subjects are associated with significant downregulation of plasma levels of circulatory mir-1, mir-26a, and mir-29c . The kinetics of the changes differed from moderate to severe ph human subjects as we observed further downregulation of the above mirnas in severe ph . Mir-21, mir-23b, mir-130a, mir-491, and mir-1246 were moderately upregulated in moderate ph subjects and were more pronounced in severe ph . Another set of mirnas that include mir-133b, mir-204, and mir-208b were significantly upregulated in all moderate ph subjects and showed further increment in severe ph subjects . Our study provided the evidence for the first time that circulating mirnas in the setting of ph may be used as early detection parameters for ph . Nevertheless, the data presented were based on limited study population and the results need to be confirmed in a larger cohort study . Furthermore, the dysregulation mirna in ph remains to be tested whether these circulatory mirnas have any influence in the target genes . Circulatory mirnas are considered to be novel promising biomarkers for detection of ph . However, at current stage, using circulatory mirna as diagnostic tool for detecting ph is still in its infancy . The key advantage of circulating mirnas is a noninvasive testing procedure and relies on the stability of mirnas in the circulation and their storage condition . We give credit to the concept of quick determination of secretory molecules (mirnas) in the blood; but the importance of mirna must be reevaluated at the clinical setting or model at multiple laboratories . Another limitation is the small number of cohort study which appears to be heterogeneous clinical sample - type . These points may consider as preliminary - type of observation and need further confirmatory studies for considering mirnas as biomarkers for clinical use . Understanding of mirnas role and function in the pulmonary circulation will offer and contribute a great potential to the pathogenesis of ph or pah . The mirna signature can be used as a diagnostic tool for the development of therapeutic strategy . The characterization of these mirnas under various experimental settings (in vitro and in vivo) should be performed along with the human subjects . The outcome will validate both physiological and pathological roles of these mirnas during the development and progression of ph prior to consider them for therapeutic intervention or future clinical trials . Furthermore, use of disease specific mouse models, conditional transgenic, and knockouts are necessary to elucidate the functional role in vivo and verify its therapeutic potential, both of which remain largely unexplored . More novel approaches such as combination of argonaute protein immunoprecipitation, deep sequencing, or proteomic profiling approaches are required to identify its potential bona fide targets responsible for its functional impact.
Pasteurella multocida was first described by revolee in 1877 and further characterized by pasteur in 1880 . Pasteurella species usually have low virulence in humans, but serious manifestations sometimes occur and can lead to bacteremia . We present a patient who developed septic shock as a result of p. multocida bacteremia, presumptively from an intra - abdominal infection . A 52-year - old african american female was brought to the emergency department for generalized abdominal discomfort, altered mental status, diarrhea, fevers and chills for 4 - 5 days . Her past medical history was significant for alcohol abuse and alcohol - related liver cirrhosis . In the emergency department, vital signs were as follows: blood pressure of 74/42 mmhg, heart rate of 112 beats / min, respiratory rate of 20 breaths / min and temperature of 98.6 f. remarkable findings on physical examination included jaundice as well as abdominal distention and tenderness to palpation, especially in the right upper quadrant . As per family, she did not have recent travels or exposure to sick people . They denied having domestic animals at home or any contact to the patient with pets . Admission diagnosis was systemic inflammatory response syndrome with sepsis and septic shock with multi - organic dysfunction syndrome, presumptively secondary to community - acquired pneumonia, possible biliary tract infection (ascending cholangitis), probable spontaneous bacterial peritonitis (sbp) and alcoholic hepatitis . Empiric intravenous antimicrobial therapy, composed by piperacillin / tazobactam (2.25 g every 8 h) and azithromycin (500 mg every 24 h), was initiated . Laboratory results on admission were: a complete blood cell count of 21,000/mm(90% neutrophils), a platelet count of 51,000/mm, a sodium level of 127 meq / l, a potassium level of 5.3 meq / l, a bicarbonate level of 15 meq / l and a creatinine level of 6.3 mg / dl . Alkaline phosphatase level was 290 iu / l, gamma - glutamyl transpeptidase level was 290 u / l and lactate dehydrogenase level was 482 total bilirubin level was 14.5 mg / dl, with a direct bilirubin level of 10.1 mg / dl . Arterial blood gases showed a ph of 7.15, a pco2 of 23, and a po2 of 132 while the patient was receiving mechanical ventilation with fio2 of 60% . After 24 h of admission, patient remained in critical condition and requiring maximum doses of vasopressors, despite of what it seems to be the appropriate empiric antimicrobial therapy . Blood culture system bact - alert 3d was used for the recovery of the organism . No minimal inhibitory concentrations for these antimicrobials were reported . In spite of all medical efforts, p. multocida is a small gram - negative non - spore forming facultative anaerobe organism that is a natural inhabitant of the normal gastrointestinal flora and the upper respiratory tract of wild and domestic animals, especially cats and dogs . It is easily overgrowing by other flora in the sputum and might be regularly missed, as it resembles haemophilus influenzae, francisella tularensis and yersinia pestis . The five species that cause the majority of pasteurellosis are: multocida, septica, canis, stomatis, and dagmatis . Human p. multocida infections have been reported to occur with and without animal exposure, the former mostly associated with the domestic cat and dog bites or scratches . Most of the infections involve the skin and soft - tissues and they can be complicated by abscess formation, osteomyelitis or septic arthritis . The respiratory tract is the second most common site of infection with a wide spectrum of diseases that includes rhinosinusitis, tracheobronchitis, epiglottitis, pneumonia, empyema and lung abscesses . The majority of p. multocida pneumonia cases occur in elderly patients with underlying chronic pulmonary disease . Invasive forms of p. multocida infection usually occur in immunocompromised patients, such as those with advanced age, chronic renal failure, solid tumors, hematological malignancies, diabetes mellitus or liver cirrhosis . P. multocida causes a wide variety of disease, including abdominal and pelvic infections, endocarditis, meningitis and endophtalmitis . Von graevenitz et al . Reviewed 21 proven cases of p. multocida bacteremia . A remarkable number of those cases involved patients with liver diseases, including liver cirrhosis of any etiology, hepatitis and infiltrating tumors . Impaired function of the reticuloendothelial system and the presence of portosystemic shunts in patients with liver disease seem to play a major role in the development of bacteremia . This case seems peculiar in that no direct contact with a domestic animal could be documented . Our patient had a history of alcohol - induced liver cirrhosis, which predisposed her to the development of bacteremia . However, retrospectively, it is difficult to establish the primary source of infection in this case . The findings of the abdominal examination may suggest that the primary source was the intra - abdominal cavity . Unfortunately, the medical team was not able to performed paracentesis to obtain ascitic fluid for cytology and cultures due to patient s medical condition . No abdominal imaging was available . On the other hand, the chest radiography findings of infiltrate and pleural effusion may point the respiratory tract as the primary source of infection . We believed that, given the history of symptoms on admission and the presence of underlying liver disease, the intra - abdominal cavity was the most likely primary source of infection in this patient . Nasopharyngeal colonization with p. multocida with transient bacteremia and seeding of the peritoneal cavity in immunosupressed cat owners could play an important role in the development of sbp . Ten of these patients were exposed to animals, nine had positive blood cultures and four patients died . However, penicillin - resistant strains in human infections have been described . In these cases, 2 and 3 generation cephalosporins, macrolides, fluoroquinolones, tetracyclines, and trimethoprim - sulfamethoxazole outcome is associated with severity of the infection, the extent of the underlying disease and the early initiation of appropriate therapy . Although cases of bacteremic p. multocida infections has been infrequently reported in the literature, clinicians should considered this organism as an important and potentially lethal pathogen in humans, where it can cause life - threatening infections . P. multocida should be included in the microbiologic differential diagnosis in patients with underlying chronic liver diseases who presents with possible intra - abdominal infection, even without history of exposure to domestic animals.
Coronary artery bypass grafting (cabg) is considered to be the gold standard in patients with multivessel disease and remains the treatment of choice for patients with severe coronary artery disease, including three - vessel or left main coronary artery disease . The use of cabg, as compared with both percutaneous coronary intervention (pci) and medical therapy, is superior with regard to long - term symptom relief, major adverse cardiac or cerebrovascular events and survival benefit [14]. However, because of the use of cardiopulmonary bypass and median sternotomy, cabg is associated with significant surgical trauma leading to a long rehabilitation period and delayed postoperative improvement of quality of life . An alternative hybrid approach to multivessel coronary artery disease combines surgical left internal thoracic artery (lita) to left anterior descending coronary artery (lad) bypass grafting and percutaneous coronary intervention of the remaining lesions [3, 68]. Ideally, the lita to lad bypass graft is performed in a minimally invasive fashion through minimally invasive direct coronary artery bypass grafting (midcab). This hybrid approach takes advantage of the survival benefit of the lita to lad bypass, while minimizing invasiveness and lowering morbidity by avoiding median sternotomy, rib retraction, aortic manipulation, and cardiopulmonary bypass [3, 8, 1014]. The purpose of the hybrid approach is to achieve complete coronary revascularization with outcomes equivalent to conventional coronary artery bypass grafting, while ensuring faster patient recovery, shorter hospital stays, and earlier return to work due to lower morbidity and mortality rates . Angelini and colleagues reported the first hybrid coronary revascularization (hcr) procedure in 1996, and several patient series using hybrid coronary revascularization have been published since then . These series support the above - mentioned presumptions and indicate that the hybrid approach is a feasible option for the treatment of selected patients with multivessel coronary artery disease involving the left main . Moreover, the introduction of drug - eluting stents (dess) with lower rates of restenosis and better clinical outcomes may make hybrid coronary revascularization a more sustainable and feasible option than previously reported [9, 15]. Nevertheless, this hybrid approach has not been widely adopted because practical and logistical concerns have been expressed . These concerns implicate the need for close cooperation between surgeon and interventional cardiologist, logistical issues regarding sequencing and timing of the procedures, and the use of aggressive anticoagulant therapy for percutaneous coronary intervention that may worsen bleeding in the surgical patient [7, 14, 16]. This review aims to clarify the place of hybrid coronary revascularization in the current therapeutic armamentarium against multivessel coronary artery disease . Second, the results of previous patient series using the hybrid approach are summarized and interpreted . The medline / pubmed database was searched in january 2012 using the medical subject headings (mesh) for coronary artery disease and angioplasty, balloon, coronary combined with the following free - text keywords: multivessel coronary artery disease, minimally invasive coronary artery bypass, percutaneous coronary intervention, and hybrid coronary revascularization . One hundred seventy - seven articles matching these search criteria were found, and the search for additional papers was continued by analysing the reference lists of relevant articles . Randomized controlled trials, nonrandomized prospective and retrospective (comparative) studies were selected for inclusion . Letters, editorials, (multi)case reports, reviews, and small studies (n <15) were also excluded . Studies examining the hcr procedure for multivessel coronary disease were included, while studies investigating the hcr procedure for left main coronary stenosis were excluded . Authors and medical centres with two or more published studies were carefully evaluated and were represented by their most recent publication to avoid multiple reporting of the same patients . A total of eighteen included studies remained eligible for analysis after applying these in- and exclusion criteria (figure 1). The primary outcome measures were in - hospital major adverse cardiac and cerebrovascular events (macces), packed red blood cells (prbcs) transfusion rate, lita patency, hospital length of stay (los), 30-day mortality, survival, and target vessel revascularization (tvr). Secondary outcome measures were intensive care unit (icu) los and intubation time, as only a limited number of studies reported these outcome measures . In addition, the period of time between pci and lita to lad bypass grafting and the cost effectiveness of hcr were examined . The long - term lita patency was not included as an outcome measure, since only a limited number of studies report this outcome measure in a clear and concise manner . In - hospital major adverse cardiac and cerebrovascular events were defined as postoperative stroke, myocardial infarction (mi), or death during hospital stay . Only the fitzgibbon patency class a (widely patent) was considered as a patent lita to lad bypass graft, while the fitzgibbon patency class b (flow limiting) and c (occluded) were defined as a nonpatent lita to lad bypass graft . Hospital los was defined as the number of days spent in hospital from operation to discharge . If the need for repeated revascularization involved a coronary artery initially treated with either bypass grafting or pci, this repeated revascularization was considered to be target vessel revascularization . One observer extracted all available outcome measures of each article and a second observer checked and supervised the first observer thoroughly . When an article did not disclose one or more of these outcome measures or reported medians and ranges as central tendency instead of means and standard deviations, the study was excluded from the analysis of that particular variable . The results were analysed using ibm spss statistics 19 software (ibm inc ., armonk, continuous data were presented as mean and standard deviation (sd), while categorical data were expressed as numbers and percentages . Nine hundred seventy patients undergoing hcr procedures were included for analysis (tables 1 and 2) [6, 7, 1114, 1728]. The most important findings are reported below . The classical indication for an hcr procedure is multivessel coronary artery disease involving lad lesion judged suitable for minimally invasive lita to lad bypass grafting but unsuitable for pci (type c), and (a) non - lad lesion(s) (most of the time right coronary artery (rca) and/or circumflex coronary artery (cx) lesions) amenable to pci (type a or b) [7, 11, 12, 14, 17, 18, 20, 22, 23, 2628]. High - risk patients especially with severe concomitant diseases (e.g., diabetes mellitus, malignancies, significant carotid disease, severely impaired lv function, and neurological diseases), who are more prone to develop complications after cardiopulmonary bypass and sternotomy, might benefit from the circumvention of cpb and sternotomy [11, 18, 20, 2224]. Exclusion criteria for hcr consist of contraindications to minimally invasive lita to lad bypass grafting or pci . Lita to lad bypass grafting in a minimally invasive fashion requires single - lung ventilation and chest cavity insufflation . Therefore, hcr procedures are contraindicated in patients with a compromised pulmonary function (i.e., forced expiratory volume in one second less than 50% of predicted) and a small intrathoracic cavity space [14, 27, 28]. Moreover, patients with a nongraftable or a buried intramyocardial lad, history of left subclavian artery and/or lita stenosis, morbid obesity (bmi> 40 kg / m), and previous left chest surgery are not well suited for minimally invasive lita tot lad bypass grafting [14, 20, 22, 27, 28]. Conditions rendering pci unsuitable include peripheral vascular disease precluding vascular access, coronary vessel diameter smaller than 1.5 mm, tortuous calcified coronary vessels, fresh thrombotic lesions, chronic totally occluded coronary arteries, extensive coronary involvement, chronic renal insufficiency (serum creatinine 200 mol / l), and allergy to radiographic contrast [7, 14, 18, 20, 22, 27, 28]. Finally, haemodynamic instability, need for a concomitant operation (e.g., valve repair or replacement), and decompensated congestive heart failure are regarded as exclusion criteria [7, 17, 20, 22, 27, 28]. Three hcr strategies can be distinguished: (i) performing pci first, followed by lita to lad bypass grafting or (ii) vice versa; (iii) combining lita to lad bypass grafting and pci in the same setting in a hybrid operative suite . In the included studies, staged hcr procedures (i and ii) were applied much more frequently than simultaneous procedures (iii). In a staged procedure, in which pci and lita to lad bypass grafting are carried out at separate locations and/or different days, both interventions can be performed under ideal circumstances (in a modern catheterization laboratory and modern operating room, resp .) [11, 18, 29]. However, patients have to undergo 2 procedures, while they remain incompletely revascularized and at risk for cardiovascular events for an extended period of time [14, 29]. When pci is performed first, a staged procedure takes place with an unprotected anterior wall, which could pose serious health risks in case the lad lesion is considered the culprit lesion . In addition, lita to lad bypass grafting is performed after aggressive platelet inhibition for prevention of acute (stent) thrombosis, which might lead to unnecessary postponement of following operation or may cause a higher than expected rate of bleeding [12, 13, 21, 29]. Moreover, stent thrombosis is risked after reversal of surgical anticoagulation and is related to the inflammatory reaction after cardiac surgery . Furthermore, the opportunity for quality control of the lita to lad bypass graft and anastomosis by a coronary angiogram is lost and, therefore, this strategy requires a reangiography [12, 13]. These repeat control angiograms increase overall healthcare costs unnecessarily and decrease cost effectiveness . First, revascularization of non - lad vessels provides an optimized overall coronary flow reserve, thereby minimizing the potential risk of ischemia and myocardial infarction during the lad occlusion for lita to lad bypass grafting [6, 12]. Second, it is possible for the interventional cardiologist to fall back on conventional cabg in case of a suboptimal pci result or major pci complications . However, failure of pci leading to emergency conventional cabg has become extremely rare with decreasing incidence since the introduction of coronary artery stenting [12, 20, 2932]. Furthermore, this strategy allows hcr in patients with the immediate need for pci in a non - lad target and no immediate possibility for emergency bypass surgery [11, 24]. Critical stenosis in the right coronary artery (rca) or the left circumflex coronary artery (lcx) or difficult pci targets are considered as clear indications for a pci first approach because these patients can undergo conventional cabg in case of pci failure . When the lita to lad bypass graft is performed first, antiplatelet therapy is routinely started after surgery to prevent antiplatelet - related bleeding complications during surgery and is present at time of pci [6, 13, 27]. These antiplatelet agents can be administered long term, which is mandatory for preventing stent thrombosis . Moreover, the quality control of the lita to lad bypass graft and anastomosis can be performed simultaneously without a further angiogram [6, 12, 13, 18, 20, 23, 25, 26, 29]. In addition, pci is performed in a protective environment with a revascularized anteroseptal wall, which probably reduces the procedural risks and gives the interventional cardiologist the ability to approach lesions that would be quite challenging without a revascularized lad [13, 20, 25, 26, 29]. However, patients undergoing this strategy could require a second, much higher - risk, surgical intervention due to complications of the pci [13, 23, 25]. Finally, the cardiac surgeon has to be aware of possible intraoperative ischemia during this hcr strategy because the collateral, non - lad vessels are unprotected . Nevertheless, combining the two procedures in one stage under general anaesthesia in a specific hybrid - operating room, which combines the potential of catheterization and cardiac surgery, has advantages compared with staged hcr procedures [7, 14, 25, 28]. This simultaneous approach represents a single procedure that achieves complete revascularization, while minimizing patient discomfort and reducing the need for anaesthetics [12, 14, 18, 20, 28]. This approach eliminates logistic concerns about timing and sequence of two separate procedures and maximizes patient satisfaction [7, 14, 25, 28]. Moreover, the quality of the lita to lad bypass graft and anastomosis can be confirmed immediately by an intraoperative angiogram, which enables direct revision of the lita to lad bypass graft [18, 25]. Complications and difficulties during pci or midcab can be dealt with immediately in the same setting by conversion to conventional, open - chest cabg . Perioperative haemorrhage can become a problem because full antiplatelet therapy and incomplete heparin reversal are necessary instantly after midcab to prevent a transient rebound increase in thrombin formation associated with stent thrombosis and ensure an optimal intraoperative des placement [7, 14, 18]. Besides, off - pump surgery may give rise to hypercoagulability and increased platelet activation during the early postoperative period, which is associated with an increased risk of stent thrombosis . Therefore, a modified antiplatelet protocol and careful patient selection seem appropriate, especially in one - stop hcr, in order to minimize the risk of stent thrombosis without increasing perioperative bleeding risk . A tried and tested protocol of dual antiplatelet therapy (dapt) includes continuous use of aspirin (100 mg / day) until the operation day and intraoperative administration of a loading dose clopidogrel (300 mg) via a nasogastric tube after confirming lita graft patency, followed by a maintenance dose of 75 mg / day for 12 months . However, caution is required when using dapt, since reversal agents for clopidogrel and aspirin are not available . Moreover, newer more potent antiplatelet agents, like prasugrel and ticagrelor, should be reserved exclusively for selected cases (high risk of stent thrombosis) and managed with even more care, since the clinical experience with these newer antiplatelet agents is limited in cardiac surgery and the bleeding risk may be increased . Furthermore, intraoperative collaboration and communication among cardiac surgeons, interventional cardiologists, and anaesthesiologists should be outstanding and ongoing to optimize continuity of care [11, 14]. Currently, this simultaneous procedure is used in only a few centres, and some authors state that this might be caused by the need to possess catheterization laboratories outfitted to accommodate cardiac surgery or hybrid operating rooms equipped with a mobile coronary angiography c - arm or permanent fluoroscopic equipment [7, 13]. The latter is reflected in the small number of patients undergoing a simultaneous procedure in our sample of included studies [7, 13, 14, 18, 24, 25, 28]. Expansion of other percutaneous and hybrid procedures like hybrid af ablation may help to make these hybrid, multipurpose operating rooms more common in the future . However, staged hcr procedures could offer a more realistic alternative for many institutions without a so - called hybrid operating room, and this is supported by the fact that staged hcr procedures are applied much more frequently than simultaneous procedures in the included studies [6, 1113, 1724, 26, 27]. Tables 3 and 4 present the period of time between both procedures in a staged hcr strategy, and this period of time varied notably from 0 to 180 days . Therefore, some patients remained incompletely revascularized and were in theory at risk for cardiovascular events for a considerable length of time, while complete myocardial revascularization should be the main goal of treatment in patients with multivessel coronary artery disease . Moreover, delhaye et al . Found that pci with clopidogrel preloading can be performed within 48 hours of lita to lad bypass grafting without increasing the bleeding risk . In addition, zenati et al . Performed pci zero to four days after lita to lad bypass grafting without increasing the prbc transfusion requirements, while lowering the hospital length of stay (2.7 1.0 days). The mean hospital length of stay was 5.5 1.8 days (range: from 2.7 to 8.2 days), and hospital length of stay seems not to be influenced by the hcr strategy used (table 2). As shown in table 1, the surgical techniques for lita to lad bypass grafting have evolved continuously since the introduction of the hcr procedure in 1996 by angelini et al . Most of the initial patient series performed the lita to lad bypass graft in a minimally invasive fashion carrying out a mini - thoracotomy on the anterolateral chest wall in imitation of angelini et al . [3, 7, 12, 1719]. In this so - called minimally invasive direct coronary artery bypass (midcab) approach the anastomosis to the lad is performed with 8 - 0 or 4 - 0 prolene sutures on the beating heart (without cpb) with the help of mechanical stabilizers . In more recent patient series, the lita was identified and harvested thoracoscopically or robotically, which decreased rib retraction, chest wall deformity, and trauma [11, 14, 21, 22, 27]. This approach significantly minimizes the typical thoracotomy - type incisional pain and wound complications of conventional midcab, while optimizing graft length and retaining the reliability of manually sewn lita to lad anastomosis [21, 22]. Some teams prefer to place the lita bypass graft to the lad through a ministernotomy (inversed l - shaped or reversed j - shaped), which makes it possible to switch to full sternotomy in case complications may occur during the original operation [20, 23, 28]. Nevertheless, this surgical technique increases surgical trauma and, therefore, may raise morbidity and mortality . In addition, some centres even decided to perform the lita to lad bypass graft through a full sternotomy on the beating heart (off - pump cabg), thereby further increasing invasiveness [6, 25, 26]. If the lita bypass graft is placed on the lad through a sternotomy on the arrested heart (on - pump cabg), circumvention of cpb is lost too [6, 25, 26]. Thus, both on - pump and off - pump cabg can be seen as suboptimal procedures to carry out the lita to lad bypass graft . This might explain the higher macce rates found by zhao et al . And delhaye et al . And the high 30-day mortality discovered by zhao et al . And gilard et al ., who decided to place the lita to lad bypass graft on the arrested heart through full sternotomy in the majority of the patients [6, 25, 26]. Lastly, some authors prefer to perform the lita to lad bypass graft in a totally endoscopic, port - only fashion using totally endoscopic coronary artery bypass grafting (tecab) [13, 24]. This most challenging form of lita to lad bypass grafting using robotic telemanipulation techniques was initially performed on the arrested heart with the use of peripherally introduced cardiopulmonary bypass with intraaortic balloon occlusion and cardioplegic arrest [13, 24]. A major disadvantage of this approach is the use of the heart lung machine, which increases the risk of stroke, bleeding, and an inflammatory response to surgery . The latter can be solved by using beating heart tecab (bh - tecab), in which cpb and its considerable drawbacks are avoided . Total endoscopic completion of the lita to lad bypass graft on the beating heart requires an additional port subxiphoidally to place a specially designed endoscopic stabilizer, which stabilizes the heart to optimize the quality of the anastomosis . This so - called beating heart totally endoscopic coronary artery bypass (bh tecab) procedure might be the least invasive approach for coronary bypass surgery without making concessions to graft patency [24, 3538]. However, the tecab procedure is an extremely challenging and a potentially expensive procedure with an extensive learning curve, which may raise concerns about widespread adoption and application . The postoperative lita patency seemed to be independent of the surgical technique of lita to lad bypass grafting, since lita patency has shown to be approximately equal for all surgical techniques (table 2). The postoperative lita patency varied between 93.0% and 100.0% (mean: 98.8% 2.3%). The mean in - hospital macce rate was 1.3% 1.9% (range: from 0,0% to 5.6%) with relatively high macce rates shown by katz et al . Strikingly, three of these authors (katz et al ., zhao et al ., and delhaye et al .) Performed lita to lad placement on the arrested heart [13, 25, 26]. The percentage of patients requiring prbc transfusion varied considerably between 0.0% and 35.4% (mean: 13.6% 11.7%). The surgical technique or hcr strategy (staged versus simultaneous) used did not appear to affect the percentage of patients requiring prbc transfusion . Overall, the 30-day mortality rate was 0.4% 0.8% (range: from 0.0% to 2.6%). Interestingly, higher than expected 30-day mortality rates were found in studies (gilard et al . And zhao et al .) Using on - pump cabg to perform the lita to lad bypass graft in the majority of patients [6, 25]. Finally, the mean overall survival rate in hybrid treated patients was 98.1% 4.7% (range: from 84.8% to 100.0%). Besides the technical improvements of lita to lad bypass grafting, innovations occurred in the field of pci . This development was supported by the increased rate of des implantation in later patient series compared to earlier patient series, which used percutaneous transluminal coronary angioplasty (ptca) only or ptca in combination with bms implantation . Application of drug - eluting stents should lower the restenosis rate, but their potentially beneficial effect on the target vessel revascularization (tvr) is not supported by data from the included studies (table 2). However, the (early and late) patency rate of new generation drug - eluting stents in non - lad lesions, provided that proper dapt is applied, may already be superior to that of saphenous vein grafts . Hard evidence is however lacking, since a head - to - head comparison of (early and late) patency rates between des (in non - lad lesions) and saphenous vein grafts is not available . Finally, the introduction of bioresorbable scaffold (brs) technology may improve sustainability, safety and feasibility of future hcr interventions . The application of brs technology can make long - term dapt redundant reducing bleeding complications without increasing the risk of stent thrombosis and may allow future reinterventions or reoperations on the same vessel if necessary due to its bioresorbable features . A relatively small number of studies in our sample (table 5) compared the hcr procedure using minimally invasive lita to lad bypass grafting with conventional cabg or off - pump coronary artery bypass (opcab) [7, 12, 27, 28]. All four of these studies selected matched controls who had undergone elective cabg or opcab with lita and saphenous vein grafts through median sternotomy during the same period using propensity score matching [7, 12, 27, 28]. Hu et al . Found that patients in the hybrid group had a statistically significant shorter hospital length of stay, icu length of stay, and intubation time compared with opcab, while de cannire et al . Reported that hospital and icu length of stay was statistically shorter in hybrid treated patients compared with patients treated with cabg [7, 12, 28]. Showed that intubation time, icu, and hospital length of stay were similar between the hybrid and opcab group . Moreover, these studies revealed that prbc transfusion requirements were reduced by the hybrid approach [12, 27, 28]. Lastly, the in - hospital macce rates were considerably lower in the hybrid groups compared with both the cabg and the opcab groups . Currently, only a few studies have explicitly explored the costs associated with hybrid coronary revascularization . De cannire and colleagues were the first to quantify costs associated with hcr and to compare these costs with costs involved in conventional double cabg . Costs were calculated using six major expenditure categories: costs of hospital admission (including intensive care unit and postsurgical cardiac ward cost as well as costs associated with delayed repeat procedures), pharmaceutical costs, surgical costs, pci - related costs, costs of blood products, and other miscellaneous fees (including physiotherapy and consultants). The extra cost associated with pci (including stents) in the hybrid group in comparison with the cabg group (2.517 288 versus 0 0), which uses autologous grafts to treat non - lad lesions, counterbalanced the cost savings on all other expenditure categories, which resulted in a nonsignificant cost difference at 2 years between both groups (10.622 1329 versus 9699 2500; not statistically significant). It is worth mentioning that the reduced icu and hospital length of stay due to faster recovery were largely responsible for the cost reduction in the hybrid group compared with the cabg group (3.033 499 versus 4.156 1.413). Showed that shorter intubation times, shorter icu and hospital length of stay, and less prbc transfusions resulted in a significant reduction in costs for hybrid treated patients in the postoperative period . Conversely, intraoperative costs were statistically significant higher in patients undergoing hcr compared with opcab, largely because of longer operative times and the use of coated stents (des) rather than autologous grafts ($14.691 2.967 versus $9.819 2.229; p <0.001). In conclusion, the difference in intraoperative costs was almost completely outweighed by the lower postoperative costs in the hybrid group . This resulted in slightly, but not significantly, higher overall costs in the hybrid group . The nonhealthcare costs after hcr will presumably be lower than after cabg or opcab because both kon et al . And de cannire et al . Showed that return to work was significantly faster in the hybrid group, leading to a marked reduction in absenteeism from work in hybrid treated patients [7, 12]. This difference in nonhealthcare costs should be able to compensate the opposite difference in healthcare costs, resulting in a negligible difference in total societal costs . Moreover, the emergency of simultaneous hybrid procedures in especially designed multipurpose operating rooms combining the potential of catheter - based procedures and cardiac surgery will reduce the unnecessary costs incurred by staged hcr procedures [12, 25]. Lastly, more experience with minimally invasive cardiac surgery will shorten operative times, which might help reduce total healthcare costs . This review is the largest and most comprehensive report to date comparing the clinical outcomes of patients who underwent either hybrid coronary revascularization or conventional on- or off - pump cabg for multivessel coronary artery disease . Three principal findings were revealed as follows: (1) hybrid treated patients showed a significantly faster recovery with lower prbc transfusion requirements and less in - hospital major adverse cardiac and cerebrovascular events than patients treated by on- or off - pump cabg; (2) staged procedures were associated with considerable period of times between both procedures, leaving patients incompletely revascularized and in theory at risk for cardiovascular events for a considerable length of time; and (3) the invasiveness of surgical lita to lad bypass grafting appeared to influence the clinical outcome, with higher macce and 30-day mortality rates in patients treated by more invasive surgical techniques using cpb and/or median sternotomy . As with any review, this report shares the limitations of the original studies . First, the initial reports especially included a relatively small number of patients, which may have resulted in biased results due to outliers . Furthermore, almost all studies were performed retrospectively with inherent patient selection bias, since the decision to perform the hcr procedure was taken on an individual and highly selective basis according to cardiac surgeon and interventional cardiologist discretion . Likewise, the inclusion and exclusion criteria used to select high - risk patients for the hcr procedure differed notably between the included studies, yielding a very heterogenic population . In addition, the used surgical techniques to perform the lita to lad bypass graft varied considerably, with learning curve issues and different levels of expertise and equipment . All these factors potentially contribute to heterogeneity, which may reduce the certainty of the evidence presented in this review . Moreover, the mean length of followup was generally short, almost never exceeding two years, which made it difficult to assess long - term clinical outcomes of hybrid treated patients . Therefore, this review relies mainly on in - hospital and short - term outcomes to assess the safety and feasibility of the hcr procedure . Another limitation was the lack of long - term systematic and routine angiographic followup of graft and stent patency in the majority of studies included in the present review, which precluded any conclusions about the graft and stent longevity of the hcr procedure . Furthermore, the comparative studies lacked randomization and nonblinded assessment of outcome, which might have led to selection bias and might have influenced outcome measures by preconceived notions about the superiority of the hcr procedure . Finally, postoperative pain, which might be higher in patients treated with conventional midcab, was not included as outcome measure in the present review, because only a limited number of studies assessed this outcome measure . Notwithstanding these weaknesses and limitations, this review selected the best evidence currently available to give a broad and comprehensive overview of the preliminary results of the hcr procedure . Larger, multicenter, prospective, randomized trials with long - term clinical and angiographic followup and cost analysis comparing hcr with both conventional on - pump and off - pump cabg or multivessel pci will be necessary to further evaluate whether this hybrid approach is associated with similar promising long - term results . In the meantime, the first prospective, randomized pilot trial to compare hcr with conventional cabg in patients with multivessel coronary artery disease has been started . These data are also needed to identify patient populations that would benefit most from this hybrid approach . Furthermore, more insights in the different surgical techniques for lita to lad bypass grafting and their clinical outcomes are necessary . Therefore, the different surgical techniques for lita to lad bypass grafting in the hcr procedure should be integrated in these large, multicenter hcr studies in order to determine the best way of lita to lad bypass grafting in hcr . Moreover, different hcr strategies (staged versus simultaneous) should be compared to decide which strategy will serve which patients best . Finally, the advantages and disadvantages of a hybrid operative suite need to be explored further . The large variability in hcr techniques makes it difficult to draw firm conclusions from the currently available evidence, but hcr appears to be a promising and cost - effective alternative for cabg in the treatment of multivessel coronary artery disease in a selected patient population . The hcr procedure was associated with short hospital stays (including icu stay and intubation time), low macce and 30-day mortality rates, low prbc transfusion requirements and tvr, high postoperative lita patency rates, and high survival rates . These promising early outcomes warrant further research with larger sample size, multicenter rcts to determine the definite place of hcr in the current therapeutic armamentarium against coronary artery disease . Until then, this review justifies the continued use of the hybrid approach, but careful patient selection and close cooperation between cardiac surgeons and interventional cardiologists will determine the clinical outcomes to a significant extent.
Globally four million deaths occur every year in the first month of life . Almost all (99%) neonatal deaths arise in low - income and middle - income countries [1, 2]. In india alone, around one million babies die each year before they complete their first month of life, contributing to one - fourth of the global burden [1, 3]. The neonatal mortality rate in india was 32 per 1000 live births in the year 2010, a high rate that has not declined much in the last decade [4, 5]. India's neonatal mortality rate dropped significantly, that is, by 25%, from 69 per 1,000 live births in 1980 to 53 per 1,000 live births in 1990 followed by a 15%, decline from 51 to 44 per 1,000 live births between 1991 and 2000 . In recent years the nmr has dropped by 15% that is, from 40 per 1000 live births in 2001 to 34 per 1000 live births in 2009 . Urban - rural differences in neonatal mortality exist with the mortality rates higher by 50% in rural (42.5/1000 live births) compared to urban (28.5/1000 live births) areas, as per the national family health survey (nfhs-3). The common causes of neonatal deaths in india include infections, birth asphyxia, and prematurity which contribute to 32.8%, 22.3%, and 16.8% of the total neonatal deaths, respectively [7, 8]. India is one of the ten countries, along with china, democratic republic of congo, pakistan, nigeria, bangladesh, ethiopia, indonesia, afghanistan, and tanzania, that account for more than 65% of all intrapartum related neonatal deaths . Despite the recognition of neonatal survival as a key to child survival, poor progress in neonatal survival in india poses concern regarding attainment of the fourth millennium development goal (mdg) target, that is, to reduce under-5 child mortality by two - thirds by 2015 . Despite having a comparatively higher neonatal mortality rate, rural india is tackling with the problem of ill equipped public health facilities . The numbers of existing peripheral health facilities fall short of what has been recommended by the government of india . The healthcare in rural areas has been developed as a three - tier structure based on predetermined population norms . The subcenter is the most peripheral institution and the first contact point between the primary healthcare system and the community . Primary health centers (phcs) comprise the second tier in rural healthcare structure envisaged to provide integrated curative and preventive healthcare to the rural population . Community health centers (chcs) form the uppermost tier and their function is mainly to provide specialized obstetric and child care . A situational analysis done by the neonatal health research initiative (nhri), indiaclen from 20072009, in 24 centers of the country, suggested that less than 20% of the chcs / phcs provide essential newborn care services . Also, the availability of a neonatal resuscitation area was relatively low in chcs (46%) and phcs (14%). As per the district level health survey (dlhs-3), newborn care equipment was available in only 27.9% phcs . Also, while around 76% of the community health centres had newborn care management facilities, just 35.1% had facilities for managing low birth weight babies . These findings underscore the critical condition of the public health facilities that are meant to cater to the health problems of the newborns in rural india . Rural public health facilities across the country are having a difficult time attracting, retaining, and ensuring regular presence of highly trained medical personnel especially the gynecologists and pediatricians that are epochal in ensuring and promoting newborn health . Statistics for 2010 suggest a shortfall of 10.3% for doctors at primary health centers (phcs). The condition of 4535 community health centers supposed to provide specialized medical care is even more appalling . As compared to requirements for an existing infrastructure, there was a shortfall of 62.6% of specialists at the chcs, 55.2% of obstetricians and gynecologists and 69.5% of pediatricians . According to the dlhs facility survey (2003), healthcare facilities with newborn care staff and a medical officer trained in newborn care were 59%, 45.0%, and 34% at district hospital, first referral units (frus) and chcs, respectively . As on march 2010, the overall shortfall in the posts of health worker (female)/auxiliary nurse midwife (anm) was 8.8% of the total requirement . Similarly, in case of health worker (male), there was a shortfall of 64.1% of the requirement . In case of health assistant (female), the shortfall was 31.9% and that of health assistant (male) was 44% . The lack of qualified child care specialists results in a majority of rural households receiving care for their ill babies from private providers, many of whom are less than fully qualified . The government of india has launched various initiatives envisaging a high priority action with regard to neonatal health . Under national rural health mission (nrhm), accredited social health activists (ashas) are being deployed and assigned the responsibility to create awareness in the community regarding maternal and child health issues . They are further expected to mobilize the community and help them in accessing healthcare services . A safe motherhood intervention named janani suraksha yojana (jsy) has been implemented under the nrhm to increase the institutional delivery rates and provide skilled care at birth for the newborn . Under the reproductive and child health program (rch - ii), the quality and reach of antenatal care is planned to be expanded and home - based newborn care using integrated management of neonatal and childhood illness (imnci) protocols is envisaged . The imnci strategy encompasses a range of interventions to prevent and manage the commonest major childhood and neonatal illnesses that cause death, that is, acute respiratory infections, diarrhoea, measles, malaria, and malnutrition . The imnci package is planned to be implemented at the level of household and subcentres (through anms) and primary health centres (through medical officers, nurses, and lady health visitors). Till october 2011 facility - based care of neonates (f - imnci) is proposed through strengthening of infrastructure, provision of extra nurses, and skills upgradation of physicians and nurses . The government, with the help of unicef, has started setting up special care newborn units (scnus) for managing sick newborns [17, 19, 20]. These units have been established at district hospitals and are expected to have a minimum of 12 to 16 beds manned by 3 physicians, 10 nurses, and 4 support staff . Further, newborn stabilization units (nbsus) are being set up in first referral units (frus) and community health centers (chcs) and they aim to provide care to sick newborns referred from peripheral health facilities . As of october 2011, 1134 nbsus have been set up . A total of 8582 new born care corners (nbccs), which are special corners within the labour room where resuscitation, infection control, and early breast feeding can be commenced, have been set up, as of 2011 . Janani shishu suraksha karyakram (jssk) was launched on 1 june, 2011 with the aim to promote institutional delivery, eliminate out - of - pocket expenses, and facilitate prompt referral through free transport . A program on basic newborn care and resuscitation, named navjaat shishu suraksha karyakram (nssk), is being launched to address important interventions at the time of birth that is, prevention of hypothermia and infections, early initiation of breastfeeding, and basic newborn resuscitation . The objective is to have one person trained in basic newborn care and resuscitation at every delivery . This training is being imparted to medical officers, staff nurses, and anms at chc / frus and 24 7 phcs where deliveries are taking place . Provision of comprehensive emergency obstetric and new born care (cemonc) services and basic emergency obstetric and newborn care (bemonc) at various levels has also been given due importance . Neonatal health is seemingly one of the priority issues in the agenda of the government which gets reflected in the various programs devised and implemented . The worrisome issue is the fact that improving health systems through facility upgradation and ensuring availability of trained manpower and logistics comprise essential prerequisites for the success of these programs / initiatives . The reluctance of trained manpower, especially doctors, to serve in rural areas has become a major impediment in the government's ability to provide quality health services . The main obstacles to improving newborn survival are that many babies are born at home without being attended by skilled personnel, faulty home - based newborn care practices are widespread, lack of awareness among care givers limits care - seeking for neonatal illness and even if that is taken care of, lack of trained health workforce adds to the problem . This deficiency in skilled manpower undermines the initiatives by the government to improve neonatal health . Another set of dilemma exists in bringing the neonates and the health system closer to each other . There are broadly two ways of doing so, either bring the health system closer to the neonate or bring the neonate closer to the health system . Both of these are feasible and hold the promise to yield positive results but the real challenge lies in their reproduction and sustainment at the national level . In order to ensure the availability of trained medical personnel in rural areas, we first need to understand the reasons behind the observed shortage . Recruiting trained doctors by all means is one of the essential components towards providing quality maternal and neonatal care services . A recent report documents that out of the 264 paediatricians (including both postgraduates and diploma holders) that are produced annually in india, only around half of them (i.e., 158) are available for public sector service, a large chunk either emigrate or get attracted towards private sector jobs in urban setups . The predominant reasons for preference to work in urban areas include adequate infrastructural facilities, high salary, and a decent standard of living [24, 25]. Further, in the recent years, there has been substantial emigration of trained doctors to developed countries, much of it coming from lower and middle income countries [2628]. Among the developing countries, india is the biggest exporter of trained physicians with india - trained physicians accounting for about 10.9% of british physicians and 4.9% of american physicians . A report of the national commission on macroeconomics and health documented that around 10% of the obstetrician(s) and paediatrician(s) that the country produces eventually emigrate . Although the recipient nations and the physicians that emigrate benefit from this migration, the home country loses its important health potentialities . There is no clear - cut solution to the problem of lack of doctors in rural setup . Interventions in education and financial incentives along with professional support probably have the potential to ease out the problem, as had been seen in rural australia where the gprip continuing medical education grants and locum grants designed to assist rural general practitioners to maintain and increase their skills in areas relevant to rural practice helped in their retention in rural areas . The provision of better financial incentives oriented specifically to doctors working in the rural areas might be crucial to attract and retain more doctors in these areas . In canada, the distribution of doctors was positively influenced by raising fees in rural and underserved areas and reducing fees in areas, but in the philippines, rural incentives had an unintended negative impact due to the fact that local governments were unable to hire healthcare professionals at the high salary levels specified [3133]. Thus, the experience with paying direct financial incentives, such as rural allowances, has been variable and usually depends on the affordability of resources but this should not undermine the potential it might offer to increase the influx of doctors in rural areas . Other key initiatives could include establishing rural doctor networks, mentorship programmes, and giving rural practitioners preference in admissions in specialty programs . Exposure to rural areas as part of the training of medical graduates, so they can understand the working conditions and acquire rural clinical skills, is essential and has the potential to yield positive results . This has been documented in thailand where a majority of graduates continued in rural practice after completing a compulsory rural residency . To prevent brain drain, international scholar exchange programmes could be thought of as an option besides improving healthcare infrastructure and creating an enabling work environment . Certain care practices can be deleterious to the health of the baby like applying ghee / oil on cord, early bathing, avoidance of colostrum feeding (considering it as harmful for the baby) and not practicing exclusive breast feeding . Realizing the presence of such traditions in the community and formulating intensive information, education, and communication (iec) campaigns to address these is required . There is a need to develop programs where there is a collective involvement of the communities in order to identify problems and their solutions . Several such programs have been implemented in other parts of the globe and have yielded positive results . Bolivia's warmi program where the key highlight was participatory planning at the community level, with an emphasis on women's participation to identify obstetric and perinatal health problems and their potential solutions . As a result of the intervention, neonatal mortality decreased from 120 per 1000 live births to 40 per 1000 live births . In rural nepal, a cluster randomized trial suggested that women's groups facilitated by a local female community worker could reduce neonatal mortality rates by about 30% . In eastern india, the ekjut trial (20052008) evaluated the impact of community mobilization on birth outcomes in three districts of jharkhand and orissa . These studies offer evidence to encourage community involvement and leverage the community resources to bring about improvements in neonatal health . Further, there is a need to make an effort to integrate community mobilization with health system strengthening . In a review of the evidence - based, cost - effective interventions for reduction of neonatal mortality, darmstadt et al . Documented that a combination of outreach and home - based newborn care at 90% coverage could avert 1837% neonatal deaths . Home - based newborn care could be explicated as a family as well as community oriented services that involve community mobilization and the empowerment of care givers to demand quality services for their sick newborns . Hbnc mainly aims at reducing the neonatal deaths by preventing or treating morbidities such as infections, asphyxia or hypothermia which largely form the preventable causes of mortality . Moreover, they are the underlying causes of nearly 55% of the neonatal deaths in india and addressing them could drastically cut down on the mortality rate . Have documented that community - based pneumonia case management can lead to a 27% decrease in all - cause neonatal mortality, which indeed is a very high achievement . The most convincing example was set out by bang et al . In rural gadchiroli where female village health workers were selected from the local population and they were also trained to manage neonatal sepsis by providing parenteral antibiotic treatment to sick neonates . In the three years of intervention, there was a 71% reduction in perinatal mortality and a 62% reduction in neonatal mortality compared with the control area . In another example from sirur, a periurban area near pune, maharashtra, india, forty female village health workers were trained to serve a population of 47,000 . The village worker identified high - risk cases that required treatment by herself and the nurse, under the supervision of the field medical officer . She also made 3 home visits: on day 1 or soon after delivery and on days 8 and 29 . As a result of the intervention, a decline in the neonatal mortality rate of 25% from 51.9 to 38.8 per 1,000 live births was recorded . Other successful examples include trials of home - based care in north india, bangladesh, pakistan, and nepal [4649]. In addition to creating awareness among community members and care givers in the family through information, education, and communication (iec) activities, a prerequisite for implementation of home - based care is the development of simple and easily comprehensible standard management guidelines . Further, it would be a challenging task to upscale the home care newborn package to the most vulnerable states such as uttar pradesh, bihar, jharkhand, madhya pradesh, orissa, and rajasthan with a high neonatal mortality rate . In rural india, most of the births (53%) occur at home largely unattended by skilled personnel . The lack of a trained personnel predisposes the newborn to a variety of birth related complications mainly birth asphyxia, birth injuries, and infections . Moreover, most of the neonatal deaths occur in the first week of life with a majority of them dying on the first day of birth, thus reflecting the poor intrapartum care that the mother receives [1, 50, 51]. With the shortage of trained personnel, nonavailability of adequate healthcare facilities, poor connectivity to a health facility, and lack of transport facilities, providing care at home through training of midwives / traditional birth attendants (tbas) would probably be a better option . They can be a vital link between women and the health system, giving advice, encouraging women to go to the clinic to deliver, and accompanying mothers to provide moral support . One such successful case study is from indonesia [52, 53]. In 2003, nearly half of all newborn deaths in the cirebon district of indonesia were due to birth asphyxia . In order to address this situation in cirebon, program for appropriate technology in health (path) supported by saving newborn lives / save the children began for training community midwives (bidan di desas). These midwives were taught a series of initial steps for assessing and managing a newborn's condition, including the use of a locally produced tube and mask resuscitation device that could be used in home birth settings . One year after the training, it was found that newborn deaths due to birth asphyxia dropped by 47 percent in the district, at a cost of only $42 per asphyxia death averted . In zambia, midwife training programs significantly decreased the seven - day neonatal death rate in community health clinics . The midwives were given training in essential newborn care (enc) and in neonatal resuscitation . After training, the all - cause, 7-day neonatal mortality rate decreased from 11.5 deaths per 1000 live births to 6.8 deaths per 1000 live births . The perinatal mortality rate decreased from 18.3 deaths per 1000 births to 12.9 deaths per 1000 births . Similar examples providing evidence for up scaling of trained midwives in order to lower down the neonatal mortality can be drawn from sri lanka, thailand, malaysia, and pakistan [5559]. Infant mortality rates (reflecting neonatal mortality as well) are one of the most important indicators of the differentials in health and socioeconomic condition in a community . A substantial progress in lowering down the high burden of neonatal mortality is unlikely unless ways can be found to enhance the economic wellbeing of the lower socioeconomic groups . A pertinent example is that of kerala, a southern state of india, where the state's achievement of stabilizing population growth, attaining high levels of literacy, and life expectancy have led to a significant decline in the infant mortality rates [60, 61]. In a study done in rural haryana to document the determinants of neonatal deaths, it was found that the occurrence of deaths was a multifactorial process with involvement of factors at community level, family level (socioeconomic), and biological level and that the socioeconomic determinants explained a large proportion of neonatal deaths . Further, rahman et al . In their study in qatar found that low - cost, community - based interventions, on the background of socioeconomic development, had a stronger impact on neonatal and perinatal survival as compared to high - cost institutional interventions . Similar findings documenting the importance of socioeconomic development in reducing the burden of neonatal deaths have been reported from studies done in chile, malaysia, malawi, and arab countries [6468]. Neonatal care in rural india is largely provided by a large number of unqualified healthcare providers [6971]. They are the early providers of neonatal care and often attract a large number of ill newborns because of their easier access and comparatively cheaper treatment that they offer . There is a wide range of quality of services provided by these doctors and it would be useful to standardize their services by providing support in the form of training and technical support . Though it does not qualify as a paragon solution, but this concept would probably score well, given the limited resources the country has . In alignment with what had been advocated by yadav et al . Let best not be the enemy of the good, it would be beneficial to engage these local healthcare providers and equip them with necessary skills to provide acceptable standards of neonatal care until constraints on the supply of qualified and motivated healthcare providers into the system can be alleviated . They could further be involved in promoting key newborn essential care practices as they are popular and acceptable in the community . In china, rural healthcare is provided by village doctors who are trained in preventive and curative medicine of both traditional chinese and allopathic schools . The skills acquired are regularly upgraded by apprenticeship and in - service courses [73, 74]. Another example is from usa where the shortage of physicians in the 1960s paved the way for the emergence of physician assistants who were licensed to practice medicine under the supervision of physicians . They made a considerable contribution by working in rural areas which otherwise would not have received any care at all [75, 76]. Successful examples of providing quality healthcare through involvement of local healthcare practitioners can also be seen in ghana, mexico, and bangladesh [77, 78]. Providing a degree of bachelor of rural medicine and surgery (brms) after three - and - a - half years of training, as opposed to five and - a - half years of training for a usual medical graduate, has recently been discussed as one of the possible options to cater to the need of quality healthcare in rural india . The government of india, in consultation with the medical council of india (mci), is planning to introduce this course in medical schools proposed to be established at district hospitals . The concept of a new degree course of a comparatively shorter duration is to encourage students from rural areas to take up medicine and subsequently provide services in their respective rural areas . The potential impact of selecting medical students of rural origin has been documented by rabinowitz et al . In a longitudinal study that evaluated the impact of the physician shortage area program (psap) in the usa . On multivariate analysis, rural origin was the single variable most strongly associated with rural practice . Studies done in south africa, southern australia, and canada have also substantiated that the doctors with rural background have more tendency to work in rural areas [8183]. Students enrolled in the proposed brms course will be taught preclinical as well as clinical subjects with more focus on paediatrics and obstetrics / gynaecology . Further, it is envisaged to impart special training in care of the newborn and vaccination . Chhattisgarh, a state in central india, has come up with the concept of awarding a degree named rural medical assistants (rmas). This three - year course was a response to a major crisis in human resources for health that the state faced . Three colleges were inaugurated in 2001 and were situated in rural / tribal districts, but with access to a large government hospital (usually the district hospital) to make it possible for clinical teaching and internship . There has been overwhelming positive response to recruitment of rmas to the most rural and tribal phc postings, where previously no trained physician existed . It will certainly improve health care delivery in rural, remote, and tribal areas by providing qualified practitioners but the training of these rural healthcare practitioners will be a major area of concern . It is doubtful as to how overworked, poorly staffed, ill - equipped district hospitals, which cater to thousands of patients, can become quality training grounds for healthcare practitioners . Ensuring that these graduates would practice only in rural areas and not shift to urban setup further, there is a need to document the difference in the quality of care provided by the new cadre of healthcare professional and mbbs graduates . The use of mobile phones to improve the quality of care and enhance efficiency of service delivery within healthcare systems is known as mobile health, or m - health . Who defines m - health as the provision of health services and information via mobile technologies such as mobile phones and personal digital assistants (pdas). M - health tools have shown promise in providing greater access to healthcare to populations in developing countries, as well as creating cost efficiencies and improving the capacity of health systems to provide quality healthcare . Studies done in kenya, sierra leone and zanzibar unleash the immense potentialities this innovative concept holds in addressing a wide variety of healthcare challenges [8688]. As earlier discussed, in rural setup, access to healthcare professionals and medical facilities is limited . Although much work has not been done in context of m - health in india, yet efforts are required to be made to implement this in the indian context based on the initial success in other developing countries . The feasibility does not seem to be highly questionable considering the recent increase in the number of mobile phone users in rural areas . According to the press release by the telecom regularity authority of india (trai), the number of telephone subscribers in india increased to 943.49 million at the end of february 2012 . The share of urban subscribers had been 65.59% whereas share of rural subscribers had reached 34.41% . Subscription in rural areas had increased from 320.29 million in january 2012 to 324.68 million in february 2012, an increase of 4.39 million in just one month . Now with the recent initiative by the government to provide a subsidy of 20 percent on bills of less than rs 300 a month to mobile users in rural india, the increase in the number of mobile users could further be expected . Mobile telephone short - message service (sms) can be used for delivering health behaviour change interventions . This service has wide population reach, can be individually tailored, and allows instant delivery, suggesting potential as a delivery channel for health behavior interventions . Researchers in korea, croatia, new zealand, and united kingdom have used sms to deliver information pertaining to diabetes and asthma self - management, smoking cessation, and increasing physical activity and this has proved to be beneficial byincreasing awareness and bringing about the desired behaviour change [9194]. Mobile technology can also be involved in better training of community health workers in using cellular short messages (sms) to encode and transmit basic health information such as vital signs and health symptoms to a monitoring computer . Algorithms on the monitoring computer could recognize emergent conditions and send system - generated notification informing the community health worker of the appropriate management of the baby related to the inputted vital signs and symptoms . Given the volume of neonatal care services that are being sought through the private sector in rural areas, one cannot hope to reduce neonatal mortality through public sector interventions alone [6971]. Because the private sector does not operate within the restrictive confines of a government bureaucracy the advantage with such a partnership could be the wider coverage and increased service utilization . Also, using strengths and skills of each partner enhances efficiency . Successful examples of improving maternal and neonatal health through public private partnerships have been documented in the literature [9598]. One such example is of pampers / unicef collaboration to eliminate neonatal tetanus . Through this collaboration, over 300 million tetanus vaccines, protecting over 100 million mothers and their babies in 25 of the world's poorest countries, have been provided . In even the poorest countries, the private sector is a major provider of goods, services, and information for maternal and child health . There could be different ways to involve the private sector, depending on the resources available and the need of services . One of the strategies could be to use microfinance to allow private sector doctors and other healthcare providers to provide quality practices . One such innovative scheme in india is the chiranjeevi scheme in gujarat [98, 100]. It is an innovative health financing scheme covered through public - private partnership for emergency obstetric care and emergency transport services, for women belonging to below poverty line (bpl) category . Have published an evaluation of a pay for performance (p4p) scheme implemented in rwanda . P4p scheme involves for - profit organizations who are provided incentives based on improvements in utilization and quality of care . Statistically significant improvements were observed in the maternal and neonatal health (mnh) indicators of institutional delivery and quality of prenatal care which increased by 21%, and 7.6%, respectively over baseline in the p4p districts [101, 102]. Introducing public - private partnerships to improve the quality of maternal and child health services is not new in india . Key examples include vande mataram scheme in west bengal which involves private sector for provision of safe motherhood and family planning services, janani express yojna in madhya pradesh for transportation in case of obstetric emergencies, and use of vouchers in uttar pradesh where reproductive and child health services for below poverty line (bpl) women and children are provided through private practitioners [103, 104]. Under national rural health mission (nrhm), several initiatives based on public - private partnerships have been or / are planned to be implemented . The key issues include sustainability of such initiatives and ensuring that quality services are being provided . To conclude, the neonatal mortality rate in india is still high and skewed towards rural areas . Much of the problem lies in the nonavailability of trained manpower and this in turn influences the quality of care the neonates receive . Bringing qualified health professionals to rural, remote, and underserved areas is a challenging task which needs to be addressed urgently to avert neonatal deaths . Other options such as training of local rural healthcare providers and traditional midwives, promoting home - based newborn care, creating community awareness and community mobilization along with strengthening public - private partnerships should be explored further, as evidence generated from previous studies and large scale projects support these strategies as a way to improve neonatal health . More research should be directed towards upcoming innovations such as m - health in order to exploit the potential they offer in terms of enhancing the quality of care . While the focus should be on devising strategies to recruit and retain trained manpower in rural areas, alternative strategies such as community mobilization, upscaling of home - based newborn care, imparting training and subsequent involvement of local rural healthcare providers and midwives should be attempted as well . More research is required to reveal the potential that innovations such as m - health, telemedicine, and public - private partnership hold in context to improving the quality of care in rural india.
The hypersensitivity syndrome, described as drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe, acute, drug reaction, defined by the presence of fever, cutaneous eruption, and systemic findings including enlarged lymph nodes, hepatitis, or hematologic abnormalities with eosinophilia and atypical lymphocytes1,2). The syndrome can involve several sites, leading to findings such as pneumonitis, renal failure, myocarditis, thyroiditis, or neurologic symptoms, but the liver is the most commonly affected internal organ3). This reaction can be life threatening, with a mortality rate of approximately 10%, most commonly secondary to liver failure1). Here we report the first pediatric case of liver transplantation for the treatment of acute liver failure caused by vancomycin - induced dress syndrome in korea . A 14-year - old girl was referred to asan medical center children's hospital on the basis of test results that indicated abnormal liver function . She had been injured by a traffic accident 10 years earlier, and had undergone plastic surgery twice to heal wounds on her forehead . Thereafter she had suffered from methicillin - resistant staphylococcus aureus (mrsa) osteomyelitis, which had been treated with intravenous vancomycin over five weeks . Following this treatment, she developed fever and whole - body pruritic erythema, and abnormal liver function tests 6 days prior to her transfer . On admission, she presented with fever, nausea, vomiting, and abdominal discomfort . A generalized erythematous rash with variously sized, discrete lesions was noted on the face, trunk, and extremities . The patient reported an itching and heating sensation, which was aggravated after vancomycin injection . Laboratory tests showed a total eosinophil count of 3,150/mm (normal, <500 mm), c - reactive protein level of 15.1 mg / dl (normal, <0.6 mg / dl), creatinine level of 2.5 mg / dl (normal, 0.7 to 1.4 mg / dl), aspartate aminotransferase (ast) level of 320 iu / l (normal, <5 to 40 iu / l), alanine aminotransferase (alt) level of 263 iu / l (normal, <5 to 40 iu / l), alkaline phosphatase level of 440 iu / l (normal, 40 to 120 iu / l), gamma glutamyl transpeptidase level of 321 iu / l (normal, 8 to 35 iu / l), lactate dehydrogenase level of 2,437 iu / l (normal, 120 to 250 iu / l), total bilirubin level of 3.3 mg / dl (normal, 0.2 to 1.2 mg / dl), direct bilirubin level of 1.8 mg / dl (normal, <0.5 mg / dl), prothrombin time (pt) international normalized ratio (inr) of 1.68 (normal, 0.8 to 1.3), and activated partial thromboplastin time of 34.5 (normal, 25 to 35). Serologic tests for hepatitis a, b, and c, as well as cytomegalovirus, epstein - barr virus, and autoimmune hepatitis were all negative . The vancomycin level was 26.9 mg / l (normal, 20 to 40 mg / l). Treatment with vancomycin was stopped, and replaced with ciprofloxacin for the treatment of osteomyelitis . Intravenous delivery of a high dose of methylprednisolone was initiated upon an initial suspicion of dress syndrome . However, her liver function worsened progressively on hospital day 7, with an ast level of 1,285 iu / l, alt level of 1,077 iu / l, total bilirubin level of 15.5 mg / dl, direct bilirubin level of 8.0 mg / dl, and pt inr of 4.8, all suggesting acute liver failure . Given the presence of aggravated hepatic encephalopathy, azotemia, and that the patient was refractory to medical treatments, she received a living - donor liver transplantation from her aunt on hospital day 9 . The detailed care of preliver and postliver transplantation and care of acute liver failure in our program was described elsewhere4,5). After liver transplantation, the skin rash disappeared, with normalization of the eosinophil count and scores in liver and renal function tests . No severe clinical and surgical complications developed postoperatively . Serial liver biopsies showed no evidence of acute rejection . Over the course of a 25-month follow - up period, there has not been any definite recurrence of dress syndrome, with the exception of persistently elevated levels of liver enzymes and intermittent eosinophilia . The patient is currently suffering from iatrogenic cushing's syndrome owing to the high dose of steroids administered to control her elevated levels of liver enzymes . We here report a pediatric case of acute liver failure resulting from dress syndrome, which was treated by liver transplantation . Most reported cases of dress syndrome show that a cure can be achieved by the immediate withdrawal of the causative agent and the administration of methylprednisolone6). Liver involvement in dress syndrome is common and may range from a transitory increase in liver enzymes to liver necrosis with acute liver failure . To our knowledge, acute liver failure caused by dress syndrome has been reported at least twice in other european countries8,9). In one of these cases9), the patient died waiting for a liver transplant, whereas in the other case8), fatal recurrence occurred after liver transplantation despite potent immunosuppression and cessation of the precipitating factors . The pathogenesis of dress syndrome is not fully understood, and may be multifactorial10). Although it is most commonly associated with antiepileptic drugs, dress syndrome has also been reported after exposure to a range of medications, including sulfasalazine, doxycycline, allopurinol, linezolid, and vancomycin1,8,10). The differential diagnosis includes stevens - johnson syndrome (sjs), life - threatening, cutaneous adverse reaction . The two syndromes overlap clinically, but have different characteristics, treatments and prognoses11). Therefore, a high index of suspicion and rapid diagnosis may be necessary to save the patient's life . The only undisputed way to treat the use of systemic corticosteroids is common, although evidence regarding their effectiveness is scant12). In our case, we replaced vancomycin with ciprofloxacin approximately 6 days after symptoms of dress syndrome were evident, and immediately began corticosteroid treatments after the switch to ciprofloxacin therapy . Although ciprofloxacin is also known to cause dress13), we finally chose that agent to control infection because other several antibiotics aggravated skin rash as well . These measures were ineffective in preventing liver failure . To our knowledge, this is the first pediatric case report to describe vancomycin - induced acute liver failure occurring as a component of the dress syndrome in korea . We highlight the need for awareness of the association between drugs, dress syndrome and liver failure . Given the absence of reports describing the outcomes of liver transplantation in patients with dress syndrome, particular attention should be devoted to identification of its possible recurrence after liver transplantation.
Despite the implementation of clinical guidelines and improvements in the critical care of patients with traumatic brain injury (tbi), the prognosis of patients with severe tbi has remained poor during the last decade.16) the mortality rate of severe tbi is known to be about 32% to 52%, and about 6% to 11% of patients become vegetative or are severely disabled.313) it has been reported that dysfunction of at least one non - neurologic organ system develop in about 89% of patients with severe tbi . Among them, the incidence of renal failure in tbi has been reported as 0.45% to 1.9%.1217) acute renal failure (arf) may occur by ischemic renal injury, sepsis or administration of nephrotoxic agents in tbi patients, and can be fatal to these patients . Therefore, renal replacement therapy is required for tbi patients with arf to decrease post - traumatic mortality . Among treatment modalities for arf, continuous renal replacement therapy (crrt) has shown to have a much lesser effect on intracranial pressure (icp) than intermittent hemodialysis (ihd).6) additionally, some authors have reported that crrt have an effect on lowering icp.89) regarding hemodynamics, crrt can be a better treatment modality for arf compared to ihd in severe tbi patients . However, little has been reported about the treatment results of crrt on tbi patients with arf . We aimed to investigate the outcomes of crrt on tbi patients in whom arf developed after trauma, and to study relevant factors which influence patients survival . Between april 2011 and june 2015, 1,190 tbi patients were treated at our institution . Among them, 29 patients underwent renal replacement therapy for arf . We retrospectively reviewed the laboratory, clinical, and radiological data of those 29 patients . Initial glasgow coma scale (gcs) score was checked when the patient was arrived to the emergency room . Injury severity score (iss) has six categories to head and neck, face, chest, abdomen or pelvic contents, extremities or pelvic girdle, and external . Iss scores ranges from 1 to 75 (iss scores of 5 for each category). The mean age at admission was 60.2 years (range, 16 - 84). The mean initial gcs score was 9.2 (range, 3 - 15), and the mean iss was 24 (range, 9 - 59). Brain computed tomography (ct) scan showed epidural hematoma (edh), subdural hematoma (sdh), intracerebral hemorrhage (ich), and traumatic subarachnoid hemorrhage (t - sah) in 5, 25, 7, and 17 patients, respectively . Eighteen out of 29 patients underwent surgical intervention for treatment of tbi (table 1). All 29 patients had normal kidney function on admission . However, 3 patients had a past history of chronic kidney disease . When the patients urine output decreased, we checked the serum creatinine level and calculated the glomerular filtration rate . Crrt was started when the serum creatinine level increased to more than twice of the basal level, and the urine output decreased to less than 30 ml per hour despite hydration and diuretics . The mean serum creatinine level was 1.03 mg / dl (range, 0.6 - 5.0) at admission and 2.79 mg / dl (range, 1.6 - 7.9) before starting crrt . Crrt was maintained until serum creatinine level was normalized (0.7 - 1.2 mg / dl). The median starting date of crrt was 3 days (range, 0 - 34) after tbi . If the patients' vital sign was stable, more than 100 ml / hour (> 2400 ml / day) of dialysis volume was removed . We investigated survival time of 29 tbi patients who underwent crrt and analyzed the relationship between survival time and the laboratory, clinical, and radiological factors . Survival factors were categorized as follows; age (65 vs. <65), 24-hours urine output (500 ml vs. <500 ml) before receiving crrt, iss (<25 vs. 25), the presence of sdh, initial gcs score (<9 vs. 9), pupil size at admission (isocoria vs. anisocoria), cranial surgery (yes vs. no), and intracranial hemorrhage (edh, sdh, ich, t - sah absent vs. present). Statistical analysis was performed using spss version 12.0 (spss inc ., kaplan - meier analysis was used and survival outcome was compared using the log - rank sum test for categorical variables . Between april 2011 and june 2015, 1,190 tbi patients were treated at our institution . Among them, 29 patients underwent renal replacement therapy for arf . We retrospectively reviewed the laboratory, clinical, and radiological data of those 29 patients . Initial glasgow coma scale (gcs) score was checked when the patient was arrived to the emergency room . Injury severity score (iss) has six categories to head and neck, face, chest, abdomen or pelvic contents, extremities or pelvic girdle, and external . Iss scores ranges from 1 to 75 (iss scores of 5 for each category). The mean age at admission was 60.2 years (range, 16 - 84). The mean initial gcs score was 9.2 (range, 3 - 15), and the mean iss was 24 (range, 9 - 59). Brain computed tomography (ct) scan showed epidural hematoma (edh), subdural hematoma (sdh), intracerebral hemorrhage (ich), and traumatic subarachnoid hemorrhage (t - sah) in 5, 25, 7, and 17 patients, respectively . Eighteen out of 29 patients underwent surgical intervention for treatment of tbi (table 1). All 29 patients had normal kidney function on admission . However, 3 patients had a past history of chronic kidney disease . When the patients urine output decreased, we checked the serum creatinine level and calculated the glomerular filtration rate . Crrt was started when the serum creatinine level increased to more than twice of the basal level, and the urine output decreased to less than 30 ml per hour despite hydration and diuretics . The mean serum creatinine level was 1.03 mg / dl (range, 0.6 - 5.0) at admission and 2.79 mg / dl (range, 1.6 - 7.9) before starting crrt . Crrt was maintained until serum creatinine level was normalized (0.7 - 1.2 mg / dl). The median starting date of crrt was 3 days (range, 0 - 34) after tbi . If the patients' vital sign was stable, more than 100 ml / hour (> 2400 ml / day) of dialysis volume was removed . We investigated survival time of 29 tbi patients who underwent crrt and analyzed the relationship between survival time and the laboratory, clinical, and radiological factors . Survival factors were categorized as follows; age (65 vs. <65), 24-hours urine output (500 ml vs. <500 ml) before receiving crrt, iss (<25 vs. 25), the presence of sdh, initial gcs score (<9 vs. 9), pupil size at admission (isocoria vs. anisocoria), cranial surgery (yes vs. no), and intracranial hemorrhage (edh, sdh, ich, t - sah absent vs. present). Statistical analysis was performed using spss version 12.0 (spss inc ., kaplan - meier analysis was used and survival outcome was compared using the log - rank sum test for categorical variables . The actuarial median survival time of the 29 patients was 163 days after trauma (range, 3 - 317). Among the 29, 22 patients died with a median survival time of 8 days (range, 3 - 55) (figure 1a). The causes of death were tbi - related in 8, sepsis due to pneumonia or acute respiratory distress syndrome (ards) in 4, and multi - organ failure in 10 . Among the various factors related to survival, 24-hr urine output before receiving crrt (500 ml vs. <500 ml) (figure 1b), the presence of sdh (figure 1c), and iss (<25 vs. 25) were significant after univariate analysis (figure 1d, table 2). Median survival time was 10 days (range, 3 - 136) when the patients urine output was less than 500 ml per day before receiving crrt, whereas more than half of the patients survived when the urine output was 500 ml per day or more (p=0.015). Of the nine patients who had a 24-hours urine output of more than 500 ml per day before receiving crrt, 4 (44.4%) patients died (tbi - related brain herniation in 1, multi - organ failure in 3), with a median survival time of 146 days (range, 4 - 317). Of the 20 patients who had a 24-hours urine output of less than 500 ml per day before receiving crrt, 18 (90.0%) patients died (tbi - related brain herniation in 7, multi - organ failure in 11), with a median survival time of 10 days (range, 3 - 136 days) (figure 1b). For the patient with sdh, the median survival time was 102 days, whereas more than half of the patients survived when sdh was absent (p=0.030) (figure 1c). Median survival time was 2529 days when the iss score was less than 25, whereas it was 73 days when the iss score was 25 or more (p=0.047) (figure 1d). In multivariate analysis, only 24-hours urine output before receiving crrt (500 ml vs. <500 ml) remained as a significant factor (p=0.026) (table 2). The actuarial median survival time of the 29 patients was 163 days after trauma (range, 3 - 317). Among the 29, 22 patients died with a median survival time of 8 days (range, 3 - 55) (figure 1a). The causes of death were tbi - related in 8, sepsis due to pneumonia or acute respiratory distress syndrome (ards) in 4, and multi - organ failure in 10 . Among the various factors related to survival, 24-hr urine output before receiving crrt (500 ml vs. <500 ml) (figure 1b), the presence of sdh (figure 1c), and iss (<25 vs. 25) were significant after univariate analysis (figure 1d, table 2). Median survival time was 10 days (range, 3 - 136) when the patients urine output was less than 500 ml per day before receiving crrt, whereas more than half of the patients survived when the urine output was 500 ml per day or more (p=0.015). Of the nine patients who had a 24-hours urine output of more than 500 ml per day before receiving crrt, 4 (44.4%) patients died (tbi - related brain herniation in 1, multi - organ failure in 3), with a median survival time of 146 days (range, 4 - 317). Of the 20 patients who had a 24-hours urine output of less than 500 ml per day before receiving crrt, 18 (90.0%) patients died (tbi - related brain herniation in 7, multi - organ failure in 11), with a median survival time of 10 days (range, 3 - 136 days) (figure 1b). For the patient with sdh, the median survival time was 102 days, whereas more than half of the patients survived when sdh was absent (p=0.030) (figure 1c). Median survival time was 2529 days when the iss score was less than 25, whereas it was 73 days when the iss score was 25 or more (p=0.047) (figure 1d). In multivariate analysis, only 24-hours urine output before receiving crrt (500 ml vs. <500 ml) remained as a significant factor (p=0.026) (table 2). Tbi patients are often exposed to multiple trauma, sepsis, and many nephrotoxic drugs . Therefore, arf can occur after hypotensive acute tubular necrosis (atn), vasomotor atn, and toxic atn after tbi.5) urea and other solutes are increased in arf patients and these solutes can pass into the brain because the blood - brain barrier (bbb) breaks down in patients with tbi ., this compensation mechanism is disrupted in tbi, thereby cerebral edema can become more worse.1211) additionally, arf can alter the concentration of neurotransmitters or circulating cytokines, acid - base balance, hemostasis, and drug metabolism . Increased circulating cytokines can lead to disruption of the bbb, allowing increased access to cytotoxic inflammatory cells, cytokines, complement, amino acids, and organic osmolytes.14) therefore, it has been reported that tbi patients with arf have a higher incidence of poor outcome when compared with tbi patients without renal dysfunction.12) renal replacement therapy for arf in the patient with tbi is challenging, because conventional ihd is known to lead to an increase in brain water content, even in non - tbi patients undergoing regular hemodialysis.5) compared to standard ihd, crrt has been shown to result in greater intracranial stability.615) during treatment with crrt, changes in osmolality and changes of urea and bicarbonate levels were much less than those during ihd.5) this intracranial stability can be achieved by improved cardiovascular stability, because various modes of crrt can be selected according to cardiovascular volume status.4) therefore, it can be stated that crrt has many advantages over conventional ihd in the treatment of arf for patients with tbi . The acute kidney injury network working group summarized the available evidence and presented absolute indications for initiation of crrt as follows; a serum urea concentration> 224 mg / dl (blood urea nitrogen [bun]> 100 mg / dl), hyperkalemia (> 6 meq / l and electrocardiogram abnormalities), hypermagnesemia (> 8 meq / l), severe acidosis (ph <7.15), lactic acidosis related to metformin use and anuria with diuretic resistant volume overload.710) however, to our knowledge, there are no previous reports regarding indications for initiation of crrt for arf in patients with tbi . In our study, it was found that starting early crrt before when the urine output is reduced to less than 500 ml per day significantly prolonged survival time of tbi patients with post - traumatic arf . Our result suggests that starting crrt in the early stage of arf is required to improve the survival rate of tbi patients who have worse outcomes compared to those without post - traumatic arf . This study has some limitations of a retrospective design, a small number of patients, and a non - comparative study . However, to the best of our knowledge, this is the first analysis of outcomes of crrt in tbi patients with post - traumatic arf . Further studies with a prospective design, many more cases, and control group with conventional ihd are necessary to prove the beneficial therapeutic effects of crrt compared to conventional ihd, and the best timing of initiation of crrt in the tbi patients with post - traumatic arf . According to our results, we suggest that early intervention with crrt before urine output is reduced to less than 500 ml per day may be beneficial in the treatment of tbi patients with impending arf . To define the therapeutic advantages of early crrt in the tbi patients with arf, a well - designed and controlled study with more cases
Systemic lupus erythematosus (sle) is an autoimmune disease of connective tissue involving multiple organs . It is currently accepted that there are several genetic, environmental, and hormonal factors responsible for complex immunological disorders contributing to its development . Recent studies have shown that abnormal stimulation of innate immunity may have a great influence on the immunopathogenesis of sle . Hence, the receptors for pathogen - associated molecular patterns (pamps) have been the source of much recent attention . One of the representatives of this group is toll - like receptors (tlrs). They are associated with innate immunity insofar as they are agents in the pathogenesis of sle and lupus - like syndromes . Tlr3, tlr7, and tlr9 seem to be involved in the development of autoimmune diseases . The ligation of a tlr activates a chain of proteins which transmit a signal to the nucleus, which in turn leads to increased production of proinflammatory cytokines, the expression of major histocompatibility complex (mhc) class i and ii antigens, and costimulatory molecules, which effectively activate antigen presentation and acquired immunity [5, 6]. Intracellular tlrs, apart from pathogen recognition and initiation of innate immunity, are capable of recognizing endogenous ligands . In sle patients, impaired apoptosis and invalid cell debris clearance lead to increased concentration of serum nucleic acids (ssrna, dsrna, and dna), which are well - known ligands for tlr3, tlr7, and tlr9 . Nucleic acid - dependent activation of endosomal tlr is mediated by bcr receptor on lymphocytes b and fc, binding immunologic complexes and inducing their endocytosis . The activation of these receptors by specific ligands is thought to initiate autoimmune processes, which has been confirmed by studies on animal sle model . Tlr stimulation leads to increased expression of proinflammatory cytokines (il-6, ifn, and tnf), which may reflect the intensity of the disease . On the other hand, synthetic oligodna with tlr receptor inhibitory properties causes the opposite effect, leading to a clinical improvement being observed in animal sle models . The aim of our study was to assess the tlr3, tlr7, and tlr9 expression on peripheral blood mononuclear cells (pbmcs), including cd3 t lymphocytes and their cd4 and cd8 subpopulations, and cd19 b lymphocytes, in patients with sle, compared to healthy controls . The original results of this study serve as the first presentation of a simultaneous analysis of the relationship between the expression of the studied tlrs and disease activity, the degree of organ damage, several clinical and laboratory parameters, and the influence of immunosuppressive treatment . Moreover, a correlation between the expression of tlrs and gender as well as pre- and postmenopausal period was evaluated . Thirty - five sle patients, diagnosed as having met at least 4 criteria according to the acr, were included in the study . All of the patients had been treated at the department of dermatology and venereology, medical university of lodz and did not present symptoms of active infection or neoplastic disease at the time of the study . The study group comprised 30 women and 5 men aged from 25 to 65 years . The average duration of sle was 7 years, ranging from 3 months to 21 years . Disease activity was assessed according to the slam (systemic lupus activity measure) scale . Patients who reached 10 and more points were diagnosed as having active sle . During the study, organ damage was then assessed with the slicc / acr (systematic lupus international collaborating clinics / american college of rheumatology) damage index . However, 22 patients received 1 point and 8 of them 2 points, indicating severe organ damage . Pbmcs were isolated by gradient centrifugation using ficoll - histopaque-1077 (paa laboratories, pasching, austria). Briefly, blood was precisely applied on the surface of the gradient and centrifuged at 1600 rpm for 20 min . The obtained buffy coat at the interphase was collected and dispersed in 5 ml of hank's medium (biomed, lublin, poland) and centrifuged at 1600 rpm for 10 min . The supernatant was collected and cells were washed twice with rpmi 1640 medium (paa laboratories, pasching, austria) at 1100 rpm for 5 min . Each time . Isolated pbmcs were divided into 1 10 cells per tube (each 100 l of pbs) and incubated with surface monoclonal antibodies against cd3, cd4, cd8, and cd19 conjugated with the fluorochromes allophycocyanin (apc), peridinin chlorophyll protein (per - cp), and phycoerythrin - cy7 (pe - cy7) (all from bd pharmingen, san diego, ca, usa) at a concentration of 20 l/1 10 cells, in darkness at room temperature for 30 min . The cells were then fixed and permeabilized using an intracellular tlr staining kit according to the producer's protocol (imgenex, san diego, ca, usa). The cells were then incubated with monoclonal antibodies against tlr3, tlr7, and tlr9 conjugated with fluorescein isothiocyanate (fitc) and phycoerythrin (pe) and their corresponding isotype controls (invivogen, san diego, ca, usa), at a concentration of 4 l/1 10 cells, in darkness, at room temperature for 30 min . Six - color, two laser flow cytometry measurements were performed using the facs canto ii cytometer, equipped with bd facs diva software (all becton dickinson, san jose, ca, usa) as previously reported . The cell fluorescence was estimated using standard fluorescence filters: fl1 (313 nm 10), fl2 (264 nm 10), fl3 (374 nm 10) and fl4 (467 nm 10), fl5 (355 nm 10), and fl6 (653 nm 10). For each sample, the lymphocyte population was discriminated from pbmcs by forward scatter (fsc) versus side scatter (ssc) distribution . Then, the percentages of cd3, cd4, cd8, and cd19 expressing tlr3, tlr7 or tlr9 were assessed . Finally, the ratios of tlr3, tlr7, and tlr9 in the whole population of pmbcs were calculated . Representative dot plots from flow cytometry measurements of tlr3 and tlr9 expression on t- and b - cells in patients and healthy controls (panel b) are presented in figure 1(a). Representative dot plots from flow cytometry measurements of tlr7 expression on b - cells in patients and healthy controls are presented in figure 1(b). For measurable characteristics, minimum and maximum values were shown; average values were calculated: the arithmetic mean, median, and mode were calculated as were the parameters describing the internal differentiation (standard deviation). The interquartile range was also calculated as the distance between the third and the first quartiles . For quality characteristics, the percentage of occurrence of the categories was determined . To determine the pattern of distribution of the quantitative variables, the shapiro - wilk test was used . The mann - whitney test was used to assess the significance of any differences in average values between two groups, as the distribution pattern was not normal, and the anova rank test and kruskal - wallis test, followed by a post hoc test of multiple comparisons of average ranks (dunn test), were performed, to evaluate the differences in average values in several groups . The assessment of the relationship between the measurable variables was based on the spearman rank correlation coefficient . In all comparisons, the level of significance was p 0.05 . Significantly higher percentages of tlr3- and tlr9-positive pbmcs and cd3 t lymphocytes, including those positive for cd4 and cd8 antigens, as well as cd19 b lymphocytes were observed among patients with sle, compared to healthy controls (figures 2 and 3). A higher percentage of cd19 b lymphocytes expressing tlr7 was found in patients with sle than in healthy subjects (p <0.006) (figure 4). With regard to pbmcs and both subpopulations of t lymphocytes, tlr7 expression did not differ between patients and healthy controls (table 2). There were no significant correlations between the proportions of various cell subsets expressing the studied tlrs and disease activity (table 2). No statistically significant correlation was observed between the expression of any of the studied types of tlr among the given cell subpopulations and the degree of organ damage according to the slicc / arc damage index . However, subjects with severe organ dysfunction presented a higher percentage of tlr9-positive pbmcs, cd4 and cd8 t lymphocytes, and cd19 b lymphocytes (table 3). A significant mutual correlation was seen to exist between the expression of tlr3 and tlr9 in pbmcs (p <0.00001). No statistically significant correlation was observed between the expression of studied tlrs and the patient's gender . A significantly higher percentage of cd19 b lymphocytes expressing tlr7 was found in premenopausal women with sle than in postmenopausal women (3.52% 6.46 versus 0.12% 0.17 resp ., p <0.03). A significantly lower count of cd4 cells with tlr9 was observed in patients with lymphopenia, compared with patients with a normal lymphocyte count (> 1000/mm) in the peripheral blood (4.59% 5.83 versus 6.86% 6.83 resp ., p <0.005). In this subpopulation of cells, there was a significantly higher count of cells among patients with hypogammaglobulinemia, representing less than 12% of all proteins in the proteinogram analysis, compared to subjects with normal concentrations of gammaglobulins (32.12% 13.78 versus 11.46% 12.05, resp ., p <0.05). Among patients with anaemia, there was a higher percentage of tlr7-positive cd3 (4.19% 5.45), cd4 (4.19% 5.45), and cd19 cells (5.87% 8.71), compared to patients with haemoglobin concentration> 12 g / dl (0.55% 1.08, p <0.05; 0.55% 1.08, p <0.03; 0.85% 2.24, p <0.02, resp . ). Moreover, an erythrocyte sedimentation rate (esr) of more than 25 was significantly more frequent in subjects with lower counts of tlr3-positive, cd19 b lymphocytes compared to esr 25 (2.55% 2.85 versus 5.10% 3.37, resp ., p <0.03). A review of the clinical findings reveals that only patients with joint symptoms have lower tlr9-positive cd19 b lymphocyte counts, compared to subjects with no joint symptoms . No statistically significant correlation was observed between the expression of studied tlrs and immunosuppressive treatment . Despite intensive research in many centres, the pathogenesis of sle remains poorly understood, and hence, the condition lacks targeted therapy . However, the discovery of tlrs in humans opened a new field in the studies of lupus, and our study of tlr3, tlr7, and tlr9 confirms their potential influence on the disease . Tlr expression has been studied on the molecular level (mrna), as well as the protein level, and involves many subsets of peripheral blood cells [1520]. Higher expression of tlr9 has been shown in sle patients, compared to healthy individuals, which is consistent with our findings . However, the results of studies concerning tlr3 and tlr7 expression are inconsistent . Most of them concentrate on tlr9 expression in b lymphocytes, probably due to the fact that these cells are the main source of pathological antibodies responsible for the propagation of the disease [16, 18, 20]. A higher count of cd19 b and cd3 t lymphocytes expressing tlr9 were seen in our study group, compared to healthy controls . This observation is similar to those obtained by wu et al ., who assessed patients with newly diagnosed, untreated sle . On the other hand, papadimitraki et al . Observed a higher percentage of tlr9-positive cd19 b lymphocytes in a group of patients with active disease, compared to those with inactive disease . What is more, they observed a decrease in tlr9 expression on b cells of as much as 50% when the patient entered remission . It is plausible that in remission, stimulation of b lymphocytes through tlr9 is less intense, and as a result of this phenomenon, the autoimmune inflammation subsides . A potential confirmation of this hypothesis is a study by wong et al ., who demonstrated a positive correlation between the concentrations of proinflammatory cytokines and chemokines produced after tlr9 stimulation and disease activity . However, in our study, no difference was observed between active and inactive sles in terms of the percentage of tlr9-positive cd19 b lymphocytes . This discrepancy between our and other centres may stem from the use of different criteria for patient selection . Patients with lupus nephritis dominated in the study by papadimitraki et al ., constituting 36% of the whole study group, whereas they only constituted 3% of our group . Other research demonstrates greater tlr9 expression in the glomeruli of patients with lupus nephritis, and that the stimulation of glomeruli with endogenous tlr9 ligands augments inflammatory reactions in the kidneys . Wong et al . Analysed tlr3, tlr7, and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes among 16 chinese women . This is the only available publication which addressed the same markers as the present study . The results regarding tlr3 and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes obtained in by both the present study and that of wong et al . . However, these results need to be confirmed by rt pcr on t cells as was done on b cells by nakano et al . . Differences between those results concerned only tlr7 population . While our report shows a markedly higher count of tlr7-positive lymphocytes b cd19 in sle patients than in healthy subjects, wong et al . Did not find any difference in tlr7 expression for any cell subset between patients and healthy controls . However, after tlr7 stimulation, they observed an increase in the production of the chemokines cxcl10 and ccl5 by pbmcs from patients with sle . The observed inconsistence of the results may be due to heterogeneous nature of the study groups used by the two studies or their different genetic background . Similar to our results, although obtained via molecular techniques, are the findings by komatsuda et al ., who report that the concentrations of mrna for tlr7 and tlr9 in pbmcs are significantly higher among patients than in healthy controls . There are several publications regarding correlations between the expression of tlrs with sle activity, but the conclusions are contradictory . Wong et al . Do not report any such correlation in terms of tlr3, tlr7, or tlr9 expression . The lack of any relationship was probably due to the relative predominance of subjects with an inactive disease, according to sledai scale (sle disease activity index). In addition, no such significant relationship was demonstrated, although the majority of patients (63%) presented with active sle . Nakano et al . Studied 19 subjects in the active sle phase and identified a positive relationship between tlr9 mfi (mean fluorescence intensity) in b lymphocytes and sledai score . Analysed a group of 35 newly diagnosed patients and found a negative correlation between the percentage of tlr9-positive b cells and sle activity . They pointed to a possible protective role of tlr9 in the development and propagation of sle . The discrepancy of published data may be explained by heterogeneous study groups in terms of clinical and therapeutic parameters . The presence of anti - dsdna antibodies and tlr expression was also noted in the present study . This type of antibody is a pathognomonic marker of sle, specific for renal involvement . Available publications concerning the relationship between the expression of tlr and the presence of anti - dsdna antibodies are inconsistent . There was a positive correlation between the concentration of anti - dsdna antibodies with the percentage of tlr9-positive cd19 b lymphocytes from patients with active sle . However, other studies, as well as our own results, do not reveal any significant relationships between these parameters [17, 21]. On the contrary, komatsuda et al . Observed a negative correlation between anti - dsdna autoantibodies and the tlr9 mrna content in cells . This may be explained by the heterogeneity of studied populations in terms of clinical presentation, accompanying diseases, treatment modalities, and occult infections, in particular ., unlike other researchers, evaluated an entire pbmc population, including b and t lymphocytes and monocytes, and subjects included in the study were untreated . Moreover, while the authors assessed the concentration of anti - dsdna antibodies, the others only noted their presence . A significant part of our study was the assessment of tlr expression with characteristic clinical and laboratory parameters . In the present study, a lower percentage of cd4 cells expressing tlr9 was seen in patients with lymphopenia, compared to those with lymphocyte counts above 1000/l . To our knowledge, there has been only one publication evaluating the relationship between lymphocyte count and tlr expression so far . Did not find any relationship between the amounts of tlr2 - 5, tlr7, and tlr9 mrna in pbmcs and leukocyte, lymphocyte, neutrophil, and platelet counts, although 18 of 21 subjects presented with hematological abnormalities . It may be plausible that the decreased lymphocyte count of cd4 t lymphocytes coexpressing tlr9 may be related to immunosuppressive treatment in this group of patients ., who report that methylprednisolone inhibits the survival of activated cd4 lymphocytes activated by specific tlr3 and tlr9 ligands in vitro but has no effect on their expression . There were 8 subjects (23%) with hypogammaglobulinemia in our group and half of them were receiving immunosuppressants . They presented with a significantly higher percentage of cd4 tlr9-positive cells, compared to individuals with gammaglobulin levels above 12% . It may be that the treatment with glucocorticosteroids and/or cytostatic agents led to a decrease of gammaglobulins but did not diminish the number of cd4 t lymphocytes expressing tlr9 . This may be due to a low number of subjects with hypogammaglobulinemia with and without immunosuppressive treatment . However, it cannot be excluded that the differences in tlr9 expression between these two subgroups (lymphopenia and hypogammaglobulinemia) may be caused by the different numbers of patients receiving immunosuppressive drugs, the number being lower in the case of hypogammaglobulinemia . Our findings warrant further studies on tlr expression in t lymphocytes from patients with sle, as they may lead to a better understanding of the complex interactions between innate and acquired immunity in the pathogenesis of sle . One profitable course of action would be to inquire into the molecular level of the cell cycle using rt - pcr . The results of the present study note a lower count of cd19 b lymphocytes with tlr3 in patients with esr> 25 . Glucocorticosteroids have a strong anti - inflammatory potential, caused by the inhibition of cytokine biosynthesis at the genome level treatment with this group of drugs may have led to a decrease in cytokines in the sera of patients with high esr, resulting in a lower percentage of b cells expressing tlr3 . Despite this, the treatment did not quench the inflammatory process and, therefore, did not lower the increased esr . The only available article by nakano et al ., where the authors evaluated the correlation between tlr9s in lymphocytes b and t with increased esr, does not confirm any significant relationship . A higher count of cd3, cd4, and cd19 cells coexpressing tlr7 was found in patients with anemia compared to subjects with hemoglobin above 12 g / dl . In sle, anemia may stem from autoimmune hemolysis or chronic inflammatory process (anemia of chronic diseases, acd). In our group, this type of anaemia develops due to a chronic inflammatory reaction, characterized by increased concentrations of tnf-, il-1, or ifn - gamma, which inhibit the secretion of erythropoietin and availability of iron, essential for efficient erythropoiesis . As a result of tlr activation, numerous proinflammatory cytokines it was indicated that proinflammatory cytokines may regulate tlr expression . Moreover, the proinflammatory cytokine - dependent expression of tlr, adaptor proteins, and kinases participating in signal transduction towards the cell interior has been proved . An increased concentration of ifn- in the serum of sle patients, combined with raised ifn - type i dependent gene expression in the mononuclear cells of peripheral blood cells, has been characterized as interferon signature . The continuous, tlr - mediated biosynthesis of ifn- by nucleic acids containing immunologic complexes may be responsible for the interferon signature phenomenon . Moreover, it has been revealed that the level of ifn alpha - dependent gene expression is correlated with sle activity and more detrimental clinical disease forms, associated with damage to the kidneys, bone marrow, or cells of the central nervous system [29, 30]. Increased inf - alpha concentration is regarded as the response to the continuous activation of tlr pathways . However, komatsuda et al . Did not confirm any correlation between tlr and ifn - alpha induced ly6e (lymphocyte antigen 6 complex, locus e) gene expression . The observed higher percentage of cd3, cd4, and cd19 cells with tlr7 among subjects with anemia may reflect the presence of chronic inflammation and increased proinflammatory cytokines . In our study group, a higher count of tlr7-positive b and t cells was seen although 78% of patients received immunosuppressive drugs . This may be due to the majority of patients experiencing active sle (89%). Exacerbation of sle may be induced by the usage of oestrogen - based anticontraceptive pills, that may also elevate the risk of a more severe disease course . However, during menopause sle tends to become milder, which is probably due to a decrease in oestrogen levels in peripheral blood . In our study, we demonstrated a statistically significant higher percentage of b lymphocytes cd19 expressing tlr7 in premenopausal women, compared to females after menopause . These observations suggest the influence of female sex hormones on tlr7 expression on lymphocytes b. this effect has been confirmed in other studies . Young et al . (2011) indicated the increased in vitro expression of endosomal tlrs, including tlr7, on pbmc cells from normal women ader estradiol stimulation, with no effect after treatment with testosteron . In another study, 17-estradiol treatment of normal postmenopausal women enhanced tlr7/9 pdc production of ifn . However, secretion of ifn by plasmacytoid dendritic cells after tlr7 activation was lower in postmenopausal than in premenopausal females . Furthermore, stimulation of tlr7 with a synthetic agonist in lupus - prone mice lacking the alpha oestrogen receptor led to a lower il-6 synthesis by lymphocytes b than in wild type animals . When the clinical symptoms were analyzed, a significantly lower count of lymphocytes b cd19 with tlr9 was found in patients with joint symptoms (75% of subjects) than in patients with no joint symptoms (25%). Some publications describe the expression of tlrs in rheumatoid arthritis [38, 39] and note that patients demonstrate higher expression of tlr2, 3, and 4 on fibroblasts from the synovial tissue . The synovial fluid contains various tlr ligands such as peptidoglycan, dsrna released from necrotic cells, lipopolysaccharides, and cpg - rich nucleic acid . Their presence stimulates the synthesis of many proinflammatory cytokines and chemokines, which sustain inflammation in joints [38, 39]. The lower percentage of cd19 b lymphocytes expressing tlr9 in patients with joint symptoms in our study group may be related to the presence of immunosuppressive treatment . However, immunosuppressive therapy was found to have no influence on tlr expression in our study, which is consistent with the results of other researchers [16, 17]. Reports a significant decrease of mfi for tlr9 in cd20 b lymphocytes in 8 out of 11 patients with sle . No correlation was observed between tlr expression and the degree of organ damage, according to slicc / acr . The lack of any relationship may be explained by the fact that the organ damage reflects the final outcome of the inflammatory process . A significant positive correlation was recorded between tlr3 and tlr9 expression in pbmcs (p <0.00001). However, it is probable that the significant correlation of tlr3 and tlr9, but not tlr7, stems from the higher lability of ssrna (the ligand for tlr7), which undergoes rapid degradation by ribonucleases and is quickly removed from circulation . Tlrs are able to recognise endogenous antigens which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases [40, 41]. These tlr ligands bind tlrs, possibly initiate intracellular signaling pathways, and may initiate autoimmunity processes . Tlrs act on the monocyte - macrophage system and activate dendritic cells, which then engage self - antigens, as the first step for the induction of autoimmunity . Tlr9 activation induces the expression of membrane - bound b - cell activating factor (baff) on human b cells and leads to increased proliferation in response to both soluble and membrane - bound baff . A sizable body of evidence suggests that the endolysosome - restricted nucleic acid sensing subset of tlrs (na - tlrs) plays an important role in the production of antinuclear autoantibodies . Recently, koh et al . Documented that na - tlrs promote the induction of antinuclear abs in sle . Their data indicates that the presence of na - tlrs in b cells is necessary to drive the initial autoimmune response and to promote the activation and escape of tolerance of self - reactive b cells . In addition, overexpression of tlr7 within the b cell compartment was found to enhance b cell tlr7 expression, permit the specific development of anti - rna autoantibody production, and exacerbate sle disease in an animal model . Moreover, the inhibition of both tlr7 and tlr9 reduces autoimmune pathology in experimental sle [48, 49]. This observation suggests that the aberrant activation of a number of tlr pathways may lead to the initiation and/or perpetuation of sle and may indicate the direction for more specific therapy of this disease . In conclusion, our results suggest that tlrs exert an influence on sle development and describe the potential roles played by tlrs in the involvement of specific organs in this disease . Even so, more targeted studies concerning the biology and function of tlrs are warranted and may lead to the development of a new class of drugs.
Neuroretinitis (nr) is considered to be an inflammatory condition which is characterized by optic disc edema and, as a result, formation of a macular star figure . Nr is an atypical presentation of toxoplasmosis infection, and such cases are quite rare . A 13-year - old girl presented with painless subacute visual loss in her right eye for a week at khatam - al - anbia eye hospital in mashhad, iran . Although toxoplasmosis nr is rare, it should be considered in the differential diagnoses of nr . Neuroretinitis (nr) is considered an inflammatory condition which is characterized by optic disc edema and, as a result, formation of a macular star figure (1). This disorder is possibly caused by an infectious process affecting the disc; in other instances, a post - viral or autoimmune mechanism seems to be a more probable cause (1). Furthermore, some instances of nr have been reported to be accompanied by a wide spectrum of infectious pathogens (1). The most common case reported so far is the result of cat scratch disease (csd), accounting for two - thirds of cases in one study (2). However, there are other infectious etiologies of neuroretinitis, including rubeola, toxoplasmosis, herpes simplex, varicella, tuberculosis, lyme disease, leptospirosis, syphilis, various fungi, and multiple viral illnesses (1). Additionally, sporadic cases of nr may occur owing to noninfectious forms of uveitis, such as sarcoidosis and periarteritis nodosa (3, 4). Optic disc edema with a macular star may also occur as a result of other factors such as diabetic papillopathy, hypertensive neuropathy, and anterior ischemic optic neuropathy papilledema (1). To determine the relevant history of patients with nr, the practitioner needs to concentrate on plausible risk factors for specific infectious parameters, such as travelling to areas where lyme disease or tuberculosis (for example) are endemic, exposure to waste material (e.g., leptospirosis), animal exposure (especially cats), and sexual contact that may have resulted in the contraction of syphilis . The clinician should investigate patients for systemic symptoms such as lymphadenopathy, headache, fever, and skin rash . Laboratory tests ought to be customized for the individuals on the basis of information from both their reported history and the examination . Serologic tests for most cases may include the fluorescent treponemal antibody absorption test (fta - abs), cat scratch titers (bartonella species), and a tuberculosis skin test (purified protein derivative, or ppd) (1). In this study, we present a case of unilateral neuroretinitis in which the serology result was apparently negative for acute infection of toxoplasmosis (negative igm titer for toxoplasmosis, but positive for igg). Based on anti - toxoplasma treatment that was administered for the positive igg levels, the condition responded well with near optimal visual recovery . A 13-year - old girl was referred to the ophthalmic emergency department of khatam - al - anbia eye hospital, mashhad, iran, which is affiliated with mashhad university of medical sciences (mums), in may of 2013 with painless subacute visual loss in her right eye for one week . She denied any focal neurologic condition, pain on eye movement or ophthalmodynia, or any other systemic symptom . She also stated that she had not engaged in outdoor camping or any other related activity . Furthermore, she had no remarkable past medical and ophthalmologic history, and she had no contact or proximity to pets, especially cats . Upon examination, the best corrected visual acuity (bcva) was 20/250 in the right eye and 20/20 in the left eye . Moreover, the patient had a grade 2 + relative afferent papillary defect (rapd) in the right eye . An examination of the anterior segment demonstrated 2 + cells in the anterior chamber (a / c) and 1 + vitreous reaction with normal intraocular pressure (iop) in the right eye . The investigation of the right fundus revealed optic disc swelling, macular star lipid (hard exudates) deposition, and peripapillary vascular sheathing (figure 1). Many tests were performed for the patient, including the erythrocyte sedimentation rate (esr), complete blood count differential (cbc diff . ), c - reactive protein (crp), toxoplasmosis serology, purified protein derivative (ppd), angiotensin converting enzyme (ace), fta - abs, brain and orbital mri, chest x - ray, and infectious and rheumatologic consultation . Bartonella henselae serology was not available in our ophthalmology center, nor in any other nearby neighborhood . Based on our clinical results, neuroretinitis etiology revealed csd as the most common infectious etiology, along with the negative systemic and ocular history, and empirical treatment commenced including azithromycin 500 mg daily, trimethoprim / sulfamethoxazole, and the application of a topical steroid . After 72 hours, all the above - mentioned laboratory indices returned to the normal level, except for the toxoplasmosis serology . Furthermore, the serologic test for toxoplasmosis was negative for igm antibody whereas the igg antibody titer was> 100 iu / ml . Relying on the positive igg toxoplasmosis serology and the absence of any response to initial treatment, we embarked on shifting to classic anti - toxoplasmosis treatment, i.e., sulfadiazine 500 mg qid (four times a day), pyrimethamine 50 mg / day, and folinic acid . After 72 hours had passed, prednisolone 1mg / kg / day was added and thereafter tapered during the treatment . The disc edema diminished over the course of a week . Also, visual acuity gradually improved and the a / c inflammatory reaction faded away . After the decrease in disc swelling, a very small hyperpigmented focus appeared in the juxtapapillary position . Gradually, over the next two months, the patient s visual acuity improved to 9/10 and the optic disc swelling and macular exudates resolved (figure 2). The clinical syndrome of idiopathic stellate maculopathy accompanied by optic nerve edema was first identified by leber in 1916 (5). Later on, this syndrome was renamed as leber s stellate neuroretinitis . As is now known, the most common form of infectious nr results from csd (1). The engagement of the optic nerve suggests the need for urgent intervention . In spite of the lack of evidence either for animal exposure or any other systemic condition for this case, empirical csd treatment was instituted as lab tests were pending and given that cat scratch serology was not accessible . Toxoplasmosic retinochoroiditis is rendered as one of the most frequent causes of posterior uveitis, specifically in young patients (6). However, toxoplasmic optic neuropathy is an infrequent condition and is often characterized by subacute visual loss and optic nerve swelling, and is sometimes associated with a macular star (neuroretinitis) (7). The engagement of the optic nerve most commonly encountered in ocular toxoplasmosis is optic nerve edema with a simultaneous distant active lesion . Other types of optic nerve involvement include pure papillitis presented as optic disc swelling, associated with peripapillary vascular sheathing close to the healed lesion, and neuroretinitis, i.e., optic disc swelling with macular hard exudate deposition . Additionally, monocular involvement has been observed in most cases with favorable visual prognosis (8). Considering the positive toxoplasmosis serology in this patient, toxoplasmosis neuroretinitis was suspected, hence prompting the specified treatment . This case disclosed optic disc involvement and neuroretinitis owing to juxtapapillary chorioretinitis reactivation . In some cases, the juxtapapillary chorioretinal scar cannot be found in the acute stage of disease due to disc and peripapillary edema . Although toxoplasmosis neuroretinitis is rare, it should be still considered in any suspected case of neuroretinitis . This case re - emphasizes the necessity of considering toxoplasmosis in the differential diagnosis of neuroretinitis . Awareness of this potential cause and prompt treatment after positive testing may therefore result in a good visual outcome.
We grew the budding yeast saccharomyces cerevisiae in 200 l batch culture on bd falcon 96-well microtest plates at 30.7 c (0.2c, standard deviation) using synthetic media (yeast nitrogen bases + nitrogen, complete supplement mixture) supplemented with 2% sucrose . Cultures were maintained in a well - mixed condition by growing on a shaker at 825 r.p.m . Serial dilutions were performed daily (23 hours of growth) with variable dilution factors . Population densities were recorded each day before the serial dilution by measuring optical density at 600 nm using a thermo scientific varioskan flash multimode reader . The calibration between optical density and cell density was based on the previous characterization of this system . In the group of connected populations, for each dilution factor there were 4 replicate arrays each consisting of 10 patches . Populations were connected by controlled dispersal between nearest neighbors (dispersal rate d=0.5, which is defined as the fraction of population going out of a patch). Reflecting boundary conditions were adopted, meaning that a population on the edge would have 75% of its cells remaining in the patch during the dispersal step . In the group of isolated populations, the experiment was performed in a similar spatial setting except that there was no dispersal (d=0); for each dilution factor there were 4 arrays each consisting of 5 patches, thus a total of 20 replicate populations isolated from each other . The dilution factors for the data presented in fig . 2 are 500, 1000, 1200, 1400 and 1600 . In the experiment to measure recovery length, populations were connected by nearest - neighbor dispersal (d=0.5, reflecting boundary conditions). The dilution factor for the bad region (1 patch) was 2500; the dilution factor for the good region (5 patches) was varied as the environmental driver . The dilution factors for the data presented in fig . 4 are 500, 750, 1000, 1133, 1200, 1266, 1350 and 1400 . Statistical indicators for the connected populations were calculated among 10 populations in one array on each day and averaged over a span of at least 5 days, after the populations stabilized . The mean value of 4 replicate arrays and sem (n=4) are shown in figure 2 . For the isolated populations, statistical indicators were calculated on each day among 20 populations over 5 days . We used bootstrap to compute ses of the indicators by resampling 1000 times the ensemble of replicate populations (for the coefficient of variation and the temporal correlation) or arrays (for the spatial correlation). The coefficient of variation (cv) was calculated as the sample standard deviation (supplementary fig . 3b) divided by the sample mean . Because the local populations in our experiment were grown in a homogeneous environment, in principle they could all be treated as replicates . Assuming the system is ergodic, the cv calculated over an ensemble of replicates can be interpreted either as spatial cv of many populations at one time point or temporal cv of a single population over many time points . The temporal correlation, defined as the lag-1 autocorrelation, was estimated by the pearson s correlation coefficient between the population densities at subsequent days . To correct for negative bias in small samples n is a fixed number for different dilution factors, so using the modified estimators would not affect the trend of indicators . The spatial correlation, defined as the two - point correlation between all neighboring pairs, was estimated by the moran s coefficient . The expectation of moran s coefficient is 1n1 in the absence of spatial correlation; we used a modified estimator with an additional term 1n1 so that the expectation is 0 . In this case, the sample size n is the number of patches in an array: n=10 for connected populations; n=5 for isolated populations . For detailed formula of the statistical indicators, see supplementary note 4 . In the analysis we ensured environmental homogeneity by removing a linear gradient of population density observed in connected populations the small gradient is presumably caused by some heterogeneity in experimental conditions (temperature, dilution errors, etc .) Across the plate . Removing gradient - type spatial heterogeneity before statistical analysis is similar to the detrending procedure commonly used in time - series analysis; it prevents spurious signals such as positive spatial correlation (supplementary fig . After the recovery profile stabilized, we tracked the population density profiles of at least 6 replicates over several days . The half - point recovery length l was estimated by performing a shape - preserving interpolation (matlab function pchip, piecewise cubic hermite interpolating polynomial) to the recovery profile and then locating the position of half recovery at which n(x = lhalf)=12n(x=5). The population density of the bad region (dilution factor 2500) in our experiment was close to 0 (fig . 4 and supplementary fig . 7). In the more general scenario with a sharp boundary between two regions of different quality (supplementary fig . 6 and 8), the position of half recovery can be defined as the midpoint between the equilibrium population density of the region of interest and the population density at the boundary . The exponential recovery length was estimated by fitting an exponential function with three parameters c1 exp(x / l) + c2 to the recovery profile n(x). The data points used for exponential fitting are from position 1 to 5 (except for dilution factor 500, the data for fitting are from position 0 to 5). We note that our definition of exponential recovery length is phenomenological, because: 1) the deviation is expected to be exponential only close enough to the equilibrium; 2) at higher dilution factors the profile can deviate from an exponential form (supplementary fig . 4c) may be due to the limited data points used in fitting or experimental errors . For both the half - point recovery length and the exponential recovery length, we used bootstrap to compute ses for the indicators by resampling the ensemble of steady - state profiles 100 times and fitting the average recovery profile . We grew the budding yeast saccharomyces cerevisiae in 200 l batch culture on bd falcon 96-well microtest plates at 30.7 c (0.2c, standard deviation) using synthetic media (yeast nitrogen bases + nitrogen, complete supplement mixture) supplemented with 2% sucrose . Cultures were maintained in a well - mixed condition by growing on a shaker at 825 r.p.m . Serial dilutions were performed daily (23 hours of growth) with variable dilution factors . Population densities were recorded each day before the serial dilution by measuring optical density at 600 nm using a thermo scientific varioskan flash multimode reader . The calibration between optical density and cell density was based on the previous characterization of this system . In the group of connected populations, for each dilution factor there were 4 replicate arrays each consisting of 10 patches . Populations were connected by controlled dispersal between nearest neighbors (dispersal rate d=0.5, which is defined as the fraction of population going out of a patch). Reflecting boundary conditions were adopted, meaning that a population on the edge would have 75% of its cells remaining in the patch during the dispersal step . In the group of isolated populations, the experiment was performed in a similar spatial setting except that there was no dispersal (d=0); for each dilution factor there were 4 arrays each consisting of 5 patches, thus a total of 20 replicate populations isolated from each other . The dilution factors for the data presented in fig . 2 are 500, 1000, 1200, 1400 and 1600 . In the experiment to measure recovery length, populations were connected by nearest - neighbor dispersal (d=0.5, reflecting boundary conditions). The dilution factor for the bad region (1 patch) was 2500; the dilution factor for the good region (5 patches) was varied as the environmental driver . 4 are 500, 750, 1000, 1133, 1200, 1266, 1350 and 1400 . Statistical indicators for the connected populations were calculated among 10 populations in one array on each day and averaged over a span of at least 5 days, after the populations stabilized . The mean value of 4 replicate arrays and sem (n=4) are shown in figure 2 . For the isolated populations, statistical indicators were calculated on each day among 20 populations over 5 days . We used bootstrap to compute ses of the indicators by resampling 1000 times the ensemble of replicate populations (for the coefficient of variation and the temporal correlation) or arrays (for the spatial correlation). The coefficient of variation (cv) was calculated as the sample standard deviation (supplementary fig . 3b) divided by the sample mean . Because the local populations in our experiment were grown in a homogeneous environment, in principle they could all be treated as replicates . Assuming the system is ergodic, the cv calculated over an ensemble of replicates can be interpreted either as spatial cv of many populations at one time point or temporal cv of a single population over many time points . The temporal correlation, defined as the lag-1 autocorrelation, was estimated by the pearson s correlation coefficient between the population densities at subsequent days . To correct for negative bias in small samples n is a fixed number for different dilution factors, so using the modified estimators would not affect the trend of indicators . The spatial correlation, defined as the two - point correlation between all neighboring pairs, was estimated by the moran s coefficient . The expectation of moran s coefficient is 1n1 in the absence of spatial correlation; we used a modified estimator with an additional term 1n1 so that the expectation is 0 . In this case, the sample size n is the number of patches in an array: n=10 for connected populations; n=5 for isolated populations . For detailed formula of the statistical indicators, see supplementary note 4 . In the analysis we ensured environmental homogeneity by removing a linear gradient of population density observed in connected populations . The small gradient is presumably caused by some heterogeneity in experimental conditions (temperature, dilution errors, etc .) Across the plate . Removing gradient - type spatial heterogeneity before statistical analysis is similar to the detrending procedure commonly used in time - series analysis; it prevents spurious signals such as positive spatial correlation (supplementary fig . After the recovery profile stabilized, we tracked the population density profiles of at least 6 replicates over several days . The half - point recovery length l was estimated by performing a shape - preserving interpolation (matlab function pchip, piecewise cubic hermite interpolating polynomial) to the recovery profile and then locating the position of half recovery at which n(x = lhalf)=12n(x=5). The population density of the bad region (dilution factor 2500) in our experiment was close to 0 (fig . 4 and supplementary fig . 7). In the more general scenario with a sharp boundary between two regions of different quality (supplementary fig . 6 and 8), the position of half recovery can be defined as the midpoint between the equilibrium population density of the region of interest and the population density at the boundary . The exponential recovery length was estimated by fitting an exponential function with three parameters c1 exp(x / l) + c2 to the recovery profile n(x). The data points used for exponential fitting are from position 1 to 5 (except for dilution factor 500, the data for fitting are from position 0 to 5). We note that our definition of exponential recovery length is phenomenological, because: 1) the deviation is expected to be exponential only close enough to the equilibrium; 2) at higher dilution factors the profile can deviate from an exponential form (supplementary fig . 4c) may be due to the limited data points used in fitting or experimental errors . For both the half - point recovery length and the exponential recovery length, we used bootstrap to compute ses for the indicators by resampling the ensemble of steady - state profiles 100 times and fitting the average recovery profile
The prediction of a patient's growth potential is of clinical significance because it determines the optimal timing of starting or stopping bracing and the timing of growth - guidance surgery with an expandable prosthesis.1 2 3 4 many clinical and radiologic methods are available to predict a patient's growth potential including the age at menarche, difference in body height growth and arm span, risser sign, and tanner and whitehouse (tw3) digital skeletal age.5 6 7 8 however, most of these measures have disadvantages in the current clinical practice.9 clinical measurements of standing height and arm span require serial measurements to determine growth trends, and its retrospective nature thus likely misses the period of peak growth spurt . The menarche age is not consistent in determining the termination of peak growth as some patients develop delayed menarche and its presence marks a stage too late for any meaningful bracing.10 similar to menarche, the risser sign has been shown by several studies to be a poor indicator of skeletal maturity, cessation of growth, and more importantly the risk of curve progression.11 12 13 14 15 up to 75.2% of patients may have persistent growth despite capping of the iliac apophysis.13 in addition, the progression of iliac apophysis has been shown to proceed in a reverse direction (posteromedially to anterolaterally) or in fragments.15 the digital skeletal age assessment using the tw36 7 8 or the greulich and pyle16 methods are more accurate in determining different growth phases, but these techniques are complex and far too difficult to apply in the clinical setting . Sanders et al showed that according to the tw3, the finger epiphysis maturation is closely related to the peak height velocity.17 the peak height velocity is noted to occur during early adolescence, and menarche and the risser sign appear after the peak growth spurt.18 in response to the limitations possessed by the previous methods in assessing skeletal maturity, the distal radius and ulna (dru) classification was created and reported by luk et al.19 this classification, which included 11 radius grades (r1 to r11) and 9 ulna grades (u1 to u9), was found to accurately determine the peak height velocity (r7 and u5) and cessation of growth (r10 and u9). However, the reliability of the classification has not been assessed . As such, the following study aimed to test the classification's reliability . A prospective radiographic study assessing patients with adolescent idiopathic scoliosis (ais) undergoing bracing during the month of july 2014 was performed to assess the reliability of the dru classification.19 ethics approval was obtained from our institutional review board . The study was performed at the duchess of kent children's hospital, pokfulam, hong kong, a tertiary referral center for spinal disorders . For the dru classification to be practical clinically, the reliability testing should be based on a wide range of patient ages, as commonly seen in our clinics . In addition, the aim of this classification was to correctly identify patients in the peak growth spurt so that timely interventions can be applied . Thus, choosing braced patients would likely encompass a larger range of grades and avoid reliability assessment of only a certain age group or dru grade . The follow - up left - hand radiographs during the study month were obtained in all subjects, and data was extracted regarding the age at which the radiograph was taken and the sex of the patient . These anteroposterior radiographs of the hand and wrist were taken with 42 peak kilovoltage and 1.6 ma - seconds of x - ray energy . K.d .- k.l . ), one junior and one senior consultant spine surgeon, for grading . There was no discussion between the two examiners about how to classify each grade prior to the initiation or during the study . Radiographs were accessed by a dicom - based radworks 5.1 (applicare medical imaging bv, zeist, the netherlands) computer software program . All data was coded and entered on a spreadsheet and was blinded to the examiners until the end of reliability measurements . Spss version 20 (chicago, illinois, united states) was utilized to perform the statistical analysis . The reliability assessment was based on intraclass correlation (icc), which was an appropriate statistical tool for this analysis.20 icc could be interpreted based on the following values: 0 to 0.29 indicated poor agreement; 0.30 to 0.49 indicated fair agreement; 0.50 to 0.69 indicated moderate agreement; 0.70 to 0.80 indicated strong agreement; and> 0.80 indicated almost perfect agreement.21 22 the 95% confidence interval (ci) bounds were assessed for precision . A p value of <0.05 was considered significant . All data was coded and entered on a spreadsheet and was blinded to the examiners until the end of reliability measurements . Spss version 20 (chicago, illinois, united states) was utilized to perform the statistical analysis . The reliability assessment was based on intraclass correlation (icc), which was an appropriate statistical tool for this analysis.20 icc could be interpreted based on the following values: 0 to 0.29 indicated poor agreement; 0.30 to 0.49 indicated fair agreement; 0.50 to 0.69 indicated moderate agreement; 0.70 to 0.80 indicated strong agreement; and> 0.80 indicated almost perfect agreement.21 22 the 95% confidence interval (ci) bounds were assessed for precision . A p value of <0.05 was considered significant . One hundred sixty - one subjects (124 females and 37 males) satisfied our inclusion criteria and were recruited for this study . The mean age was 13.3 (standard deviation: 1.5; range: 8 to 18). The interobserver reliability was icc = 0.97 (95% ci: 0.96 to 0.98) and icc = 0.97 (95% ci: 0.96 to 0.98) for the radius and ulna, respectively . Was icc = 0.93 (95% ci: 0.91 to 0.95) for the radius and icc = 0.95 (95% ci: 0.930.96) for the ulna; for k.d .- k.l ., icc = 0.94 (95% ci: 0.91 to 0.95) for the radius and icc = 0.95 (95% ci: 0.93 to 0.96) for the ulna ., there were 18 one - grade disagreements (11.1%) and 1 two - grade disagreement (0.6%). For the ulna, there were 39 one - grade disagreements (24.1%) and 1 two - grade disagreement (0.6%). Was as follows: for the radius, there were 28 one - grade disagreements (17.3%) and 5 two - grade disagreements (3.1%). For the ulna, there were 42 one - grade disagreements (25.9%) and 2 two - grade disagreements (1.2%). The intraobserver disagreement for k.d .- k.l . Was as follows: for the radius, there were 26 one - grade disagreements (16.0%) and 4 two - grade disagreements (2.5%). For the ulna, there were 40 one - grade disagreements (24.7%) and 5 two - grade disagreements (3.1%). For the radius, there were 18 one - grade disagreements (11.1%) and 1 two - grade disagreement (0.6%). For the ulna, there were 39 one - grade disagreements (24.1%) and 1 two - grade disagreement (0.6%). The intraobserver disagreement for j.p.y.c . Was as follows: for the radius, there were 28 one - grade disagreements (17.3%) and 5 two - grade disagreements (3.1%). For the ulna, there were 42 one - grade disagreements (25.9%) and 2 two - grade disagreements (1.2%). The intraobserver disagreement for k.d .- k.l . Was as follows: for the radius, there were 26 one - grade disagreements (16.0%) and 4 two - grade disagreements (2.5%). For the ulna, there were 40 one - grade disagreements (24.7%) and 5 two - grade disagreements (3.1%). From this study, we found that the dru classification had excellent reliability among a wide range of radius and ulna grades.19 although we did not have patients young enough to present with radius stages r1 to r4, interventions for ais such as bracing are only applicable during the peak growth spurt . The r6 to r9 and u5 to u8 were important grades because they represented the period of peak growth spurt, and these grades were well represented in our testing . After all measurements were made through this vigorous process of testing, the two examiners thoroughly discussed each grade description and were able to form a consensus about which aspects of the classification were more representative and easier to apply during reading . The difficulties encountered by both readers during testing mainly arose from the grading of r7 to r9 and u5 to u8 . Both reviewers agreed that the grading of r7 to r9 and u5 to u8 were the most difficult because they were based on descriptions of multiple parameters . Each clinician might focus on something different, such as the appearance of the physis narrowing, the definition of capping, and when the epiphysis acquired the same width as the metaphysis, among others . Disagreements in the ulna grades appear larger than the radius grades, likely due to a larger range of ulna grades measured as compared with the radius . For the radius, the main identifying factor between r7 and r8 was the appearance of capping over the medial side in r7 (fig . 1) and over the lateral side in r8 (fig . 2). Both readers agreed that capping was defined as a hornlike structure at the ends of the epiphysis like a sharp bony outgrowth . A simple step to confirm this observation on the radius was to trace the proximal border of the epiphysis to find any dipping of the line toward the metaphysis at the medial or lateral ends . For r9 (fig . 3), strong capping was not always a consistent finding, thus narrowing of the physis was the main determinant description for this grade . Examiners should focus on the medial side capping and the absence of lateral side capping (red arrow). Note the hooklike structure / sharp outgrowth at the medial physis facing the metaphysis that deviates from the physeal line . This radiograph also represents the u6 grade with the appearance of an epiphysis and metaphysis of the same width but no narrowing of the medial physeal plate (white arrow). Examiners should focus on the lateral side capping (red arrow). Note the hooklike structure / sharp outgrowth at the lateral physis facing the metaphysis that deviates from the physeal line ., the ulna is graded u7 as there is narrowing of the medial physeal plate (black arrow) but incomplete fusion . Generally, grading of u1 to u4 was not difficult due to some distinct features such as the size of the epiphysis for u3 and the appearance of the styloid for u4 . The difficulties were noted for descriptions in u5 to u8 . For u5 (fig . 4), the appearance of a denser ulna head was not easily observed and so flattening of the radial half of the ulnar epiphysis was the main determinant . For u6 (fig . 1), we found that the overlapping of the metaphysis with the epiphysis at the center third was inconsistent in most radiographs, and thus we often relied on the appearance of the epiphysis being as wide as the metaphysis . To differentiate u7 (fig . 5), we usually used narrowing of the medial ulnar physis (u7) and fusion of the medial ulnar physis (u8) as the determining factor . The descriptions of the smooth curve line articulation in u7 might not be very easily understood . Examiners should focus on the flattening of the radial epiphysis (white arrow) and should note that the epiphysis is not as wide as the metaphysis, indicated by the vertical white line touching the metaphysis but not the epiphysis . Interestingly, the interobserver reliability was somewhat better than the intraobserver reliability, which suggests that the readers may become more competent in the measuring skills with increased grading experience from measuring these 161 radiographs, demonstrating a learning curve effect . Hence, this classification scheme should be useful as a guide for decision making and as a communication tool between clinicians . Despite not having any prior discussions or training into how each grade should be correctly assessed, the results showed that both examiners used similar grading methods in approaching each radiograph . Thus, the difficulties and suggestions raised during the consensus meeting were important to help less experienced examiners with this classification scheme . The dru has been shown to be strongly associated with the peak growth phase and growth cessation,19 which can help clinicians decide between bracing and surgery . The results from this study suggested that the dru classification was a reliable method to assess skeletal growth . Although the most appropriate time to get these radiographs still requires further investigation, the authors suggest a yearly radiograph with consideration of radiographs every 6 to 9 months during the period of peak growth spurt . In the future, prospective studies are being developed to apply this classification scheme on patients with ais to determine its sensitivity and specificity in identifying the peak growth spurt, predicting curve deterioration, as well as detecting skeletal maturity . In addition, it can be used to help decide the appropriate timing for initiation and cessation of bracing, when to use growth guidance surgery, and when to recommend fusion . By more accurately predicting a child's remaining growth potential, we can potentially narrow the duration of effective bracing without jeopardizing the deformity control, thereby reducing any unnecessary psychological burden.23 this knowledge could also guide the duration and interval of follow - up and the number of x - rays needed, thus reducing radiation exposure . Additional studies are needed to validate this dru classification in other ethnicities and populations and to further directly compare it with other new and established skeletal maturity schemes.24 25 26 27 this study has shown that the dru classification is a reliable tool for measuring skeletal growth . Further prospective studies are required to test its predictability of peak growth spurt and growth cessation and to compare it with other skeletal growth measures such as the risser sign, tw3 grading, and age of menarche.
Neospora caninum is an apicomplexan protozoan, infecting a large range of mammals . In cattle, several studies focussing on the specific immune response to n. caninum in cattle have shown that the time of gestation is important with regard to the outcome of the infection . Proinflammatory cytokines, produced by lymphocytes, are crucial for controlling a variety of intracellular pathogens, including n. caninum . These cytokines are produced by natural killer (nk) cells, as well as by cd4 t - cells and cd8 t - cells . Cd4 cells mediate the humoral response and their involvement is associated with increased igg1 levels, whereas cd8 cells are involved in the cellular immune response, which is characterized by increased production of igg2a . N. caninum is an intracellular parasite and resides within a specialized compartment, a parasitophorous vacuole, surrounded by a parasitophorous vacuole membrane (pvm). Following egress from a host cell, these parasites immediately search for a new host cell to invade, and the direct accessibility for components of the immune system to the parasite within the circulation is rather limited . Thus, extracellular tachyzoites have a low chance to be detected by t - helper-2-(th2-) type cells . A humoral immune response is therefore not sufficient to clear a n. caninum infection . For more efficient protection against intracellular pathogens such as n. caninum, the host usually generates a cellular, t - helper-1-(th1-) type response [6, 7]. This is possible since secretory parasite molecules pass through the pvm into the host cell cytoplasm, where they interact with and manipulate host cell functions . Once in the cytoplasm these molecules can be processed and corresponding peptides are presented on the host cell surface via major histocompatibility complex (mhc) class i molecules . Cd8 lymphocytes will recognize the peptides presented on these mhc - i molecules and activate th1 cells, which then produce cytokines such as interleukin-12 (il-12), interferon gamma (ifn-), and tumour necrosis factor (tnf-). The production of these cytokines leads to the activation of pathways that generate free oxygen radicals and nitric oxide (no) and its metabolites among other factors, which are potentially lethal for many protozoa . However, these processes can also be deleterious to the fetoplacental interface and potentially induce abortion and/or fetal resorption, especially in the first trimester of pregnancy, when levels of pregnancy hormones, such as progesterone, which counteracts these effects, are relatively low . As a result, there is no or only little th2 cytokine polarization and the dam will generate a th1 immune response, which also affects the placental and foetal tissue . Since a strong th1 response is incompatible with successful pregnancy, the infection can lead to the loss of the unborn foetus . Progesterone promotes th2 cell proliferation the production of il-4, il-5, and il-10 is known to inhibit no and tnf- production and impairs nk cell activity [1113]. However, since the immune response is not capable of controlling the parasite, the risk of transplacental transmission of n. caninum is relatively high . Thus, resistance to n. caninum seems to rely, at least partially, on a functional th1 response . In this study, inbred balb / c, cba / ca, and c57bl/6 strains of mice (with different mhc - i haplotypes) were compared for their capacity to cope with a n. caninum infection . In addition, we show that cba / ca mice were the by far most resistant, and protection correlated with the induction of a th1-biased response . The results demonstrate that a meaningful evaluation of the efficacy of, for example, vaccine candidates and/or chemotherapeutically interesting compounds against experimental infection with n. caninum in mice requires standardization with regard to the mouse strains used for such experiments . Female balb / c, cba / ca, and c57bl/6 mice aged 812 weeks were obtained from harlan (horst, the netherlands). Mice were randomly divided into groups as soon as they came to hand and marked individually . The conditions in the animal housing facilities were negative air pressure, air exchange of 300 m / hr, temperature of 22c 2c, and artificial day / night cycle with 12 hrs light per day . Five mice were housed per group in macrolon type iii cages (820 cm) enriched with polyvinyl chloride tubes and tissues . Mice received ad libitum standard rodent feed (rmhb2118, ab - diets woerden, the netherlands). Water was supplied in bottles and refreshed at least three times a week . Prior to challenge, mice were given an acclimatization period of 7 days, during which they were observed daily for normal behaviour, wellness, food and water intake, and posture . N. caninum tachyzoites (nc - liv isolate) were maintained by continuous passages in vero cells grown in rpmi 1640 medium supplemented with 10% foetal bovine serum (fbs), 2 mm l - glutamine and 25 mg / l gentamycin sulphate . Cultures were grown in a humidified incubator at 37c with 5% co2 . To liberate the tachyzoites from the vero cells, the culture was taken up through a 20 g needle and extruded through a 26 g needle . For the preparation of parasites for infection, tachyzoites were purified on a pd10 column (ge healthcare, diegem, belgium) centrifuged and resuspended in sterile 0.04 m isotonic pbs . Tachyzoites were counted in a neubauer chamber . In order to prepare n. caninum tachyzoite antigen lysate, purified tachyzoites were subjected to one freeze - thaw cycle followed by sonication on a branson sonifier s-250a (branson ultrasonics, danbury, usa) for 60 sec, output 4, and a cycle duty of 50% . Mice were challenged by intraperitoneal injection of 1 10, 5 10, or 25 10 live nc - liv tachyzoites in 0.5 ml 0.04 m isotonic pbs . Mice were then closely monitored daily for neosporosis disease symptoms according to a clinical scoring system that included hunched back, rough hair coat, impaired movement, and spinning . For each feature the average clinical score per group was determined by dividing the total score per group by the amount of mice in that group . As soon as the health status of the animals deteriorated, the frequency of observations was increased to two times a day . Blood samples were collected by orbital puncture at day 0 and day 34 postinfection (at the time of euthanasia) or, whenever possible, at the time of inter - current death . Latest at day 34 postinfection (pi), surviving mice were sacrificed by cervical dislocation and brains and spleens were harvested . For each mouse, sections of 3.5 m thickness were cut longitudinally from nervus i to medulla oblongata with cerebrum, hippocampus, and cerebellum on a brand microm h355 s microtome (adamas, rhenen, the netherlands), and were placed onto glass slides . Serum antibody levels were analysed using a commercially available elisa kit based on antigens of n. caninum (idexx neospora ab test, idexx laboratories, westbrook, usa) according to the instructions of the manufacturer . On each plate, sera were tested in 2log titrations, and negative and positive sera were tested in octaploid . Sera of nave swiss outbred mice (charles river, sulzfeld, germany) were used as a negative control . As a positive control for igg1 and igg2a detection, sera of balb / c mice vaccinated with the chimeric antigen r - mic3 - 1 and challenged with n. caninum (nc-1 isolate) sera of c57bl/6 mice taken 34 days after infection with 5 10 nc - liv tachyzoites were used as a positive control for igg2c analysis . Horseradish - peroxidase-(hpr-) conjugated goat antimouse igg1, igg2a, and igg2c were obtained from southern biotechnology (birmingham, usa) and diluted in enzyme immune assay (eia) buffer containing 0.05% polysorbate 80 . Results were analysed on a tecan sunrise device (breda, the netherlands) using xfluor4 software at 650 nm . Antibody titres were determined using caspex software; abendvertical version 0.11 v1 (msd animal health, proprietary software). The cutoff in antibody end titres was defined at bmin2, where bmin is the negative control . Spleens from surviving mice were washed twice in rpmi 1640 medium supplemented with 100.000 iu penicillin / streptavidin, 1 mm sodium - pyruvate, and 2 mm l - glutamine and dissociated using the gentlemacs dissociator (miltenyi biotec, leiden, the netherlands) according to the instructions of the manufacturer . Suspensions were filtered through a 100 m cell strainer and centrifuged at 300 g for 10 min at 4c . Samples were depleted of erythrocytes with erythrocyte lysis solution containing 829 g / l ammonium chloride, 100 g / l sodium hydrogen carbonate, and 3.7 g / l disodium edetate . Splenocytes from mice of the same group were pooled in a 1: 1 ratio and a total of 1 10 splenocytes were cultured for 72 hrs in 100 l complete rpmi medium containing 10% fbs in a humidified incubator at 37c with 5% co2 . 100 l of an antigenic extract of either 2 10, 1 10, or 5 10 nc - liv tachyzoites prepared as described above was added to the cells . Complete rpmi medium and a phorbolmyristateacetate (pma)/ionomycin mixture (9.74 ng / ml/0.26 mm) were used as negative and positive controls, respectively . Cytokine levels in supernatants of stimulated splenocytes were analyzed using the bendermedsystems flowcytomix mouse / rat basic kit combined with the cytokine mouse simplex kits for ifn- (bendermedsystems, vienna, austria). The samples of the cytomix assay were interpolated in the standard curve by selecting the 5p logistic fit function: the bendermedsystems flowcytomix pro 2.4 software fits the best curve according to y = d + ((a d)/(1 + (x / c))). The mean fluorescence intensity (mfi) of each standard point is blank - corrected by division of the blank - mfi (b_b0 = mfi / mfi of blank100). The maximum acceptable bias, which displays the variation for the ideal standard curve defined by the theoretical standard concentrations, was set at 30% . Neospora - specific quantitative real - time pcr to determine the number of tachyzoites that has reached the cerebral tissue was performed as previously described [2, 3, 15]. Dna extraction from brain tissue was performed using the dneasy blood & tissue kit (qiagen, hilden, germany) according to the manufacturer recommendations . The dna concentration in each sample was determined by uv spectrophotometry (nanodrop, thermo scientific, delaware, usa) and was adjusted to 5 ng/l with sterile dnase - free water . Quantitative real - time pcr was performed using the light cycler instrument (roche diagnostic, basel, switzerland). The parasite counts were calculated by interpolation from a standard curve with dna equivalents from 1000, 100, and 10 parasites included in each run . Mice survival was analyzed according to kaplan - meier and the survival curves between groups were compared with the logrank test followed by regression coefficient analysis . The weight of the survivors, cerebral parasite burden, lesion score, and the serological data were compared using kruskal - wallis one - way anova followed by the kruskal - wallis multiple comparison z - value test . The p value between two significantly different groups was calculated by mann - whitney u test . The proportion of animals with clinical signs, proportion of animals with brain lesions and data on n. caninum - dna - positive animals were organized in a contingency table and compared by a chi - square test . Balb / c mice (figures 1(a) and 1(b)) challenged with 1 10 tachyzoites were not affected, with no decrease in body weight following infection . However, challenge with 5 10 nc - liv tachyzoites resulted in neosporosis symptoms in balb / c mice, starting on day 18 pi . Hunched back, ruffled coat, and weight loss of ~25% were observed in these mice . Two mice reached the point where severe neurobiological symptoms made it necessary to get them euthanized on day 23 pi, while the other three mice, although exhibiting clinical symptoms, but less severe, survived throughout the observation period of 34 days (figures 1(a) and 1(b)). Balb / c mice were not able to control the highest infectious dose of 25 10 tachyzoites, resulting in death of all five animals within that group between days 7 and 12 pi . Cba / ca mice (figures 1(c) and 1(d)) exhibited a high degree of resistance against n. caninum at an infection dose of 1 10 and 5 10 tachyzoites, with no mortality and no body weight changes during the entire experimental period (figures 2 and 1(d)). However, none of the cba / ca mice could control the infection at the highest dose and they all were euthanized within 9 days pi (figures 1(c) and 1(d)). In comparison to the other two mouse strains, c57bl/6 mice (figures 1(e) and 1(f)), all mice started to exhibit clinical symptoms including weight loss of ~10% and two out of five mice had to be euthanized at day 29 pi (figure 2). Strong variations were observed in the group of c57bl/6 mice challenged with 5 10 tachyzoites . Three mice of this group demonstrated severe symptoms of neosporosis and were euthanized on days 7, 8 and 13 pi, while the other two mice showed only very mild symptoms on day 8 pi and returned to being clinically normal 9 days after the challenge . Shortly after a challenge with 25 10 tachyzoites, c57bl/6 mice showed a hunched back and ruffled hair coat (figures 1(e) and 1(f)). Symptoms progressed to include impaired movements and spinning when picked up by the tail, requiring euthanasia of four of these mice on day 8 pi . The remaining mouse of this group completely recovered by day 11 pi and survived till the end of the experiment . Statistically, cba / ca mice exhibited a significantly higher survival rate than c57bl/6 mice at an infection dose of 1 10 tachyzoites (p <0.001, cox regression analysis) and than c57bl/6 and balb / c mice at an infection dose of 5 10 tachyzoites (p <0.001, cox regression analysis). The mean weight of the survivors of the balb / c mice infected with 1 10 tachyzoites was slightly higher than those infected with 5 10 parasites, although the difference was not significant (kruskall - wallis multiple comparison test). The number of symptomatic mice in the cba / ca mice at an infection dose of 5 10 tachyzoites was significantly lower than in the two other groups (p <0.01, chi - square test) while no significant differences between strains were observed for the other infection doses (table 2). The number of mice presenting brain lesions as well as the lesion score per group at an infection dose of 5 10 parasites was significantly lower in the cba / ca mice (p <0.01, chi - square test and p <0.001, mann - whitney u - test, resp .) Than in the two other groups (table 2). No significant difference was observed regarding the overall lesion score per strain (table 2). The surviving balb / c mice infected with 1 10 tachyzoites (n = 5) and cba / ca mice infected with 5 10 tachyzoites (n = 5) were challenged again with 25 10 n. caninum tachyzoites at day 34 . However, they all succumbed to infection within 24 hrs, indicating that they had not built up any form of protective immunity . In order to obtain information on the type of immune response induced by n. caninum infection, the production of igg1-type antibodies is primarily induced by a humoral immune response, whereas igg2a subclasses indicate the involvement of a cellular immune response . Since c57bl/6 mice poorly produce igg2a antibodies, the igg2c levels were measured in serum of these mice . In none of the groups n. caninum - specific antibodies experimental infection of balb / c mice with 1 10 or 5 10 tachyzoites resulted in high igg2a and igg1 serum levels, indicating the presence of a mixed cellular and humoral immune response (table 1). In cba / ca mice challenged with 1 10 or 5 10 tachyzoites, predominantly igg2a antibodies were detected . Igg2a and igg1 were undetectable in cba / ca mice challenged with 25 10 tachyzoites, most likely because mice succumbed to the infection before a humoral response was mounted (table 1). In general, sera of mice surviving until day 34 pi contained higher levels of igg2c compared to sera of their group mates that died at earlier time points (table 1). The difference igg2a / c - igg1 postinfection was highly significantly higher in the cba / ca mice than in the two other strains (p <0.01, mann - whitney u - test) and significantly higher in the c57/bl6 mice than in the balb / c mice (p <0.05, mann - whitney u - test) at an infection dose of 1 10 parasites . At an infection dose of 5 10 tachyzoites, both cba / ca and c57/bl6 mice had a significantly higher igg2a / c - igg1 difference than the balb / c mice (p <0.01, and p <0.05 respectively, mann - whitney u - test), and cba / ca mice also had a higher igg2a / c - igg1 difference than c57/bl6, although not significant (table 2 and figure 4). The production of ifn-, indicating the occurrence of a cellular immune response, was measured in the supernatants of spleen cells derived from survivors of balb / c mice (infected with 5 10 tachyzoites), cba / ca mice (infected with 1 10 tachyzoites), and all surviving c57bl/6 mice . Splenocytes were stimulated with either n. caninum antigens or pma / ionomycin for 72 hrs, or were left unstimulated . Stimulation with a lysate corresponding to 2 10, 1 10 or 5 10 purified nc - liv tachyzoites resulted in increased production of ifn-. Ifn- production was a dose - dependent stimulus in all groups (figure 3). The strongest ifn- response was observed for the cba / ca mice, as was especially evident using low and medium tachyzoite stimulus doses . A lower ifn- production was observed for the balb / c mice, while splenocytes from c57bl/6 mice demonstrated large variations in ifn- production upon antigen stimulation . Data from balb / c mice and cba / ca mice infected with 25 10 parasites is missing, since these mice succumbed to infection prior to 34 days postinfection . Spleen cells of balb / c mice infected with 1 10 tachyzoites (n = 5) and cba / ca mice infected with 5 10 tachyzoites (n = 5), which were used for rechallenge, were not assessed . The cerebral parasite load in all groups was measured by real - time pcr and compared with histopathological findings . Corresponding results are summarized in table 2 . In three out of five balb / c mice challenged with 1 10 tachyzoites, no parasite dna could be detected by pcr, while high numbers were detected in the other two samples . However, in only one of these mice a brain lesion was detected by histology . Challenge of balb / c mice with 5 10 tachyzoites resulted in high cerebral parasite load in all mice, and brain lesions were detected upon histopathological inspection in four out of five mice . After infection of balb / c mice with 25 10 tachyzoites, parasites were detected at moderate to high numbers in all brain samples investigated, but histopathology failed to detect any lesions at all . Real - time pcr detected n. caninum dna in one of 10 brain samples of cba / ca mice challenged with 1 10 and 5 10 tachyzoites . In contrast, lesions were observed by histopathological means in two mice infected with 1 10 tachyzoites . However, these were samples that were not positive by real - time pcr . In the cba / ca mice infected with 25 10 tachyzoites, lesions in the central nervous system (cns) were observed by histopathology for three out of five animals, and real - time pcr showed high numbers of parasite load in all 5 members of the group, which all died between days 8 and 9 pi . In three c57bl/6 mice challenged with 1 10 tachyzoite lesions in the brain were observed . In two of them infection with 5 10 tachyzoites resulted in parasites and lesions in three mice on days 13 and 34 pi . In the c57bl/6 mice infected with 25 10 tachyzoites, four out of five mice died 8 days after challenge, with real - time pcr - positive brain tissue but no lesions detectable upon histopathology . One mouse survived until day 34 pi, and this mouse exhibited a high cerebral parasite load and also brain lesions . Thus, a high cerebral parasite load as determined by real - time pcr did not always correspond with the detection of lesions in the brain and did also not always match with the occurrence of clinical signs of neosporosis . At an infection dose of 1 10 and 25 10 tachyzoites, no significant difference in the number of pcr positive mice between the groups was observed (chi - square test). At an infection dose of 5 10 tachyzoites, the number of pcr - positive mice was significantly lower in the cba / ca mice than in the balb / c and c57/bl6 mice (p <0.01 and p <0.05, respectively, chi - square test). No significant difference regarding the parasite load of infected mice was observed between the groups (kruskal - wallis multiple comparison test). In this study, we compared the capacities of three inbred mouse strains expressing different mhc - i molecules to cope with a n. caninum infection . Significant differences in the responses to n. caninum infection in inbred and outbred mice have been demonstrated previously, and these differences have been postulated to be due to varying abilities to present n. caninum antigens on their mhc molecules . When using mouse models to investigate the outcome and immunological parameters of infection, the selection of the n. caninum isolate as well as inoculum doses are important variables that will influence the results . The present study showed high variations in morbidity and mortality in the three inbred mouse strains balb / c, cba / ca, and c57bl/6 after inoculation with different numbers of n. caninum (nc - liv) tachyzoites, demonstrating that the choice of mouse strain plays a crucial role for the assessment of experimental infections and agents that could potentially interfere therein . While balb / c mice did not suffer from clinical signs of neosporosis after a challenge dose of 1 10 n. caninum tachyzoites, infection with 5 10 tachyzoites resulted in severe clinical symptoms starting at day 20 pi . This is in accordance with several vaccination studies, which have used the balb / c and c57bl/6 models to investigate the protective effects of vaccine candidates (reviewed in [2, 3]). In contrast, only limited research has been performed using cba / ca mice as a model to investigate the immune responses against n. caninum infection . Our investigations demonstrated that this mouse strain is clearly more resistant against neosporosis compared to balb / c and c57bl/6 mice . Previously, these features of cba / ca mice with regard to n. caninum infection had been highlighted by rettigner et al ., who described this mouse strain to be resistant against an infection of 5 10 tachyzoites of the nc-1 isolate of n. caninum following treatment with immunosuppressing agents and suitable for the generation of cerebral tissue cysts . This is surprising, since the nc-1 isolate, in contrast to nc - liv, does hardly convert into bradyzoites under in vitro conditions such as high ph treatment or incubation with sodium nitroprusside [19, 20]. With respect to c57bl/6 mice, ramamoorthy et al . Observed no clinical signs of neosporosis throughout an observation period of 21 days after an infection with 5 10 nc-1 tachyzoites but also showed these mice to be highly susceptible to an infection with 2 10 nc-1 tachyzoites . Others failed to observe clinical signs in c57bl/6 mice after a challenge with 5 10 nc-1 tachyzoites as long as 44 days pi . In this study, after a challenge dose of 1 10 nc - liv tachyzoites disease symptoms occurred after 26 days but only after 8 days when 5 10 tachyzoites were inoculated, with three out of five mice succumbing to infection . This illustrates that, besides the mouse strain used, the selection of the neospora isolate also makes a big difference . In any case, an infection dose of 25 10 tachyzoites of the nc - liv isolate was far too high for any of the mouse strains to cope with, requiring euthanasia of all but one mouse between days 712 pi . Since n. caninum is an intracellular parasite, the protective immune response is likely to involve cell - mediated immunity; thus th1-mediated responses are important . However, in balb / c mice it has been demonstrated that besides cellular immunity also humoral immune responses are crucial in controlling n. caninum infection and limiting the pathological changes caused by an excessive proinflammatory response [23, 24]. Teixeira et al . Described that intraperitoneal injection of n. caninum tachyzoites (nc-1 isolate) in balb / c mice induced a parasite - specific, nonpolyclonal, b - cell response . A strong bias towards a th2-type response resulted in increased susceptibility to n. caninum and enhanced the corresponding pathologic effects . However, vaccination of mice with recombinant rhoptry antigen rop2 emulsified in saponin adjuvants induced an igg1-biased humoral immune response, while formulating the same antigen in freund's incomplete adjuvants resulted in an igg2a - biased antibody response, and both were protective in the nonpregnant balb / c mouse model . Protection associated with an igg2a - biased humoral immune response was also demonstrated for recombinant protein disulfide isomerase applied for vaccination intranasally [2729]. Vaccination of mice with native ncsrs2 induced a th2-biased protective response that reduced congenital infection of offspring balb / c mice . Reduction in congenital infection, also associated with a th2-biased immune response, was demonstrated by vaccination experiments in balb / c mice employing combined ncmic1, ncmic3, and ncrop2 antigens . Taken together, it is probably rather a suitable balance in th1/th2-type immune responses than a strict th1 response that allows the host to deal with n. caninum infection . Similar to earlier studies in balb / c mice [25, 26], levels of igg1 antibodies in animals infected with 1 10 and 5 10 tachyzoites were generally high, and it is interesting to note that a challenge dose of 5 10 tachyzoites in balb / c mice resulted in more pronounced igg1 responses compared to the igg1 antibody levels found in mice inoculated with 1 10 tachyzoites, while igg2a titres were basically equal in both groups . In the balb / c mice inoculated with 5 10 tachyzoites, titres for igg1 and igg2a were similar, which is in agreement with previous observations reported by lundn et al . . In addition, we found that splenocytes obtained from balb / c mice inoculated with 5 10 tachyzoites and stimulated with n. caninum antigen produced ifn-, albeit at a lower level compared to cba / ca mice . The results of rettigner et al . On n. caninum infection in cba / ca mice showed no evidence of differential isotype secretion but a high expression of ifn-, while in our experiments sera of infected cba / ca mice exhibited a high igg2a response, and splenocytes of cba / ca mice infected with 1 10 n. caninum tachyzoites also secreted high levels of ifn-. This indicated that the induction of a cellular immune response is a prerequisite for successful dealing with the infection . The importance of the humoral immune response during n. caninum infection was demonstrated by eperon et al . Who showed increased susceptibility to n. caninum (nc-1 isolate) infection in mt b - cell - deficient mice when compared to wild - type c57bl/6 mice . They also described the predominant presence of parasite - specific igg2a isotypes and an absence of igg1 isotypes in c57bl/6 mice at various time points ., we did not observe any igg2a antibody titres in c57bl/6 mice and analysed the igg2c antibody isotype levels instead . Our results showed the major parasite - specific igg isotype to be igg2c . In agreement with a shift from a th1- to a th2-biased response, increasing titres of n. caninum - specific igg1 were detected in mice sera collected at later time points after infection . While n. caninum obviously induces differential humoral and cellular immune responses in different mouse strains during the acute phase of infection, one could imagine that chronic infection would be characterized by the production of tissue cysts . Therefore, in order to mimic the infection in cattle, a mouse model able to form tissue cysts, and also exhibiting recrudescence during pregnancy, is required . However, only few reports have actually been published on the production of n. caninum tissue cysts in mice . Tissue cyst production had been demonstrated in outbred mice and immunosuppressed cba / ca mice inoculated with nc-1 or nc - liv tachyzoites but not all of the mice investigated harboured cerebral tissue cysts and rettigner et al . Showed that immunosuppressed female cba / ca mice given 5 10 nc-1 tachyzoites were able to survive and to consistently develop cerebral tissue cysts . More recently, tissue cyst production, marked by positive staining with an antibody directed against the toxoplasma bradyzoite antigen bag5, has also been demonstrated in an experimentally infected carnivorous marsupial, the fat - tailed dunnart sminthopsis crassicaudata . It is also very likely that n. caninum tissue cysts are not necessarily formed predominantly in the cns but also at other locations such as muscle tissue [34, 35], and this should be taken into account in future studies . In conclusion, we comparatively assessed balb / c, cba / ca, and c57bl/6 mice as models for n. caninum infection and demonstrated that cba / ca mice exhibited the highest degree of resistance at low and medium infection doses but rapidly developed acute signs of neosporosis when challenged with the highest dose of n. caninum nc - liv tachyzoites . This mice strain has previously been shown to be suitable for tissue cyst production, which renders them an interesting model for investigations on protective effects and potential tools for vaccination . In contrast, the use of balb / c mice, although extensively employed, is debatable due to their inherent th2 bias, resulting in increased susceptibility to n. caninum . Thus, there is clearly a need for a standardisation of experimental murine infection models for n. caninum in order to achieve comparable results between research groups that work with different tools for immunoprevention and chemotherapy.
The 16s r rna gene sequences were deposited in genbank under the following accession numbers: bacillus dakarensis marseille - p3515 (lt671589), bacillus sinesaloumensis marseille - p3516 (lt671591), halobacillus massiliensis marseille - p3554 (lt671593), lentibacillus massiliensis marseille - p3089 (lt598549), oceanobacillus senegalensis marseille - p3587 (lt714172) oceanobacillus timonensis marseille - p3532 (lt671597), virgibacillus dakarensis marseille - p3469 (lt631544) and virgibacillus marseillensis marseille - p3610 (lt714170). The strains were deposited in the collection de souches de lunit des rickettsies (csur, wdcm 875) under numbers bacillus dakarensis marseille - p3515 (p3515), bacillus sinesaloumensis marseille - p3516 (p3516), gracilibacillus timonensis marseille - p2481 (p2248), halobacillus massiliensis p3554 (p3354), lentibacillus massiliensis marseille - p3089 (p3089), virgibacillus dakarensis marseille - p3469 (p3469) and virgibacillus marseillensis marseille - p3610 (p3610).
Cardiovascular disease is the predominant cause for mortality in patients with type-2 diabetes mellitus, accounting for 5060% of deaths . Metabolic syndrome (mets) refers to a conglomeration of cardiovascular risk factors that identifies a cohort of individuals with increased risk of cardiovascular morbidity . Asian indians are ethnically predisposed to increase cardiovascular morbidity . Increased adiposity (overall and especially visceral) in spite of lower body mass index (bmi), as compared to other ethnic groups (like caucasians) may contribute to this increased risk . Bmi, waist circumference (wc), waist - hip ratio, and waist - height ratio (whtr) are simple anthropometric indices to measure this generalized and central adiposity . Specific challenges associated with diabetes in india, the single most important contributor to cardiovascular morbidity and mortality, is the high prevalence of diabetes (9%) and also prediabetes (1214%), young age of disease onset, with significantly higher rates of disease progression (prediabetes to diabetes conversion rate in india, china, finland, and the usa being (1418%, 11%, 6%, and 2.5%, respectively). Hence, there is an urgent need of easy to measure anthropometric marker, specific to the indian population, which is a good marker for early diagnosis of this high - risk cohort of individuals, who are predisposed to mets, diabetes, and cardiovascular morbidity . Few studies have suggested that subcutaneous adipose tissue in the upper body distribution (upper body obesity), to have an independent role in predicting cardiovascular morbidity . Neck circumference (nc) has been validated to be a simple measure of upper body subcutaneous fat deposition and being a predictor of cardiovascular risk factors . Data on the evaluation of nc as a predictor of cardiovascular risk factors in scant from india . Furthermore, the predictive potential of nc based indices for predicting mets and its components, as compared to the traditional anthropometric indices (bmi, wc, and whtr) among indians are not known . Neck height ratio (nhtr) has also been suggested to be a measure of upper body adiposity like nc . Nhtr has the advantage over nc, as it adjusts for the difference in nc attributable to differences in heights . Nhtr has not been previously evaluated as an index for predicting mets and other cardiovascular risk factors . Hence, the aim of this study was to evaluate the role of nc and nhtr as independent predictors of mets and its components among asian indians as compared to traditional anthropometric indices (bmi, wc, and whtr). Individual attending the biannual health camps conducted by the department of endocrinology and metabolism across the city of calcutta were considered . Screening of apparently healthy individuals that included family members of patients with diabetes was done during these camps by either fasting blood glucose (fbg) if they came after an overnight fast, or a non - fbg using a glucometer (accu - chek active; roche, mumbai, india). Individuals, 3080 years age, without any co - morbid states, were considered for the study . Patients not metabolically stable with uncontrolled blood glucose values on glucometer screening, viz . Fasting / random blood glucose> 300 mg / dl were excluded . For those patients having blood glucose values in the diabetes range, only those who were recently diagnosed with diabetes (within last 6 months) patients on medications that can interfere with body composition and lipids such as anti - depressants, glucocorticoids, and anti - lipid medications were excluded . Pregnant women were not considered for the study . The study protocol was explained, and only those who gave informed written consent included individuals attended the camp again the next day after 12 h fast, underwent anthropometric assessment, blood samples collected, serum separated which was transported to the department in cold chain and stored at 80c . Height (to 0.1 cm) was measured in all individuals using a charder hm200pw wall - mounted stadiometer (calibrated using a 36 calibration rod [perspective enterprise, portage, michigan, usa]), and body weight (to 100 g) measured using an electronic calibrated scale (tantia, japan, model - ha521, lot number-860525). Bmi was calculated as weight in kilograms divided by the square of height in meters (kg / m). Nc was measured using a calibrated plastic tape, with the head positioned along the frankfurt plane, at mid - neck height, between the mid - cervical spine and mid - anterior neck, to within 1 mm . In men with a laryngeal prominence, wc was measured at the end of a gentle expiration midway between the lower rib margin and iliac crest with the patient standing with feet 2330 cm apart . The collected blood samples were used for estimation of fbg, glycosylated hemoglobin (hba1c%) and fasting lipid profile using clinical chemistry analyzer (daytona, serial number-58260536, furuno electric, nishnomeya, japan). The presence of mets was ascertained using the modified national cholesterol education program adult treatment panel (ncep atp) iii criteria (ethnic - specific cutoffs for wc viz .> 90 cm in males and> 80 cm in females) with the presence of three or more considered diagnostic . The prevalence of mets in india has been reported to be 31.6% . Keeping a power of 80% and type - i error at 5%, it was calculated that we needed to evaluate at least 235 individuals with mets in our study . Anova with post hoc analysis and kruskal wallis nonparametric anova with dunn's postcorrection were performed for normally and nonnormally distributed variables, respectively . Pearson's or spearman's correlation coefficient was calculated for normally and nonnormally distributed variables, respectively . For categorical data, frequencies, and percentages the associations between metabolic risk factors and anthropometric parameters were assessed using partial correlation analysis . Receiver operating characteristic (roc) analyses were performed to assess the accuracy of the anthropometric parameters as diagnostic tests for detecting mets and determine optimal sex - specific nc cut - offs in relation to mets . The youden index, defined as (sensitivity + specificity)-1 was used to determine the optimal cut - off points . Spss version 16 (chicago, il, usa) was used for statistical analysis . Anova with post hoc analysis and kruskal wallis nonparametric anova with dunn's postcorrection were performed for normally and nonnormally distributed variables, respectively . Pearson's or spearman's correlation coefficient was calculated for normally and nonnormally distributed variables, respectively . For categorical data, frequencies, and percentages the associations between metabolic risk factors and anthropometric parameters were assessed using partial correlation analysis . Receiver operating characteristic (roc) analyses were performed to assess the accuracy of the anthropometric parameters as diagnostic tests for detecting mets and determine optimal sex - specific nc cut - offs in relation to mets . The youden index, defined as (sensitivity + specificity)-1 was used to determine the optimal cut - off points . Spss version 16 (chicago, il, usa) was used for statistical analysis . A total of 867 individuals were screened of which 451 individual who fulfilled all inclusion and exclusion criteria, gave informed consent, and came for detailed anthropometric and biochemical evaluation were included in the study and analyzed [figure 1]. In our study, 228, 55 and 168 individuals were classified as having normoglycemia, prediabetes, and diabetes respectively as per fbg and hba1c values . Males had significantly lower bmi, but higher nc as compared to females [table 1]. The occurrence of hypertension, dysglycemia (prediabetes or newly diagnosed diabetes) was significantly higher in males [table 1]. Total cholesterol and low - density lipoprotein cholesterol (ldl - c) were significantly higher in females [table 1]. Patients with mets in both the sexes had significantly higher nc, nhtr, hba1c, fbg, and dyslipidemia (higher triglycerides, total cholesterol / high - density lipoprotein cholesterol (hdl - c) ratio, ldl - c / hdl - c ratio, and lower hdl - c), as compared to those without mets [table 1]. Ckd: chronic kidney disease; cld: chronic liver disease; cva: cerebrovascular accident; tia: transient ischemic attack; cad: coronary artery disease baseline characteristics of the study population with regards to sex distribution and occurrence of metabolic syndrome (n=451) males and females were divided into subgroups based on nc tertile [table 2]. In both the sexes, individuals in the highest tertile of nc had significantly greater central obesity, significantly more likely to have bmi in the overweight and obese range, lower hdl - c and significantly higher occurrence of mets [table 2]. Males, but not females in the highest tertile of nc had significantly higher occurrence of hypertriglyceridemia [table 2]. Nc had a strong significantly positive correlation with other evaluated anthropometric parameters (wc, bmi, and nhtr) in both the sexes after adjusting for age [table 3]. Nc had significant positive correlation with serum total cholesterol and triglycerides in males [table 4]. A significant inverse correlation was observed between nc and hdl - c in both the sexes [table 4]. Wc, bmi, and nhtr had significant positive correlation with total cholesterol, triglycerides, and ldl - c in males [table 4]. Anthropometric and biochemical characteristics of the study subjects as per the distribution of neck circumference relation between anthropometric indices after adjusting for age in males and females correlation between anthropometric indices and cardio - metabolic risk factors after adjusting for age the areas under the roc curves (area under the curves [aucs]) were constructed to evaluate the predictive values of anthropometric indices for mets and its components [table 5]. The auc for nc for predicting mets in males and females was 0.753 and 0.768 respectively, which was statistically significant [table 5]. This auc for nc was lower than that for wc but higher than that for bmi with regards to predicting mets in both the sexes [table 5]. Nhtr had a higher auc than nc for predicting mets in males (0.77 and 0.753, respectively) [table 5]. Among all the 4 anthropometric parameters evaluated, nhtr had the highest auc with regards to predicting mets, hypertension, hypertriglyceridemia, and low hdl - c in males [table 5]. In females, wc had the highest auc with regards to predicting mets and hypertension, whereas nc had the highest auc with regards to predicting low hdl - c . Area under the roc by different anthropometric indices as predictor of metabolic syndrome and cardio - metabolic risk factors (n=451) a nc of> 34.9 cm (sensitivity 78.6%; specificity 59.3%) for men and> 31.25 cm (sensitivity 72.3%; specificity 64.4%) for women were the best values of combined sensitivity and specificity in identifying mets . A logistic regression analysis, using mets as the dependent variable, showed that the relationship between nc and mets after adjusting for sex and age was statistically significant (odds ratio 1.52 [95% confidence interval [ci]: 1.371.68]; p <0.001). Similarly a nhtr of> 21.17 cm / m (sensitivity 80.7% and specificity 64.6%) for men and> 20.48 cm / m (sensitivity 80.4% and specificity 60%) for women were the best values of combined sensitivity and specificity in identifying mets . A logistic regression analysis, using mets as the dependent variable, showed that the relationship between nhtr and mets after adjusting for sex and age was statistically significant (odds ratio 1.96 [95% ci: 1.672.29]; p <0.001). Twenty - four out of 258 males (9.3%) and 8 out of 148 females (5.4%) with mets had normal wc . Ten (41.66%) and 18 (75%) of these 24 males had nc> 24.9 cm and nhtr> 21.17 cm / m, respectively . Three (37.5%) and 4 (50%) of these 8 females had nc> 31.25 cm and nhtr> 20.48 cm / m, respectively . Wc a measure of abdominal subcutaneous and visceral fat traditionally has served as the standard index to identify patients with mets . However, it has been observed that a significant proportion of patients with cardiovascular disease with normal wc, thereby highlighting its shortcomings . Studies have suggested that upper body subcutaneous fat is responsible for a much larger proportion of systemic free fatty acid release than visceral fat, as it is lipolytically more active than lower body adipose tissue . Hence, this fat may have a significant contribution to genesis of insulin resistance and dyslipidemia . Nc has been demonstrated to be an index of upper body subcutaneous fat, with correlation with indices of obesity and individual cardiovascular risk factors . In a longitudinal follow - up study, it was shown that the change is nc correlated with changes in cardiovascular risk factors and that using wc alone was a simplification and did not account for all the changes in risk factors . Followed up participants from the framingham heart study over a period of 10 years prospectively and noted that nc correlated with development of multiple cardiovascular risk factors . The same group also estimated visceral adiposity by computed tomography along with anthropometry and metabolic parameters and demonstrated that nc was associated with cvd risk factors even after adjustment for vat and bmi, thus, suggesting that upper body subcutaneous fat may be a unique pathogenic fat depot . In our study, patients in the highest tertile of nc had significantly higher occurrence of mets, central obesity, and dyslipidemia parameters that are established predictors of cardiovascular morbidity . Nc had a strong positive correlation with traditional anthropometric indices of central obesity (bmi and wc), which are also established predictors of cardiovascular risk . Nc had significant positive correlation with total cholesterol, triglycerides, and significant negative correlation with hdl - c in males ., wc had the largest auc for predicting mets in both males and females, followed by nhtr, nc, and bmi . Nhtr had the largest auc for predicting hypertriglyceridemia and low hdl - c in males . Nc, in contrast, had the largest auc for predicting low hdl - c in females . Our study demonstrated that among south asians, an nc of> 34.9 cm for men and> 31.25 cm for women were the best predictors for identifying mets . These cut - offs are lower than that previously reported in literature from other parts of the globe . Suggested cut - offs of 39 cm and 35 cm for diagnosing mets among turkish population . The same cut - off was proposed by yang et al . For diagnosing mets in the chinese population . Zhou et al . In another study from a different part of china suggested lower cut - offs for 37 cm and 33 cm for identifying mets in males and females, respectively . The lower cut - offs observed in our study in part can be explained by the lower median nc among our study subjects . Different ethnicity and body composition of different populations evaluated in different studies may explain this difference . Our study proposed for the 1 time cut - offs for nhtr in predicting mets . A nhtr of> 21.17 cm / m for men and> 20.48 cm / m for women were the best predictors for identifying mets . Overall nhtr had a better odds ratio for predicting mets as compared to nc . Among patients of mets with normal wc, 75% and 50% of males and females respectively had nhtr above the suggested cut - offs, highlighting that nhtr may be a better predictor of mets . Limitations of this study include the cross - sectional design, which prevents us from establishing causality . Nc was used as surrogate for upper body subcutaneous tissue, which however was not quantified by an imaging modality in our study . The higher prevalence of mets observed in our study, as compared to previously reported literature may in part be explained by the opportunistic screening done during patient recruitment . To summarize it may be said that our study reinforced the previous observation of nc being a good predictor of mets and other related cardiovascular risk factors in asian indians . Our study demonstrated for the 1 time the reliability of nhtr as a predictor of mets and its components and highlighted that nhtr is perhaps an even better index than nc with regards to cardiovascular risk prediction . This study was funded by a research grant from research society of study of diabetes in india, west bengal chapter . This study was funded by a research grant from research society of study of diabetes in india, west bengal chapter.
Pityriasis rosea is considered to be a benign cutaneous condition [14]. However, in the setting of pregnancy, adverse effects on the newborn may be observed . Craniosynostosis is a congenital abnormality, which can occur as an isolated finding or as part of a syndrome with other associated features . A woman who developed pityriasis rosea during her first trimester of pregnancy and who subsequently delivered a healthy baby with craniosynostosis is described, and observations of infants born to women who are diagnosed with pityriasis rosea during their gestation are summarized . A 28-year - old healthy woman presented at 10 weeks gestation with a skin rash . Two weeks earlier, at eight weeks gestation, she had noticed an initial skin lesion on her abdomen (figure 1). Her husband, a 29-year - old man, had presented two months earlier with similar - appearing annular lesions on his neck (figure 2). A larger lesion had initially appeared on his left neck . Within the next five days, additional lesions appeared on the remainder of his neck and subsequently, a few lesions appeared on his distal upper extremities . A diagnosis of inverse pityriasis rosea was established based on the clinical history and lesion morphology . His lesions resolved over the next two weeks after treatment with triamcinolone 0.1% cream twice daily . Cutaneous examination of the patient s lesions showed multiple annular plaques with peripheral scaling on the abdomen and back (figure 1). A diagnosis of pityriasis rosea was established based on the appearance of the lesions and history . Cetaphil cream was applied twice daily to the patient s lesions; the lesions resolved during the next eight weeks . She was classified as a high - risk pregnancy and was followed closely for the remainder of her gestation . After 15 hours of labor, the baby had not descended into the pelvis; there was no fetal distress, and a decision for c - section was made . His apgar scores at one and five minutes were 9/9, weight 3827.1 g, and height 50.8 cm . Prior to discharge from the hospital, it was noted that the infant had a curved head . Neurosurgery consultation confirmed the diagnosis; at 9 weeks postpartum, endoscopic repair was performed successfully with no adverse sequelae . Pityriasis rosea classically presents as annular plaques with peripheral scale, typically located between the neck and the groin, and may be seasonal in occurrence . Less commonly, it can present with lesions on the neck and extremities (inverse pityriasis rosea) [1011] or during pregnancy . However, associations with human herpes virus (hsv)-6 and hsv-7 have been observed [1418]. Several studies have found that patients with pityriasis rosea have higher levels of hsv-6 and hsv-7 detected in their skin, suggesting that infection by these viruses may have a causal effect on the development of pityriasis rosea . Occasionally, pityriasis rosea has been documented in siblings or in spouses (table 1) [1921]. In these circumstances, our patient s husband developed and cleared inverse pityriasis rosea two months prior to his wife developing classic pityriasis rosea . Similar to our patient, in the majority of cases of pityriasis rosea occurring in couples, the lesions appeared in the husband prior to the wife (table 1). The interval between onset of pityriasis rosea in the wife after occurrence in the husband ranged from seven days to one year (median: 2 months). Our review of the literature, including the patient in this report, discovered 54 women who developed pityriasis rosea during their pregnancy (table 2 and table 3 [6,1214,23,24]). The onset of pityriasis rosea ranged from week 8 of gestation (3 patients: cases 7 and 8 in table 2 and case 14 in table 3) to week 32 (1 patient: case 7 in table 3). The median number of weeks of pregnancy at the onset of pityriasis rosea was 19 . Twenty - five women ages 24 to 34 (median age 29)had no prior pregnancies . However, pityriasis rosea occurred during either the second (20 women) or the third pregnancy (6 women) for the other women . Most of the women (66%, n=35) developed pityriasis rosea during the second trimester of gestation (1328 weeks). Nineteen percent (10 women) had the onset of their dermatosis during the first trimester (012 weeks). Only 10% (5 women) experienced it in the third trimester (2940 weeks). Several retrospective studies have observed adverse events affecting the newborn in women who develop pityriasis rosea during pregnancy (table 2). In these individuals, the adverse events predominantly included stillbirth at 11 to 28 weeks (median 16 weeks), premature delivery (<37 weeks), hypotonia, weak motion, and low birth weight . Less common adverse effects were hydramnios and foramen ovale . Some investigators have discovered that pityriasis rosea occurring earlier in pregnancy, such as in the first trimester, have been more often associated with a poorer prognosis, compared to women who developed the dermatosis during the second or third trimesters . However, our review of the literature showed that the majority of women (16/25, 64%) who experienced adverse events had the onset of pityriasis rosea that occurred during the second trimester . The onset of pityriasis rosea occurred during the first trimester in 9 women (36%) and none in the third trimester . Additional studies looking at the association between hsv-6 and hsv-7 dna and the occurrence of pityriasis rosea in pregnancy have also been performed . Some of the studies found reactivation of hsv-6 during pregnancy . However, a positive correlation between viral infection and clinical features of pityriasis rosea was not established . Individual case reports, including the patient in this report, have described 29 women who developed pityriasis rosea during pregnancy and have delivered healthy newborns (table 3) [6,1214,23,24]. Indeed, the literature shows a ratio of 6:5 with regards to healthy newborns versus newborns with adverse events being delivered to women with gestational pityriasis rosea . However, the number of publications regarding gestational pityriasis rosea on the outcome of the newborn may not accurately reflect the incidence of normal newborns whose mothers had gestational pityriasis rosea, since clinicians may not report these women or journals may elect not to publish the papers . It can occur as an isolated incidental event or as part of syndrome (table 4). As an isolated incidental finding, sagittal craniosynostosis, as observed in our patient s newborn, is the most common form . If left unrepaired, craniosynostosis may lead to a deformed skull, elevation of intracranial pressure, and cognitive impairment . Our patient s infant was evaluated shortly after delivery and had repair of the sagittal craniosynostosis at 9 weeks, with no complications and subsequent normal development . The incidence of sagittal craniosynostosis is about 1 in 5,000 live births . To the best of our knowledge, isolated craniosynostosis has not been observed in newborns of women who developed pityriasis rosea during their gestation . Indeed, the occurrence in our patient s child may merely be a coincidence and not associated with her episode of pityriasis rosea during her first trimester . However, the true incidence is not known, since gestational pityriasis rosea is not frequently reported . Some researchers noted that pityriasis rosea occurring earlier in pregnancy had a greater probability of resulting in adverse events for the fetus, including stillbirth, low gestational weight, hypotonia, and/or premature delivery . However, there are a similar number of reports of women who developed pityriasis rosea during their gestation and delivered normal newborns . Our patient developed pityriasis rosea during her first trimester beginning at 8 weeks gestation and lasting through 18 weeks . Her son was carried to term and delivered at 40 weeks and 6 days with apgar scores, weight, and height in normal range . Whether the presence of craniosynostosis was associated with our patient s development of pityriasis rosea during her pregnancy remains to be determined.
Bile cast syndrome (bcs) is a complication of orthotopic liver transplantation (olt). We describe 2 patients with bcs who were successfully diagnosed with endoscopic retrograde cholangio pancreatography (ercp) and one of whom was successfully treated endoscopically . A 65-year - old female underwent olt 6 months prior to presentation for chronic hepatitis c and hepatocellular carcinoma . Her intraoperative course during liver transplantation was complicated by a significant amount of blood loss from hepatic veins . She had a complicated postoperative course including hemorrhagic shock, abdominal compartment syndrome and acute renal failure which required hospitalization for 2 months after the transplantation . She developed an anastomotic bile leak which was treated with placement of a 10 fr stent . At 4 weeks and 8 weeks after initial ercp, the cbd stents were exchanged and secondary sclerosing cholangitis was suspected based on cholangiographic appearance . Her immunosuppression regimen included tacrolimus 9 mg bid and mycofenolic acid 180 mg bid . Laboratory data showed bilirubin of 1.1 mg / dl (reference range: 0.2 mg / dl-1.3 mg / dl), alanine aminotransferase of 53 iu / l (reference range: 13 iu / l-69 iu / l), aspartate aminotransferase of 60 iu / l (reference range: 15 iu / l-46 iu / l), alkaline phosphatase of 257 iu / l (reference range: 42 iu / l-140 iu / l), -glutamyl transferase of 466 iu / l (reference range: 5 iu / l-55 balloon sweeps yielded a small amount of debris, but cholangiographic findings persisted that were concerning for filling defects or marked ductal irregularity in the region of the common hepatic duct and bifurcation . Cholangioscopy (fig . 2) was performed with the spyglass single operator biliary visualization system (boston scientific, natick, ma, usa). A tubular biliary cast, in the shape of the bile duct, was seen in the donor bile duct extending from the anastomosis into the intrahepatic ducts . Attempts to extract the cast with a snare, basket, mechanical lithotripter, balloon, spybite(boston scientific, natick, ma, usa) biopsy forceps, and standard egd forceps were all unsuccessful due to inability to grasp the cast and tethering of the cast to intrahepatic extension . A 10 fr 12-cm cbd stent was placed . In subsequent months, it appears to contain filling defects which retrospectively are related to presence of biliary cast material in the right and left main hepatic ducts . A 58-year - old male with a history of hepatitis c and cirrhosis presented with fevers, chills, and a headache . The patient s immunosuppression regimen included mycophenolate 360 mg bid, prednisone 5 mg daily and tacrolimus 8 mg bid . Laboratory data showed bilirubin of 2.2 mg / dl (reference range: 0.2 mg / dl-1.3 mg / dl), alanine aminotransferase of 162 iu / l (reference range: 13 iu / l-69 iu / l), aspartate aminotransferase of 159 iu / l (reference range: 15 iu / l-46 iu / l), alkaline phosphatase of 1277 iu / l (reference range: 42 iu / l-140 iu / l) and white blood cell count of 8.17 10 cells / l . The left main hepatic duct was dilated with a 4-mm dilating balloon to facilitate clearance of the filling defects . Cholangiogram prior to intervention shows diffuse stricturing of the left and right hepatic ductal system in patient 2 . B cholangiogram after removal of the biliary cast showing a widely patent left hepatic duct in patient 2 . Subsequent to removal of the biliary cast, endoscopic treatment for the anastomotic stricture resulted in resolution of the stricture . A 65-year - old female underwent olt 6 months prior to presentation for chronic hepatitis c and hepatocellular carcinoma . Her intraoperative course during liver transplantation was complicated by a significant amount of blood loss from hepatic veins . She had a complicated postoperative course including hemorrhagic shock, abdominal compartment syndrome and acute renal failure which required hospitalization for 2 months after the transplantation . She developed an anastomotic bile leak which was treated with placement of a 10 fr stent . At 4 weeks and 8 weeks after initial ercp, the cbd stents were exchanged and secondary sclerosing cholangitis was suspected based on cholangiographic appearance . Her immunosuppression regimen included tacrolimus 9 mg bid and mycofenolic acid 180 mg bid . Laboratory data showed bilirubin of 1.1 mg / dl (reference range: 0.2 mg / dl-1.3 mg / dl), alanine aminotransferase of 53 iu / l (reference range: 13 iu / l-69 iu / l), aspartate aminotransferase of 60 iu / l (reference range: 15 iu / l-46 iu / l), alkaline phosphatase of 257 iu / l (reference range: 42 iu / l-140 iu / l), -glutamyl transferase of 466 iu / l (reference range: 5 iu / l-55 balloon sweeps yielded a small amount of debris, but cholangiographic findings persisted that were concerning for filling defects or marked ductal irregularity in the region of the common hepatic duct and bifurcation . Cholangioscopy (fig . 2) was performed with the spyglass single operator biliary visualization system (boston scientific, natick, ma, usa). A tubular biliary cast, in the shape of the bile duct, was seen in the donor bile duct extending from the anastomosis into the intrahepatic ducts . Attempts to extract the cast with a snare, basket, mechanical lithotripter, balloon, spybite(boston scientific, natick, ma, usa) biopsy forceps, and standard egd forceps were all unsuccessful due to inability to grasp the cast and tethering of the cast to intrahepatic extension . A 10 fr 12-cm cbd stent was placed . In subsequent months, it appears to contain filling defects which retrospectively are related to presence of biliary cast material in the right and left main hepatic ducts . A 58-year - old male with a history of hepatitis c and cirrhosis presented with fevers, chills, and a headache . He underwent deceased donor liver and kidney transplantation two months prior to his presentation . The patient s immunosuppression regimen included mycophenolate 360 mg bid, prednisone 5 mg daily and tacrolimus 8 mg bid . Laboratory data showed bilirubin of 2.2 mg / dl (reference range: 0.2 mg / dl-1.3 mg / dl), alanine aminotransferase of 162 iu / l (reference range: 13 iu / l-69 iu / l), aspartate aminotransferase of 159 iu / l (reference range: 15 iu / l-46 iu / l), alkaline phosphatase of 1277 iu / l (reference range: 42 iu / l-140 iu / l) and white blood cell count of 8.17 10 cells / l . The left main hepatic duct was dilated with a 4-mm dilating balloon to facilitate clearance of the filling defects . 4). Following extraction, cholangiograms revealed a widely patent left main duct (fig . Cholangiogram prior to intervention shows diffuse stricturing of the left and right hepatic ductal system in patient 2 . B cholangiogram after removal of the biliary cast showing a widely patent left hepatic duct in patient 2 . Subsequent to removal of the biliary cast, endoscopic treatment for the anastomotic stricture resulted in resolution of the stricture . Bile casts occur in 4% to 18% of olt recipients 1 . More than 70% of casts are identified in the first 16 weeks after transplant 1 . Bile casts are dark and hard in appearance and formed by lithogenic material confined to the bile duct dimensions 3 4 . Although the exact mechanism of injury is unknown, ischemic injury to the biliary epithelium is most common 5 6 7 . Other mechanisms including anastomotic and non - anastomotic strictures leading to accumulation of biliary sludge can lead to formation of casts 5 . Bacterial infection and stones are also predisposing factors for biliary ductal injury and cast formation 6 . Microcirculatory dysfunction is also suggested, given the higher prevalence of renal failure and need for renal replacement therapy in patients with bcs 7 . A combination of endoscopic interventions is considered including sphincterotomy, balloon and basket extraction, lithotripsy and stent placement . In one study, re - transplantation and 12-month mortality rates were significantly higher with bcs (30% vs. 7%) and bcs patients also required a longer intensive care unit stay post - olt 7 . Success rates with endoscopic therapy for bcs have been reported to be 70% recently 7 . Previously in a large study of post - olt adverse events, biliary cast syndrome occurred in 4/260 patients and a median of 3.5 ercp treatments attempted over a median of 21 weeks achieved a success rate of 25% 8 . Percutaneous drainage has also been advocated, especially in the setting of cholangitis or with roux - en - y choledochojejunostomy . Percutaneous transhepatic choledocoscopy via a tract created with radiographic assistance is a novel approach used for management of bcs . When there is intrahepatic biliary system involvement, management becomes even more challenging . Retransplantation is considered when complete removal is not feasible and if the patient s clinical condition deteriorates 2 7 . A combined endoscopic and percutaneous method was able to successfully clear the casts in 60% of patients in one study 9 . Surgical intervention including hepaticojejunostomy, choledochojejunostomy and revision of choledochocholedocostomy reportedly has had success rates above 85% 10.
A 27-year - old malay male patient presented two weeks after alkali splashed into his left eye while working . Prior to reporting to us, the patient consulted a general practitioner who prescribed some topical eye drops (details of the treatment were not available) and the eye condition worsened . Anterior segment examination revealed a central corneal ulcer (1 1 mm) with stromal infiltration (4 6 mm), limbal ischemia, normal anterior chamber depth and visual acuity of counting fingers . A gentle b - scan ruled out any posterior segment abnormality . A clinical diagnosis of left corneal ulcer with limbal ischemia secondary to alkali injury with secondary infection was made . Culture from corneal scraping was negative . Despite intensive therapy with oral (tab ciprobay 500 mg twice daily, bayer) and topical ciprofloxacin (gutt ciloxan 0.3% hourly, alcon) (no topical steroids used) with other supportive therapy (tab vitamin c 1 g daily, pharmaniaga), further deterioration led to formation of central corneal perforation with surrounding stromal thinning and flat anterior chamber . We performed a gunderson conjunctival flap under local anesthesia (la), which retracted and failed after one week . Slit - lamp examination revealed a large central corneal perforation (4 5 mm) with surrounding stromal thinning involving two- thirds of the cornea without any signs of infection . In order to maintain the shape of the eyeball and due to the problem of non - availability of donor cornea, we decided to use processed bp xenograft (lyolemb) supplied by national tissue bank, universiti sains malaysia after obtaining the consent of the patient . A 360 peritomy was done and the graft was sutured with 7 - 0 polyglycolic and polylactic acids (vicryl) to the perilimbal area with conjunctival advancement . Topical (gutt ciloxan 0.3% hourly, alcon) and oral ciprofloxacin (tab ciprobay 500 mg twice daily, bayer) was prescribed in the postoperative period [figure 1]. Nine months follow - up showed a well - taken graft without any exposure / dehiscence and minimal inflammation [figure 2]. Slit - lamp examination could not reveal the details of the anterior chamber and beyond due to conjunctival advancement and vision was noted as perception of light with inadequate projection of rays . Conjunctival flap is unlikely to seal large corneal perforations associated with corneal melting following chemical injury . Efficacy of amt alone or with glue in treating small corneal perforation has been well established but found unable to promote corneal stability in patients with severe corneal thinning and therefore, an additional urgent tectonic procedure has to be undertaken.3 non - availability of donor cornea poses a considerable problem in some countries . Processed bp is a tough lyophilised collagen sheet used as a surgical armamentarium in wrapping orbital implants.4,5 it renders the material antigenically inert and adds to its strength, pliability and longevity.5 it has been shown to be well tolerated with minimal inflammation in animal studies.4 although theoretic concerns about prion disease can be raised, no evidence of this problem has been reported from over 90,000 human implantations.6 devron reported no significant ophthalmic reactions due to this xenograft in his cases of orbital implants.7 at the national tissue bank, universiti sains malaysia, the procured pericardium sheet was washed using chemical agent (sterile phosphate buffer at ph 7.4) and frozen to 40c . It was then freeze - dried and packaged in double - layer plastic bag . The processed bp was radio - sterilized at 2.5 mega rads using gamma radiation and was then ready for use.8 we used this locally processed bp in our case . Its use in complicated ocular surface diseases such as large corneal perforation with corneal melting can be promising . Its use needs to be further studied more extensively as an alternative graft material in large corneal perforations.
The desynchronization of central and peripheral circadian systems contributes to the decline in optimal functioning of bodily systems . This includes changes in neuroendocrine circadian rhythms, insulin sensitivity, altered thermoregulation and acceleration of tumor growth (cincotta et al ., 1993; touitou and haus, 2000; hastings et al ., 2003; straub and mocchegiani, 2004; cretenet et al ., 2010; heller et al ., 2011 these are complex interactions and dysfunction can be due to targeted disruption of neurons, neurotransmitter or neuropeptide production, transport or secretion . It is reasonable to expect that the neuronal activity or expression of circadian clock genes be reduced or rhythms phase shifted (kolker et al ., 2003). Additionally, the connections between central and peripheral oscillators may be degraded or less functional (morales et al ., 1987). Any one or a combination of these abnormalities may result in the decoupling of the circadian oscillators and the ensuing pathologies . The master central pacemaker is the suprachiasmatic nucleus (scn) which controls circadian rhythmicity . Rhythmicity can dampen and/or elongate / shift with age but the number or cell size of neurons in the scn does not change ., 1993; madeira et al ., 1995). Additionally, glucose uptake decreases in the scn in aged animals (wise et al ., 1988) and the expression of neuropeptides also diminishes . Vasoactive intestinal polypeptide (vip) expressing neurons of the scn are retinorecipient and vasopressin (avp) expressing neurons of the scn modulate rhythmicity (wu et al ., 2007). In aged male humans, the number of scn neurons that express vip decreases and in aged female rats the expression becomes arrhythmic (zhou et al . Both genders also express less avp protein, less mrna (roozendaal et al ., 1987; zhou et al ., 1995) and the normal daytime peak of avp is reduced (hofman and swaab, 1994; liu et al ., 2000). Decreases in the neuroendocrine output of the scn may directly or indirectly affect the coupling of central and peripheral oscillators . In the scn, clock genes constitute the core clock mechanism of the mammalian timekeeping system . Though the system is fairly complex, for brevity the simplest model is described . Bmal1 and clock proteins dimerize and induce the transcription of period (per) and cryptochrome (cry) genes . In a negative feedback loop, levels of per and cry proteins increase and at a certain threshold form heterodimers which turn off the clock bmal1 regulated transcription of per and cry genes . This process takes roughly 24 h (ko and takahashi, 2006). In aged animals the expression of certain clock genes changes in the scn . Per1, per2, and cry1 expression does not change significantly with age, but the normal photic stimulation of per1 expression is reduced (asai et al . . Additionally, the free - running period of per1luc rhythmicity is shortened in aged animals (yamazaki et al ., 2002) and the amplitude of clock and bmal1 expression is decreased (kolker et al ., 2003). Changes in clock gene expression in peripheral tissues do not always reflect what is seen in the scn (yamazaki et al ., 2000), and may result from a disruption of signals to these tissues or the tissues themselves, which are more susceptible to the aging process . Furthermore, projections to and from the scn including peripheral oscillators may change with age . In motoneurons, aging results in a shortening in delay of spike potentials between axon and soma, as well as decreases in axon conduction velocity and increases in input resistance (morales et al . The output from circadian and peripheral oscillators do not only influence the sleep / wake cycle, but regulates metabolism and reproduction . Studies indicate that some tissues retain the ability to oscillate, even if connections from the master pacemaker have been degraded . Peripheral tissues in vitro that have become arrhythmic can be chemically induced to oscillate (yamazaki et al . However, in aged animals exhibiting a decreased photic response, retinal projections to the scn are not degraded and must be related to either the retina or scn clock functions (zhang et al ., it is increasingly evident that determining the source of age - related sleep / wake or circadian dysfunctions is rather complex . Another source of sleep / wake changes can likely be attributed to age - related neuronal dysfunction in the arousal and sleep promoting areas of the brain . The scn directly or indirectly communicates with the sleep and wake promoting systems (abrahamson et al ., 2001; aston - jones et al ., 2001 orexinergic (or hypocretinergic) neurons are known to stabilize or maintain wake (saper et al ., 2001, 2005). These cells receive input from the scn via the dorsomedial hypothalamus (dmh) and are localized in the perifornical and lateral hypothalamus (lh). There are two forms of the neuropeptide orexin / hypocretin (a and b) and two receptors . Though the neurons are limited to a discrete area, both orexinergic fibers and receptors are widely distributed throughout the brain . In diurnal and nocturnal rodents, orexinergic neurons are most active during the active phase (martinez et al ., 2002). Hypothalamic microdialysate analysis shows orexin-1 levels increase during wake and rem in adult animals (kiyashchenko et al ., 2002). Mammals with no orexin or dysfunctional orexin / hypocretin receptors have disrupted sleep / wake cycles and narcoleptic symptoms . When orexin is decreased, the circadian rhythm of the sleep / wake cycle is disrupted . The flip flop model of sleep / wake control suggests that there is a mutual inhibition between the areas that control sleep and the areas that control the wake state (saper et al ., 2001). In short, the ventrolateral preoptic area (vlpo) controls sleep and the brainstem cholinergic and monoaminergic systems control waking . Flipping weight between these areas blocking or destroying these neurons or the orexin receptor 2 may flip the animal s state quickly from waking to sleep and vice versa, such as what occurs in narcoleptic individuals, orexin knockout mice, and canine narcolepsy (chemelli et al ., 1999; lin et al ., 1999; peyron et al ., 2000; a disruption in orexin function or a reduction in orexin levels leads to less stable sleep / wake cycles such as that seen in many elderly patients with sleep disorders (porkka - heiskanen, 2003). Additionally, decreases in excitatory orexin innervation to the noradrenergic locus coeruleus (lc) is thought to be a contributing factor of poor sleep / wake quality in aged cats (zhang et al . Orexin b immunoreactive (-ir) axon density was determined to be significantly lower in the lc of aged macaques than that observed in the young or adult animals (43 and 35% decrease respectively; downs et al ., 2007). Real time pcr studies showed that preprohypocretin mrna does not change in the aged hypothalamus (terao et al ., 2002) but in situ hybridization studies show that at the single cell level, preproorexin gene expression does decrease in cell count and optical density (porkka - heiskanen et al ., 2004). Furthermore, orexin a and b protein expression as measured by radioimmunoassay was decreased in the lh (porkka - heiskanen et al ., the number of orexinergic - ir neurons as well as the optical density of respective fibers in the lh is reduced in aged animals (brownell and conti, 2010; sawai et al ., 2010). It is interesting to note that orexinergic innervation of the cholinergic basal forebrain, which modulates wake and rem sleep, is reduced in aged guinea pigs (zhang et al ., 2005). Hypocretin receptor 1 mrna is reduced in the hippocampus and hypocretin receptor 2 mrna is significantly reduced in thalamic areas, hippocampus, and the brainstem (terao et al ., 2002). Neural activity measured by c - fos immunoreactivity is reduced in orexinergic neurons of mice at 24 months (naidoo et al ., 2011). Changes are also seen in the cholinergic and monoaminergic wake active areas of aged animals . Nicotinic and muscarinic receptors of the acetylcholinergic system decrease in the scn with age (van der zee et al ., 1991). In young animals, the noradrenergic neurons of the lc are important in wake promotion, receive direct input from the scn and follow a circadian pattern of activation (aston - jones et al ., 2001). In aged rats, lc projections to the frontal cortex and dentate gyrus decrease but axonal branching increases depending on the target and age . This is suggested to be a compensatory mechanism (shirokawa et al ., 2000). In the ventral periaqueductal gray (vpag) the wake active dopaminergic neurons we have recently reported a reduction in the neural activity of these dopaminergic neurons of the vpag and the noradrenergic neurons of the lc in aged mice (naidoo et al ., 2009, 2011). The wake active histaminergic system originates in the tuberomammillary nucleus (tmn) and sends widespread projections to areas that include the cortex, thalamus and brainstem . Histamine levels were found to be increased in middle aged rats when compared to young, and the level of histamine methyl transferase was decreased (mazurkiewicz - kwilecki and prell, 1984). Histamine h1, h2 and h3 receptor mrna is decreased in the aging brain (terao et al ., 2004). Given these changes in the aging wake promoting neurotransmitter systems as well as wake maintaining systems, it is clear that therapies to alleviate or attenuate these changes need to be developed ., 1996; szymusiak et al ., 1998) and when lesioned results in insomnia (lu et al ., 2000). Gabaergic and galaninergic inhibitory neurons from this area project to wake active histaminergic neurons (sherin et al ., 1996). Interestingly, the number of activated vlpo neurons during sleep does not change in old rats (shiromani et al ., 2000) although connections between these areas may become dysfunctional or degraded with age . The scn has a minor input into the vlpo, but substantial direct and indirect inputs to the dmh (novak and nunez, 2000; chou et al ., 2002). The dmh heavily inputs the vlpo and it would be beneficial if these pathways were examined during aging . Age - associated changes in the serotonergic system affect the function of respiratory motor output during sleep . Serotonergic input to the hypoglossal nucleus decreases, which is thought to lead to a decline in upper airway muscle performance (behan and brownfield, 1999). In aged rhesus monkeys, serotonin receptor 2 density reduces in the occipital and parietal cortex including the deep layers of the motor cortex (wenk et al ., 1989; bigham and lidow, 1995). Serotonin levels also decrease in the occipital areas but do not change in the cingulate cortex in aged monkeys (beal et al ., 1991). It is likely with normal aging that changes in any neurotransmitter system affecting sleep vary across the brain . In many patients afflicted with neurodegenerative diseases the physical and mental consequences lead to sleep disorders (table 1). For example, sleep fragmentation can occur if the patient cannot move well or insomnia may develop due to depression or feelings of helplessness . Medications used to alleviate some of the motor or cognitive symptoms such as levodopa in parkinson s disease (pd) can also contribute to disruptions in normal sleep / wake behaviors . However, some research indicates that sleep disturbances may predict manifestation of neurodegenerative diseases (postuma and montplaisir, 2009). Sleep disturbance or loss also affects metabolic and immune function (krueger et al ., 1998; knutson et al ., 2007 chronic sleep loss could lead to neuronal damage resulting in altered hypothalamic pituitary adrenal axis function, cognitive deficits and memory loss . Increases in the number of patients with neurodegenerative diseases may be related to or the result of a society that does not sleep . Da, dopamine; inos, inducible nitric oxide synthase . Sturrock and rao (1985), zhang et al . (1982), foley et al . (2007), magri et al . (1997), touitou (1995), whitehead et al . ((1998), feinberg et al . (1967), myers and badia (1995), brun and englund (1981), teipel et al . (1985), kremer et al . (1991), arnulf et al . Initially there is an increase in nighttime arousals and a decrease in sws (vitiello et al ., 1991). In the later stages, circadian disruption, severe daytime wakefulness and a reduction in rem sleep occurs, likely due to a reduction in acetylcholine (dykierek et al ., circadian rhythm dysfunction has been proposed to be due to changes in scn and pineal functions (wu et al ., 2007). Degeneration of cholinergic input from the nucleus basalis of meynert to the cortex may be responsible for some of the sleep / wake changes (montplaisir et al ., 1995). Neurofibrillary tangles found in the histaminergic tmn of ad patients and amyloid- peptide (a) aggregation also contributes to the ad pathology . Normally in the interstitial fluid a has a diurnal fluctuation with low levels during sleep and peak levels during wake . Recently one study showed that prolonged wake and/or orexin administration increased levels of the a in the interstitial fluid of the brain in mice (kang et al . Administration of an orexin antagonist reduced amyloid deposits in several brain areas suggesting that manipulating sleep or the orexin system in ad patients could improve symptoms (kang et al ., 2009). Although the research is sparse, melatonin, phototherapy and exercise have all had positive effects in the treatment of circadian and sleep / wake disorders of ad patients (wu and swaab, 2007). As one in three americans develops ad, there is a crucial need for more research in these therapies . Rem sleep behavior disorder (rbd) has been associated with pd and thought to be an early manifestation (schenck et al ., 1996; boeve et al ., 2003; postuma and montplaisir, 2009). Sleep attacks and excessive daytime sleepiness (eds) are also commonly seen in patients with pd (factor et al ., 1990; diederich et al ., the degeneration begins at the brainstem and progresses rostrally, although degeneration of the dopaminergic neurons of the substantia nigra pars compacta is the main contributor to pd characteristics (braak et al ., rbd results from pedunculopontine dysfunction and likely explains rbd manifesting previous to pd (rye, 1997; boeve et al ., 2007). Some studies have successfully seen bright light therapy or sleep modifications reduce the symptoms of pd (hogl et al ., 1998; willis and turner, 2007). Huntington s disease (hd) is a genetic disorder characterized by a polyglutamine (cag) repeat (scherzinger et al ., 1999). Neurodegeneration is extensive throughout the brain, affecting cortical and subcortical areas but primarily affects the basal ganglia (vonsattel et al ., 1985). Sleep and wake regions of the brain including the brainstem, thalamus, hypothalamus and cortex are also affected in hd (kremer et al ., 1991). The scn pacemaker is functional in mouse models of hd, so a dysfunction of the circadian circuitry is proposed to contribute to circadian abnormalities (pallier et al ., 2007). Central and peripheral clock gene expression is altered as well (morton et al ., 2005; maywood et al ., 2010). The sleep / wake cycle is disrupted in hd patients characterized by self - reported eds, sleep fragmentation at night, and delayed sleep phase (arnulf et al ., 2008; videnovic et al ., 2009; aziz et al ., sleep is lighter with an increase in stage 1 and a decrease in rem sleep (arnulf et al ., 2008). Disruptions in the circadian and sleep / wake cycles of these patients exacerbate symptoms, increasing depression, cognitive deficits and metabolic dysfunctions (aziz et al ., 2010). It is important to note that pharmacological and behavioral manipulation of sleep and wake reduces disease progression and improves cognitive function and circadian gene expression in a mouse model of hd (hockly et al ., 2002; pallier et al ., 2007; pallier and morton, 2009; maywood et al ., 2010) amyotrophic lateral sclerosis (als) is considered an age - associated neurodegenerative disease with the age of onset ranging from 40 to 70 . Most als cases are sporadic and about 10% are familial . Also called lou gehrig s or motor neuron disease (boillee et al ., 2006), both upper and lower motor neurons are affected . Motor neurons of the motor cortex, brainstem and spinal cord gradually degenerate leading to muscle weakness, sleep disordered breathing (sdb) and paralysis (kimura et al ., 1999). Additionally, sleep is reduced in both rem and sws stages with resulting eds (barthlen and lange, 2000; lo coco et al ., 2011). Some patients find relief using assisted breathing such as continuous positive airway pressure (cpap) or bilevel positive airway pressure (bipap; howard et al ., 1989; david et al ., 1997; barthlen and lange, 2000). Amyotrophic lateral sclerosis is a complex disease of many subtypes with various genetic and environmental contributing factors . One such factor is glutamate toxicity which is decreased using the drug riluzole (shaw and ince, 1997). Levels of serotonin are decreased in als patients and compensatory increases in glutamate lead to excitotoxicity . It has been suggested that motor neurons with a high density of serotonergic innervation are more susceptible to degeneration (sandyk, 2006). As melatonin has antioxidant properties and inhibits glutamate release, this reduction would further exacerbate degeneration . Indeed, melatonin supplements slowed disease progression when given to a mouse model of familial als (weishaupt et al ., 2006). Normally the scn is coupled to peripheral oscillators, although studies have shown that scn control is not necessary for sustaining oscillatory activity . If signals from central oscillators reduce in strength due to age or neurodegeneration, other cues may entrain the peripheral oscillators (weinert, 2005). Unmasking mechanisms within sleep / wake systems is difficult due to the many checks and balances that ensure homeostasis . Although compensation and plasticity occurs to a lesser extent in older animals, a relatively high degree is preserved (van someren et al ., 2002). This may not always be advantageous as epigenetic methylation of circadian genes has been associated with dementia (liu et al ., 2008). Understanding how the aging brain can compensate and remain plastic will be beneficial to focus on more effective treatments for sleep / wake and neurodegenerative disorders . The co - morbidity of sleep disorders with neurodegenerative diseases suggests that changes in many of these neural areas manifests in sleep / wake and circadian dysfunction . Effects on the sleep / wake and circadian systems may result from, or contribute to, the increasing pathology . Some research has shown the benefit of pharmacologically or behaviorally restoring rhythms and sleep / wake for delaying pathologies (table 1). This is important to understand in a society where sleep is not considered a priority . A few points worth considering are as follows: sleep is a basic need that is made secondary to work schedules and some leisure activities for many adults . In developing children and adolescents, early school start times and late night extracurricular meetings contribute to a culture of sleep deprived, cognitively unhealthy americans . If restoring our circadian and sleep / wake cycles can ward off the deterioration of the brain, it is imperative to educate the public about the very real damage of abnormal sleep / wake cycles not only in aging individuals but at every age . The fragmented sleep / wake pattern seen in aging individuals can be due to the degeneration or dysfunction of the circadian and sleep / wake networks . Uncoupling of the central and peripheral oscillators may exacerbate dysfunction via altered feedback signals or signaling pathways . It is likely that several brain regions are affected and that there are individual differences in how the sleep / wake and circadian networks degrade . Additionally there may be differential plasticity and compensation in the integration of these neural systems, making the identification of applicable therapies very difficult . However, if mechanisms contributing to the normal aging process of these networks are identified, this may elucidate a general therapy for restoring sleep / wake and circadian homeostasis . It is crucial that we immediately invest our energies and resources in understanding these mechanisms as well as in the dissemination and implementation of current knowledge and therapies to the public . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
It is well known that whole breast irradiation (wbi) after breast conserving surgeries for patients with early infiltrating breast carcinoma (bc) significantly reduces the likelihood of local recurrence (lr). There are several evidences that lr is a predisposing factor for systemic metastasis [24] and, within this scope, radiotherapy (rt) is very useful for treating residual tumor cells after the surgery . The most used schedule for wbi is 50 gy delivered in 5 weeks using conventional fractionation . There is no consensus, however, regarding whether the entire breast needs to be irradiated . The accelerated partial breast irradiation (apbi) concept, based on confining the irradiation to the vicinity of the tumour bed, shortening the course of the treatment and allowing more convenience for patients, has contributed to changes in the rt paradigms [6, 7]. A variety of apbi techniques, including low- or high - dose rate brachytherapy, balloon brachytherapy, localized external beam rt (using either three - dimensional or intensity - modulated), and intraoperative electron or photon beam treatments, have been used with encouraging results [813]. Nondedicated linear accelerators (linacs) capable of delivering treatment with electrons have been used for intraoperative irradiation of many other tumors [14, 15]. These types of equipment are available in almost every rt facility and are used for daily patients' treatments . The possibility of delivering intraoperative radiotherapy (iort) with electrons, without dedicated equipment, is very attractive . Addressing this issue, the purpose of our paper was to assess the efficacy, toxicity, and cosmetic outcomes of iort delivered by standard linacs, during breast conserving surgeries for the treatment of early breast cancer . A prospective phase ii cohort study started in may 2004 at the sirio libanes hospital in sao paulo, brazil . As of july 2012, 187 women with diagnosis of bc by percutaneous biopsy were enrolled . Patients were eligible if they had unicentric invasive ductal carcinoma, with less than 3.0 cm at the largest diameter confirmed by mammography, ultrasonography, and magnetic resonance imaging (mri). Patients were considered ineligible if any of the following features were present: skin involvement, history of bc in the contralateral breast, or intraoperative microscopic findings of involvement of surgical margins or sentinel node (sn). Invasive lobular carcinoma subtype was also an exclusion criterion due to its high rate of multicentricity and multifocality . The breast conserving surgeries were performed at an operating theater located inside the radiotherapy department, contiguous to the linac suite . The quadrantectomy consisted of an en bloc resection of the parenchyma and pectoralis fascia, with at least a 2 cm macroscopic margin around the tumor . The skin over the tumor was generally removed by a circular incision, with its conservation being possible in small, deeply located tumours (t 1.0 cm). After verification of clear margins by intraoperative histopathologic and cytologic exams, sn radioguided biopsy was generally performed by the unique breast incision, as previously described [16, 17]. As for surgical aspects, the same maneuvers standardized for electron intraoperative therapy (eliot) by veronesi et al . Once the wide local excision and the sn biopsy were performed, the glandular tissue was detached from the pectoralis major muscle, to an extension of 3 cm margin around the resected area, and the skin flaps were detached from the parenchyma at the level of the adipose lamina for 2 cm circumferentially . The surgical bed was filled with a wet compress, the wound was covered, and the patient was transferred to the rt room, where all of the materials needed for maintaining anaesthesia, including gases, were available . A three - layer disk made of lead (down), aluminium (middle), and silicon (up) was inserted underneath the gland over the muscle, to protect the normal tissue below the irradiated area and absorb the backscattered radiation . The shielding disks (0.5 cm thick each) were available in three diameters (6, 8, or 10 cm), and the largest one fitting the space was placed . The parenchyma was approximated over the disk by separated stitches, exposing the area to the electron beam . Irradiation was performed using one of two standard models of siemens linear accelerators: primus or kd2 . Both machines produce electrons and are able to generate photon and electron beams with energy ranging between 6 and 21 mev . A single total dose of 21 gy prescribed at the 90% isodosis was delivered directly to the parenchyma at a rate of 300 cgy / min . The electron beam energy was chosen after measuring the gland thickness by inserting a needle perpendicularly to the parenchyma . A sterile, round collimator was connected to the gantry of the linac and gently placed into the surgical bed by appropriated mobilization of the couch and gantry (figure 1). The choice of collimator diameter was made according to each case but was usually up to 6 cm . A portal film was taken placing the film below the accelerator couch, orthogonally to the collimator, to guarantee the exact positioning of the disks . This procedure was repeated, if necessary, until the disk was considered well positioned . Afterwards, the staff left the room; the irradiation was delivered during in average 8 minutes, according to the chosen energy, under video surveillance of the vital signs of the anaesthetized patient (figure 2). The breast tissue was then reconstructed using oncoplastic techniques, preferentially outside the linac room, in the operating room [1921]. Adjuvant systemic therapy was at the discretion of the physician, in accordance with current guidelines . More than half of the patients received hormone therapy alone (51.3%), 8.5% of the patients received only chemotherapy, 38.1% had both, and 1.9% patients had no adjuvant systemic therapy . Follow - up was performed every 3 months in the first year and every 6 months thereafter . Lr was considered as a true relapse (tr), which represents regrowth of residual malignant cells in the same region of the primary tumour, or a second primary tumour (spt), representing tumor growth in another quadrant, suggesting a distinct clonal origin . The presence of seromas, hematomas, fat necrosis, wound infections, and dehiscences was investigated at all time points after surgery . Cosmesis evaluation was scored by the physician at least 12 months after irradiation, in accordance with the harvard criteria . Briefly, the treated breast is compared with the contralateral one and the result is classified as excellent (minimal or no difference in the size or shape); good (mild asymmetry in the size or shape); fair (obvious differences in the size and/or shape); and poor (marked change in the appearance involving more than 1/4 of the breast). The cumulative incidence of lr, overall survival, and bc survival were calculated using the kaplan - meier method . The spss package version 17.0 (chicago ii) medcalc package, 11.3.3.0 version (mariakerke, belgium), was used for statistical analysis . Of the 187 enrolled patients, 35 (18.7%) were intraoperatively excluded because of sn positivity (18 patients), difficulty in obtaining clear margins (11 patients), multicentricity / multifocality (3 patients), muscle infiltration (1 patient), t3.0 cm (1 patient), and no sn identification (1 patient). The cumulative incidence of lr as the first unfavorable event was 3.2% (95% ci: 0.88.1) (figure 3). Among the 5 cases of lr in the entire cohort 4 were considered to be a tr, and one had a failure in a quadrant other than the index lesion at 30-month follow - up, consistent with spt . Regarding other failures, two patients (1.3%) developed distant metastases, three had axillary failure (1.9%), and one patient had a contralateral tumor (0.6%). It is worth noting that among the cases with trs one had sn micrometastasis and one had lobular carcinoma, both identified only in the definitive histopathological analysis . One hundred and nine cases were followed up for at least 36 months with an estimated lr rate of 4.6% . Kaplan - meier estimates of efficacy at three years were lr of 4.6%, contralateral breast tumor 0.9%, distant failure 1.8%, cancer specific survival 98.2%, and overall survival 98.0% . The cumulative incidences of first unfavorable events are outlined in table 2, and the 3-year actuarial rates of recurrences are presented in table 3 . There were three deaths (1.9%): two related to breast cancer (one secondary to pulmonary metastasis and another due to chemotherapy toxicity) and one nononcologically related death . Overall survival is shown in figure 4 . In the first month after surgery, 6 cases of skin erythema (3.9%), 2 wound dehiscences (1.3%), and 1 case of hematoma (1.9%) were observed . Evidence of late toxicity, observed after at least 6 months of follow - up, was seen in 45 patients (29.6%) in a median time of 8 months (range 824). There were 21 cases (13.8%) of breast fibrosis (13 mild and 8 severe) and 15 cases of fat necrosis (9.8%). Among these cases, there were also 3 cases of breast lymph edema and 2 cases of nipple retraction . The esthetic outcomes have shown 70.3% excellent, 14.4% good, 3.9% regular, and 3.2% of bad results . From the entire cohort, cosmetic outcomes are listed in table 4 . In figure 5 a case with an excellent esthetic result breast conserving surgery followed by external wbi is a well established treatment for most women with early infiltrating bc [24, 25]. Currently, more sophisticated rt techniques are available, allowing better target coverage with better normal tissue sparing . In this context, apbi is a rapidly evolving strategy, with a widespread support for its use [7, 27, 28]. The main biologic rationale for intraoperative partial breast irradiation is that 85% of the lr (almost 100% of tr) occurs in the vicinity of the tumour, next to the scar, as a consequence of the persistence of neoplastic cells that most likely possess aggressive cancer stem cell properties [29, 30]. Experimental data indicate hierarchical organisation of bc with a small number of cancer - initiating cells (cics) that have ability to self - renew and exhibit multilineage potential . Cics, in contrast to their tumorigenic counterparts, can survive fractions of sublethal doses of rt, retaining self - renewal capacity over several generations [3234]. Some properties of cics could make them a more vulnerable target to a single lethal irradiation dose, soon after the breast resection, without allowing postoperative hypoxia and time for cell repopulation . Effects of iort on tumor microenvironment could improve outcomes, as it impairs local proliferation caused by surgical manipulation, inflammation, and simulation of the epithelial mesenchymal transition [36, 37]. Different rt techniques can be used with this purpose and, given that intraoperative rt with standard linacs has previously been used to treat abdominal tumors, we decided to use this form of treatment during breast conserving surgeries . The surgeries were performed at an operating theatre in the rt department, close to the linac room where the patients were transferred to receive the irradiation . This geographic characteristic by itself turned out to be a feature that helped the better feasibility of the method . However, it is still possible to transport the patient from an operative theatre far from the linac suite (usually out of business hours), previously prepared to be used as an operating room . The patient transport from the operating room to the linac may be regarded as a disadvantage of the method, when compared to the treatment with a dedicated linac . But one must realize that the use of a nondedicated linac, mainly in developing countries, may represent a cost - benefit strategy . We have previously reported our outcomes with focus on technical aspects, and the highlights of the use of nondedicated machine were to explore its capability of producing higher electron beam energies rather than dedicated machines and to check the possibility of misalignment between the collimator and the shielding disks by obtaining portal films using photon beams . Other advantages of iort with electrons are accurate targeting of rt and a precise definition of the tumour bed volume under direct guidance, offering very good dose homogeneity and more effectively sparing of the heart and lungs when compared to external beam rt . At the moment there are two published randomised trials focusing on single dose of rt during breast - conserving surgeries . Using localized photon beams delivered by the intrabeam device (carl zeiss meditec, oberkochen, germany), concluded that such approach is as efficient as conventional fractioned external beam rt for carefully selected patients . They employed two types of dedicated linear accelerators: novac 7 (hitesys, latina, italy) and liac (info and tech, rome, italy). Although they found that the rate of lr in the eliot group was within the prespecified equivalence margin of results, it was observed that this rate was significantly greater than with external radiotherapy, pointing out the necessity of defining the optimal patient selection criteria . By far, the most important benefit of iort with electrons is shortening the rt duration from the traditional 5 - 6 weeks to 58 minutes, thereby eliminating the delay in receiving rt, alleviating emotional distress, avoiding logistical difficulties in travelling to the radiation facility and ensuring 100% compliance . The rate of undertreated patientsdue to incomplete fractioned adjuvant wbi is far from ideal, especially in developing countries, being such women exposed to a higher risk of bc recurrence [42, 43]. The key feature for the development of eliot by the italian group was the estimation of dose equivalence between the standard 60 gy divided into 30 fractions and the single dose of 21 gy . In a landmark paper, veronesi et al . Presented a large phase ii study that included 1,822 cases treated with eliot using dedicated machines . After a mean follow - up period of 36.1 months, 42 women (2.3%) developed a tr, 24 women (1.3%) had a new primary ipsilateral tumour, and 26 women (1.4%) had distant metastases as the first event . Compared with conventional rt, eliot was considered a safe procedure for women with tumours measuring less than 2.5 cm, with a slightly higher lr rate . Our results presented here are very similar to the results obtained by veronesi et al . The widespread use of apbi motivated the american society of therapeutic radiology and oncology to define a suitable group of patients for whom apbi is acceptable outside of clinical trials, including the following: women older than 60 years, with t1 idc, clear margins, and the absence of multicentricity, multifocality, and axillary nodes involvement . The european society for therapeutic radiology and oncology also proposed suitable conditions for apbi: age 50 years, unicentric and unifocal t1 - 2 (3.0 cm), pn0 nonlobular invasive cancer, the absence of an extensive intraductal component and lymphovascular invasion, and negative surgical margins of at least 2 mm . Currently it is also known that estrogen receptor negativity is associated with increased risk of lr following apbi . This study has started before the publication of these recommendations, and part of our cases should be considered not suitable for apbi . However, some other results have pointed out that even patients who do not meet the ideal conditions may be locally treated with success [47, 48]. Anyway, since the publication of the recommendations (2009), women under 50 years of age were no longer accepted in our study . Although it might be tempting to offer iort to a large number of patients, at this time, a careful selection of suitable patients is paramount . For this reason we advocate preoperative mri which was performed in all of our patients, to better select the cases for partial breast irradiation . Most likely, the traditional wbi reduces the rate of spt in the treated breast solely if they were present and occult at the time of the primary treatment . Mri could potentially contribute to the more precise detection of multifocal or multicentric disease, with improvement of operative outcomes and decreased recurrence rates, although, besides the mri high diagnostic accuracy, it is always desirable to have pathological verification of the findings because of the mri high false - positive rates . The confirmation of intraoperative clear surgical margins is also mandatory, since the objective of iort is to reduce lr by treating residual malignant cells that may persist in tumour - bearing areas . With regard to efficacy moreover, complications due to local toxicity were scarce, and this form of iort led to a favorable impact on body image, as already observed by other authors . Also, when oncoplastic maneuvers are required, including immediate breast reconstruction with prostheses, they are feasible and safe [19, 21]. We consider as limitations of this study the facts that there was not a control group and that it was performed at a single institution on relatively small number of patients . In spite of these caveats, the technique was demonstrated to be feasible and was successfully implemented, with a very short learning curve . Iort with electrons delivered by conventional linacs, immediately after a wide local excision, presented the expected results until now, with very good local control and cosmetic outcomes and a low toxicity rate . Selected patients with early infiltrating breast carcinomas may benefit from the technique, which may represent an interesting option for developing countries.
Heart disease, including coronary heart disease, cardiomyopathy, and heart failure, causes functional deterioration and/or failure as well as myocardial cell death . It is one of the leading causes of death in advanced countries because adult cardiomyocytes are highly differentiated and have a limited regenerative capacity; therefore, significant loss of myocardium is mostly irreversible . Cell regeneration therapy is a promising new approach for myocardial repair, [1, 2] and in this context, there has been considerable basic research on the mechanisms of cell development into cardiomyocytes using somatic and embryonic stem (es) cells as well as embryonic carcinoma cells [36]. P19 embryonic carcinoma cells are one of the first among such cells to demonstrate differentiation into cardiomyocytes and have contributed extensively to the elucidation of the development mechanisms from stem cells into cardiomyocytes [4, 5, 8]. P19 cells are derived from a teratocarcinoma in ch3/he mice and can differentiate into all 3 germ layers . Culture and differentiation of the cells is simple, and this advantage has enabled their extensive application for decades . They continuously grow in serum - supplemented media, can retain an undifferentiated cell state without a feeder cell layer unlike es cells, and their differentiation can be controlled by nontoxic reagents . Primarily, cell aggregate formation in suspension culture under 0.51.0% dimethyl sulfoxide (dmso) followed by the reagent application in adhesion culture has been used to induce cardiomyocyte differentiation of p19 cells [7, 8, 10]. We examined the differentiation of p19 cells into cardiomyocytes by the above general method; however, it resulted in large variations in the differentiation rate and low differentiation efficiencies among individual experiments, even though high differentiation efficiencies have been reported previously [7, 8, 10]. P19cl6 cells, clonal derivatives of p19 cells, were established by habara - ohkubo . These subline cells can be differentiated into spontaneously beating cardiomyocytes by treatment with 1% dmso in adhesion culture over a period of 10 days or weeks without cell aggregate formation in suspension culture and more efficiently as compared to the parent cells . Therefore, p19cl6 cells may be more useful to examine the differentiation mechanisms of cardiomyocytes in vitro . Recently, ohtsu et al . Introduced a double stimulation method for cardiomyocyte differentiation from p19cl6 cells . They demonstrated that cells exposed to 10 m 5-azacytidine (5-aza) for 24 hours prior to treatment with 1% dmso differentiated into cardiomyocytes more effectively than the widely used single stimulation method described above . In order to produce a large number of cardiomyocytes, we induced differentiation of p19cl6 cells into cardiomyocytes by the double stimulation method to examine the differentiation efficiency; however, it resulted in large variations in the differentiation rates among individual experiments, as in the case of p19 cells . However, notably, the double stimulation method elicited differentiation from p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory . We also noticed large variations in the differentiation rates among previous reports on p19cl6 [1214] as well as p19 cells [8, 10, 15, 16] and this may imply a fundamental problem of the cells in themselves and/or the differentiation methods . To overcome this difficulty, we attempted to establish a subline from p19cl6 cells, and accordingly, we could introduce a new subline of cells, that is, p19cl6-a1 cells, that could efficiently and stably differentiate into cardiomyocytes by a differentiation method based on the double stimulation method with many modifications . In the present study, we also introduce a software program, visorhythm, which can analyze the temporal variations in the beating rhythms on moving images and chart correlograms displaying the oscillated rhythms on a windows computer . Because the spontaneously beating cardiomyocytes differentiated from p19cl6-a1 cells form large cell clusters, their contractions are large and strong . This feature may prove to be quite suitable for oscillated rhythm analysis based on moving images, and accordingly, we used this software to successfully display the increase in the cardiomyocyte beating rates on correlograms and dose - response curves on treatment with serially diluted cardiotonic reagents . This may indicate that p19cl6-a1 cells and the above software are useful tools for pharmacological screening tests . Balb / c mice of either sex maintained under standard housing and feeding conditions were used . The ventricles of the hearts dissected from mice aged 8 weeks and 16.5 days post coitum (dpc) fetuses were examined by reverse transcription pcr (rt - pcr). The cells were cultured in a tissue culture - grade dish and grown in alpha - modified eagle's minimal essential medium (-mem; sigma - aldrich japan k.k ., tokyo) supplemented with l - glutamine (4 mm; gibco brl, carlsbad, ca) or dulbecco's modified eagle's minimal essential medium (dmem; sigma - aldrich japan k.k .) Containing 510% heat - inactivated fetal bovine serum (fbs; jrh biosciences, lenexa, ka) and penicillin and streptomycin (100 u / ml and 100 g / ml, resp . ; sigma - aldrich japan k.k . ). Several subclones were isolated from the p19cl6 cells by the common cloning ring technique . Each clonal colony was expanded increasingly into larger wells and dishes and frozen until use . Two types of cell colonies were derived from a single cell: one, growing horizontally to form a single cell layer on a dish and the other, growing both horizontally and vertically to form cell aggregates of multicell layers like embryoid bodies or cell aggregates in suspension culture prepared using bacterial - grade dishes [5, 8, 13]. Both cell types were collected for the cardiac differentiation experiment; however, the latter were considerably more efficient in differentiating into cardiomyocytes . Among the several differentiation methods, the one proposed by ohtsu et al . Elicited most efficient cardiomyocyte differentiation from parent p19cl6 cells as long as the experiment was in vitro in our laboratory; however, it also led to a fundamental difficulty, that is, high variations in the differentiation efficiencies among individual experiments . Hence, we modified the above method in order to adapt it to our subclonal cells as well as to the parent cells . The differentiation conditions that we primarily considered were the concentration and/or exposure duration of the differentiation reagents, the concentration of fbs in the culture medium (-mem or dmem), and coating reagents . They were as follows: 5-azacytidine (5-aza; sigma - aldrich japan k.k . ): concentration: 5, 10, 15, 20, 30, and 40 m and exposure duration: 24, 48, and 72 hours; dimethyl sulfoxide (dmso, sigma - aldrich japan k.k . ): concentration: 0.5, 1.0, 1.5, and 2.0%; fbs in the differentiation medium: concentration: 2.5, 5.5, 6.5, 7.5, and 10%; and the use of a culture dish coated with or without collagen (3.0 g / cm, type i - a; nitta gelatin inc ., osaka) or matrigel (3.0 g / cm; bd biosciences, san jose, ca). Accordingly, we found a subclone, designated a1, which is the most efficiently differentiated into cardiomyocytes under the differentiation conditions below and used it for further experiments to evaluate the differentiation efficiency . We also found the optimum conditions that could be applied to the parent p19cl6 cell line, and the use of these conditions yielded almost the same results as those yielded by the method of ohtsu et al . ; the description has been provided below . The differentiation efficiencies of the subline, p19cl6-a1, and the parent cell line, p19cl6, were compared . The cells were plated at a density of 5.0 10 cells / well on 6-well plates or on a 35-mm tissue culture - grade dish coated with collagen (3.0 g / cm) and containing the growth medium (-mem with 6.5% fbs for p19cl6 cells or dmem with 7.5% fbs for p19cl6-a1 cells) and were incubated in a 5% co2 atmosphere at 37c . The next day, when cells reached ~95% confluence, the medium was replaced with a growth medium containing 10 m 5-aza in order to induce differentiation . The p19cl6 and p19cl6-a1 cells were treated with 5-aza for 24 and 72 hours, respectively, and the 5-aza - containing medium was changed every 24 hours . After treatment with 5-aza, the cells were incubated in the growth medium containing 1.0% dmso for more than 16 days . The dmso - containing medium was also changed everyday in order to remove the cell debris resulting from cell death . The experimental days were numbered consecutively beginning from the day after the first day of treatment with 5-aza (day 0). P19cl6 and p19cl6-a1 cells were cultured on glass coverslips coated with collagen (3.0 g / cm). Cells on day 16 were washed twice with hanks' balanced salt solution (hbss; sigma - aldrich japan k.k .) And then fixed with 4% paraformaldehyde in pbs for 30 minutes at 4c . After the fixation, cells were rinsed twice in cold pbs, treated with 0.1% triton x-100 in pbs for 20 minutes at room temperature and again rinsed twice in pbs . In order to reduce autofluorescence, cells were treated with 50 mm nh4cl in pbs for 10 minutes at room temperature . Then cells were blocked in a humid chamber with pbs containing 3% normal goat serum (ngs; sigma - aldrich japan k.k .) For 30 minutes and then incubated with the anticardiac -actin antibody (sigma - aldrich japan k.k . ; 10 g / ml in pbs containing 3% ngs) for 16 hours at 4c . After washing with pbs, cells were incubated with alexa488-conjugated goat antimouse igg (molecular probes, inc ., eugene, or) for 30 minutes at room temperature, cells were photographed with fluorescence microscopes (ix71, olympus co., tokyo). The specificity of the immunofluorescence staining was verified by incubations without the primary or secondary antibodies and no specific fluorescence was observed in the control . For quantitative analysis of rates of differentiation into cardiomyocytes, we measured ratios of cardiac -actin - positive cell areas to total areas of microscopic fields using image j software (ver . 1.41; national institutes of health, bethesda, md). Autofluorescence from multilayer cell regions was rather strong but -actin - specific fluorescence was easily discriminated from the autofluorescence because -actin - positive cells emerged as high - intensity cells in cell clusters (see figure 2). In this way we counted out autofluorescence regions . Sixty fields were examined in each cell type and the results from six independent experiments were summarized as mean se . Unpaired t - test was carried out to compare the differentiation efficiency between the two cell types . Total rna was isolated from the cultured p19cl6 and p19cl6-a1 cells on days 0, 3, 7, and 16, and from the cardiac ventricles of 8-week - old adult mice and 16.5 dpc fetuses using the trizol reagent (invitrogen japan k.k ., one microgram of total rna was transcribed into first - standard cdna using m - mlv reverse transcriptase, rnase h (promega, madison, wi), and oligo (dt) primer according to the manufacturer's instructions . For detection of gata4 and nkx2 - 5, the specific transcription factors for cardiac differentiation, and of the alpha myosin heavy chain (-mhc), which is indispensable for cardiomyocyte contraction, 1 l of the reaction mix (out of the total 25 l) as the reverse - transcribed dna template was amplified by pcr40 cycles for gata4 and nkx2 - 5 and 25 cycles for -mhc . After amplification, the pcr products were separated on 1.2% agarose gel and visualized by ethidium bromide staining . The expression levels of gata4, nkx2 - 5, and -mhc mrnas were compared between p19cl6 and p19cl6-a1 . They were determined from 3 independent experiments and normalized by reference to the expression levels of gapdh mrna (23 cycles of pcr). All analyses were performed on excel 2007 . The values were expressed as the mean se that was represented by ratios to the mean values of the p19cl6 cells in the basal protocol on day 16 as 1.0 according to the differentiation protocol . The following primer pairs were used: gata4, 5-gttgtggtggtgggtttttc-3 (forward) and 5-tttgatgttcctgggagagg-3 (reverse); nkx2 - 5, 5-tctccgatccatcccactttattg-3 (forward) and 5-ttgcgttacgcactcactttaatg-3 (reverse); -mhc, 5-tacctcatggggctgaactc-3 (forward) and 5-cgaacatgtggtggttgaag-3 (reverse); gapdh, 5-gacttcactcacggcaaatt-3 (forward) and 5-tcctcagtgtagcccaagat-3 (reverse). The 35-mm dish with the differentiated cells were placed in a stage top microscope incubator (oni - inu - f1; tokai hit, shizuoka, japan) mounted on an inverted microscope (dmirb, leica microsystems ltd ., heerbrugg, switzerland) and incubated at 37c in a 5% co2 atmosphere . Phase - contrast moving images of spontaneously beating cells were recorded through a ccd camera (coolpix4500; nikon co., tokyo) and digitalized to mpeg2 video files (8 bits / channel; 704 pixels 480 pixels / frame; 30 frames / sec) using an encoder (mtu2400 fx; canopus co. ltd ., the contraction rhythm of the spontaneously beating cells was analyzed based on the moving images using the following method based on the data analysis algorithm on the calculation of correlation coefficient as reported by yamauchi et al . . A small area of interest where brightness clearly oscillated due to the beating was selected from a moving image, and then, a reference frame was arbitrarily chosen from the moving image frames . Temporal variations in the correlation coefficient were calculated between the values of mean brightness of the reference frame and those of other frames in the selected area, and accordingly, a correlogram representing the contraction rhythm of the beating cells in the area of interest was displayed by the vertical coefficient and horizontal time axis . We developed a software program, visorhythm, which calculates the temporal variations in the correlation coefficient up to 6 rectangle areas (150400 pixels / area) simultaneously on a single moving image on a windows computer . The data can then be directly output into excel to chart the correlograms, and the beat - to - beat intervals are represented by the intervals between the upward and downward peaks in each correlogram in the selected areas . We examined the pharmacological reactions of spontaneously beating cardiomyocytes differentiated from the p19cl6-a1 cells to evaluate whether they are useful cellular tools for pharmacological screening tests . We used 3-isobutyl-1-methylxanthine (ibmx; sigma - aldrich japan k.k .) And ouabain (sigma - aldrich japan k.k . ), which are well - known potent cardiotonic reagents, for this purpose . Phase - contrast moving images of spontaneously beating cells were recorded before and after addition of serial dilutions of each reagent (10 nm, 20 nm, 70 nm, 200 nm, 700 nm, 2 m, 7 m, 20 m, and 70 m) into the medium . The beat - to - beat intervals on exposure to certain concentrations (10 nm, 30 nm, 100 nm, 300 nm, 1 m, 3 m, 10 m, 30 m, and 100 m) were obtained from the correlograms calculated on visorhythm . The median effective dose (ed50) was obtained from the dose - response curve based on the beating rates on which the basal and maximum rates were represented by 0 and 100%, respectively . We selected the differentiation protocol suggested by ohtsu et al . For the p19cl6 cells because this method was the most efficient for cardiomyocyte differentiation from among the several differentiation methods as long as it was in vitro in our laboratory . The undifferentiated populations of the cells, however, considerably proliferated even in the differentiation medium containing 1% dmso as a differentiation inducer and 10% fbs; hence, we reexamined the optimum concentration of fbs . We thus obtained a resultant concentration of approximately 6.5%, and accordingly, the protocol for the cells was as revised as follows . Subconfluent p19cl6 cells in -mem containing 6.5% fbs were treated with 10 m 5-aza for 24 hours followed by treatment with 1% dmso for more than 16 days (basal protocol). In case of the p19cl6-a1 cells, we modified the basal protocol by reexamining the conditions in each step according to the criteria of (1) rates of appearance of the beating cells per field and (2) dimensions of the beating area under a microscope . The cells showed a relatively high resistance to 5-aza, and the optimum conditions were as follows . Subconfluent p19cl6-a1 cells in dmem containing 7.5% fbs were treated with 10 m 5-aza for 72 hours followed by treatment with 1% dmso for more than 14 days (developed protocol). We also examined the differentiation efficiencies in culture dishes coated with or without collagen or matrigel and found the differentiation to be more effective on collagen coated dishes . The conditions for each protocol and for each cell type are summarized in table 1 . We observed the differentiation processes under an inverted microscope to compare the morphological differences between the p19cl6 and p19cl6-a1 cells . Both cell types grew similarly in the beginning (days 05) of the differentiation protocols . The p19cl6 and p19cl6-a1 cells reduced in number during treatment with 5-aza due to the damage by the reagent and proliferated slightly everyday thereafter during treatment with dmso . The cells started to grow vertically due to overconfluence, and multilayer cell regions appeared around day 5 . On the p19cl6 cells multilayer cell regions were generally small and appeared like scattered islands in monolayer cell regions . However, on p19cl6-a1 cells, they were connected with each other to form mesh - like structures and expanded to comprise approximately 7080% of the total area of the dishes during the procedure . This feature of the multilayer cell regions was similar to that of embryoid bodies or aggregates on culture with dmso in the nonadhesive bacteriological - grade dishes . Beating cells first appeared among the p19cl6 cells differentiated by the basal protocol on day 10 and among the p19cl6-a1 cells differentiated by the developed protocol on day 11 . The clusters of beating cells increased in number and size thereafter and peaked on day 16 in both cases . The beating cell clusters from the p19cl6 cells were observed generally in the monolayer cell regions and less frequently on the boundary between the monolayer and multilayer regions (figure 1(a)). The clusters, composed of small - sized beating cells densely arranged, were generally small in the monolayer regions (figures 1(c) and 1(e)). On the other hand, the beating cell clusters in the p19cl6-a1 cells appeared mostly in the multilayer and boundary regions (figure 1(b)). They were composed of elongated cells and formed mesh - like sheet structures (figures 1(d) and 1(f)). They were relatively large and sometimes extended beyond the visual field under the inverted microscope through a 10 objective lens, and accordingly, the contractions were large and strong . Therefore, the beating cells differentiated from the p19cl6-a1 cells on day 16 or later were suitable for contraction rhythm analyses based on moving images . In order to morphologically identify cardiomyocytes differentiated from p19cl6 and p19cl6-a1 cells clearly and to compare the differentiation efficiencies between the two, we immunocytochemically examined those cells on day 16 using anticardiac -actin antibody under immunofluorescence microscopy . Cardiac -actin - positive cells from p19cl6 were various in shape (oval, polygonal, short spindle - shaped) and generally formed round clusters (figures 2(a) and 2(c)). Those from p19cl6-a1 were relatively large and elongated, and formed mesh - like sheet structures (figures 2(b) and 2(d)). We measured ratios of cardiac -actin - positive cell areas to total areas of microscopic fields; the positive cell areas were 7.0 1.1% (mean se) of the total areas on p19cl6 while those were 38.3 5.7% on p19cl6-a1 . The value on p19cl6-a1 was 5.5 times and significantly higher than that on p19cl6 (p = .002; figure 2(e), table 1). To compare the differentiation efficiencies between the p19cl6 and p19cl6-a1 cells, we examined the expression of the cardiac - specific transcription factors, gata4 and nkx2 - 5, and of the cardiac - specific myosin heavy chain, -mhc, by rt - pcr on the respective cells with the respective differentiation protocols listed in table 1 on days 0, 3, 7, and 16 . Both gata4 and nkx2 - 5 were already expressed in both p19cl6 and p19cl6-a1 cells on day 0, that is, in the untreated cells (figure 3(a)). The expression of the former increased from day 0 to day 16 during treatment in both groups (figure 3(b)), while that of the latter dipped on day 3, but increased thereafter (figure 3(c)). On day 16, when the appearance rates of the beating cells per field and the dimensions of the beating area peaked as observed under a microscope, gata4 expression did not differ significantly between the p19cl6 and the p19cl6-a1 cells; however, nkx2 - 5 expression in the latter was significantly and 1.45 times higher than that in the former (figure 3(c)). Alpha - mhc was not expressed till day 7, although it was clearly expressed on day 16 in both groups (figure 3(d)). Its expression in the p19cl6-a1 cells was significantly and 1.77 times higher than that in the p19cl6 cells (figure 3(d), table 1). These results indicated that p19cl6-a1 cells were more efficient than the parent in terms of cardiomyocyte differentiation . We also examined -mhc expression in the ventricles of 8-week - old adult mice and 16.5 dpc fetuses by rt - pcr to compare -mhc expression in the tissues with that in the p19cl6-a1 cells differentiated by the developed protocol on day 16 . The expression levels were higher in the adult tissue than in the other tissue, but were almost equal between the fetal tissue and the cells on day 16 (figure 3(e)). We examined the pharmacological reactions of the spontaneously beating cells differentiated from p19cl6-a1 cells by the developed protocol . For this purpose, we employed ibmx and ouabain as cardiotonic reagents, and both of them increased the beating rates . The moving images of the beating cells were recorded before and after addition of serial dilutions of each reagent . To determine the temporal variations in the beating rhythms in detail, we developed the software, visorhythm, to analyze them based on moving images and chart correlograms displaying the oscillated rhythms . Figure 4(a) is an example of a series of correlograms corresponding to the temporal variations in the contraction rhythms of the beating cells treated with 10 nm, 300 nm, and 10 m; the beating rate was 1.30 times per second at the basal level and increased up to 2.30 times per second after the addition of 10 m ibmx . Figure 4(b) shows the dose - response curve to ibmx corresponding to the data in figure 4(a), and its ec50 was 950 nm . Figures 4(c) and 4(d) illustrate another series of correlograms and a dose - response curve to ouabain, respectively . These data indicate that the p19cl6-a1 cells and visorhythm are useful tools for pharmacological screening tests . P19 embryonal carcinoma cells have been extensively studied for cardiac differentiation in vitro and have contributed to the elucidation of the early events in differentiation, such as the involvement of bone morphogenic proteins and their subsequent intracellular signal cascades and oxytocin and its subsequent activation of the transcription factors gata4, nkx2 - 5, and so on [5, 7, 8, 10, 19]. For their efficient differentiation into cardiomyocytes, prior formation of cell aggregates in suspension culture under 0.51.0% dmso followed by reagent application in adhesion culture is required; however, molecular events occurring during aggregation and the necessity of their aggregation for differentiation have not yet been entirely understood . High cell densities at least can trigger spontaneous differentiation from p19 cells without the formation of cell aggregates and reagent treatment [9, 10]. On the other hand, p19cl6 cells, the clonal derivative of the p19 cells, efficiently differentiate into cardiomyocytes on dmso treatment in adhesion culture without prior formation of cell aggregates, although high cell densities are required for effective differentiation . In the present study, we introduced p19cl6-a1, a clonal derivative of p19cl6 cells, which differentiated into cardiomyocytes most efficiently among the 3 cell lines subjected to double stimulation with 5-aza and dmso in adhesion culture as reported by ohtsu et al . The p19cl6-a1 cells extensively formed multicell layers in adhesion culture under stimulation with dmso after they reached overconfluence . They were highly packed in multicell layers like the cell aggregates of p19 cells in suspension culture . P19cl6-a1-derived cardiomyocytes, which generally formed large clusters of beating cells, almost always appeared in the multilayer regions and on the boundary regions adjacent to the monolayer regions . These findings, in addition to the above differentiation characteristics in the other 2 cell types, may indicate that cell - to - cell contact all around, that is, not only from side to side but also from top to bottom, is a key event in the differentiation of p19 cells and their derivatives . Because three - dimensional contact can be achieved by the formation of multicell layers, a clonal derivative differentiating more efficiently into cardiomyocytes than p19cl6-a1 may be established when the dimensions of the multilayer regions is employed as a criterion for clonal selection from p19cl6 cells . Further investigations are necessary to elucidate whether multilayer formation is a crucial trigger for differentiation and to determine the molecular events occurring in the multilayer regions in this instance . We employed the double stimulation method using 5-aza and dmso as suggested by ohtsu et al ., as the basal protocol for cardiomyocyte differentiation because it elicited differentiation from the parent p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory . To apply it to the clonal derivative, that is, to p19cl6-a1 cells, we examined the optimum concentration and exposure time of the inducers and accordingly, the protocol for efficient differentiation was modified as summarized in table 1 . The optimum concentration of each inducer is the same as that in the basal protocol, but the exposure time of 5-aza is 3-fold longer than that for the parent cells . An almost equal and fair percentage of p19cl6 and p19cl6-a1 cells were necrotic after treatment with 10 m 5-aza for 24 and 72 hours, respectively, in the monolayer culture, and therefore, the p19cl6-a1 cells were more resistant to more prolonged exposure to the dna demethylating reagent than the p19cl6 cells . Both cell types were much more resistant to the reagent than the p19 cells because the p19 cells treated with 510 m 5-aza in the monolayer culture became totally necrotic . In the present study, dmem with 7.5% fbs of the composition of the differentiation medium elicited differentiation from both p19cl6-a1 and p19cl6 cells more efficiently than -mem with 6.5% fbs and much more than -mem with 10% fbs (data not shown), which has been generally used for the differentiation of p19 and p19cl6 cells [7, 1115, 2026]. After the 5-aza treatment, p19cl6-a1 cells proliferated slightly under dmem containing 1% dmso and 7.5% fbs and could form multicell layers in adhesion culture more extensively than p19cl6 cells under -mem containing 1% dmso and 6.5% fbs . P19cl6-a1-derived beating cells appeared mostly in the multilayer regions and formed clusters more extensively in the multilayer regions than in monolayer regions . These may indicate that more extensive multilayer formation induces the cardiomyocytes to differentiate more efficiently . However, using 10% fbs instead of 7.5% or 6.5% could not elicit more efficient differentiation of the cells probably due to stable proliferation of the undifferentiated populations in the cells even in the differentiation media containing 10% fbs . In the present study, we found that 1% dmso is optimum for the differentiation of p19cl6 and p19cl6-a1 cells . It is generally used for differentiation of p19 and p19cl6 cells [1115, 20, 2226]. Moreover, a previous report has demonstrated that the efficiency of colony formation of p19 cells in the presence of dmso indicates the absence of toxicity at concentrations of less than 1% under -mem with 10% fbs . Based on these data, we suppose that the serum concentration in combination with 1% dmso is a considerable factor for efficient differentiation . In fact, the source and lot number of fbs and calf serum used in differentiation are thought to be crucial for successful cardiac differentiation from p19 cells as well as from p19cl6 cells (riken bioresource center, personal communication). In the present study, cardiac - specific transcription factors gata4 and nkx2 - 5 were expressed in untreated cells . In most previous reports p19cl6 cells untreated with inducers such as dmso and 5-aza expressed neither gata4 nor nkx2 - 5 [12, 14, 20, 22, 24, 27]. However, there are a few reports in which gata4 and/or nkx2 - 5 expressed in p19cl6 cells before the treatment [23, 26]. In the present study we used a medium containing 10% dmso for cell cryopreservation and cells cultured for 1 day after thawing as untreated cells (day 0) for the experiments . We suppose this caused the clear expressions because the untreated cells contain cells already exposed to the dmso as a cardiac differentiation inducer probably at a high rate . There were no differences in the differentiation efficiencies under an inverted microscope between p19cl6-a1 cells with and without subcultures after thawing (data not shown) and the same finding was obtained on p19cl6 cells (dr . There are 2 isoforms of cardiac mhcs, namely - and - mhc . The former is characterized by a high atpase activity and a quick contraction velocity as compared to the latter . Alpha- and/or - mhc are often used as cardiac - specific markers for determining the efficiencies of cardiac differentiation of p19 and p19cl6 cells [8, 11, 13, 14, 20, 25]. In the present study, we showed that the expression level of -mhc mrna in the p19cl6-a1 cells treated with the inducers was significantly and 1.77 times higher than that in the p19cl6 cells and almost equal to that in the heart ventricles of the mouse fetuses . We also compared the differentiation efficiency between the two cells by using cardiac -actin immunofluorescence staining and showed that the ratio of cardiac -actin - positive cell areas to total areas of microscopic fields in p19cl6-a1 cells was 38.3% and 5.5 times higher than that in p19cl6 cells . This indicates that the differentiation efficiency estimated by the immunofluorescence staining is 3.1 times higher than that by the rt - pcr . This discrepancy is likely to be caused by the multicell layer formation of p19cl6-a1 cells; the two - dimensionally microscopic analysis may overestimate the differentiation efficiency due to the elimination of a factor in rate of formation of multicell layers and the estimation by the rt - pcr may represent the differentiation efficiency close to a real value . P19- as well as p19cl6-derived cardiomyocytes display an electrophysiological phenotype of embryonic ventricular cardiomyocytes based on the studies in which (1) they display a complete set of functional ion channels, but only limited amount of functional sodium channels, (2) the maximal diastolic potential is rather low (approximately 40 to 60 mv), and (3) the shape and characteristics of the action potential of these cells resemble those of primary isolated embryonic cardiomyocytes [5, 21, 24, 29]. Thus, p19cl6-a1-derived cardiomyocytes may have similar properties because they are clonal derivatives of p19cl6 cells . They rhythmically and spontaneously beat and form clusters much larger than p19cl6-derived cardiomyocytes, and accordingly their contractions are larger and more obvious as observed under an inverted microscope . Therefore, p19cl6-a1-derived cardiomyocytes may be suitable models for studies on the cardiac structural and functional properties during normal physiological and pathological states in vitro, particularly studies on contraction rhythm analyses based on moving images . For this purpose, that is, to figure out the temporal variations in the beating rhythms in detail, we developed the software, visorhythm, to analyze them based on moving images and chart correlograms displaying the oscillated rhythms . To illustrate the availability of p19cl6-a1-derived cardiomyocytes as physiologically functional cardiomyocytes and the software as an analytical tool for detailed analysis of beating rhythms, we employed ibmx and ouabain as potent cardiotonic reagents [30, 31]. Additionally, we found that the nonbeating cells started to contract rhythmically after the addition of these reagents (data not shown). This finding is supported by a previous study in which p19-derived nonbeating cells generated action potentials upon electrical stimulation and indicates that the actual ratio of p19cl6-a1-derived cardiomyocytes is higher than that of the beating to nonbeating cells under the microscope . There are largely two different methods to analyze beating rhythms of cardiomyocytes in vitro: one is an electrophysiological method to measure electrical potential of cardiomyocytes and the other is a microscopic method to measure oscillated motions of cardiomyocytes . As for the former method, a culture plate equipped with microelectrode arrays having 64-channels are used to record electrical potentials on cultured cardiomyocytes [32, 33]. It can analyze electrical activities of cardiomyocytes such as conduction velocities and synchronization times . As for microscopic methods, micheletto et al . Introduced a free - running scanning near - field optical microscopy setup to observe live cardiomyocytes . Weisensee et al . Also introduced an image analysis system using the video capture board for real - time subtraction between frames of moving images to analyze motions of beating cardiomyocytes . Both tools can analyze beating rates and the relative strength for inotropic force while they monitor only one object at a time . Here we have introduced visorhythm to analyze temporal variations in beating rhythms of cardiomyocytes based on moving images and chart correlograms displaying the oscillated rhythms . Yamauchi et al . Had developed a similar software on which the temporal variations in beating rhythms could be analyzed in 1 fixed area (20 20 pixels) chosen arbitrarily . In contrast, visorhythm can analyze up to 6 such areas (150400 pixels / area) on each frame of one moving image simultaneously . Therefore it can also analyze synchronization between cardiomyocytes . In conclusion, we used double stimulation with 5-aza and dmso in this study and showed that p19cl6-a1 cells, a new clonal derivative of p19cl6 cells, differentiated into cardiomyocytes more efficiently than the parent cells . We introduced a new software, visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms up to 6 areas on a single moving image simultaneously . Using p19cl6-a1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents . These indicate that a combination of p19cl6-a1 cells and visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.
Single incision laparoscopic surgery (sils) for cholecystectomy procedure has been introduced as early as 1999 to achieve less pain, less scarring, and less hospitalization period . One of the major difficulties of this procedure seems to be the traction of the gallbladder in order to expose the tissues during operation without additional ports . The use of transabdominal 20 nylon sutures attached to keith needles and the use of a kirschner wire hook introduced through subcostal area are reported methods used for traction of gallbladder and better exposition of the calot triangle . However, all these techniques consist puncture of the gallbladder with sharp needles especially if it is distended and carry the risk of bile leakage and contamination afterwards . Herein, we describe our technique in establishing single port access for cholecystectomy in five patients that involves the use of a 2 mm in diameter grasper, karl storz 27290f, that is generally used by urologists for percutaneous nephrolithiasis intervention . After induction of general anesthesia, an umbilical skin incision, 1.5 cm in length, is made vertically and a covidien sils port having 5 mm12 mm four holes is inserted to the peritoneal cavity under direct vision and pneumoperitoneum is created . Thereafter, a minigrasper, karl storz 27290f, 2 mm in diameter (figure 1), was inserted thoroughly an incision, 2 mm in length, created at the transaction point of the midclavicular vertical line and umbilical horizontal line (figure 2). It is used to hold the fundus of the gallbladder gently and push the organ in an cephalad direction to visualize calot's triangle (figure 3). This atraumatic device helped to mobilize gallbladder whenever needed during surgery without causing any wound or leakage . Then, the dissection of the gallbladder was completed in a standard fashion from bottom to top and after the organ was dissected free from the liver, it was removed directly through the umbilical incision . No complications including gas leakage around the minigrasper were encountered during and after the surgery . Followup was done in the first month and minimal scarring was observed in the umbilicus and the minigraspers' small incision was almost invisible . Single incision laparoscopic cholecystectomy (silc) was first described by navarra et al . In 1997,, silc technique has been performed by many clinicians with promising results and many advantages like few complications, acceptable operation period, less scarring, less pain, and quicker recovery . There are many technical challenges in silc surgery as one of them seems to be the retraction of the fundus cephalad and traction of the hartmann's pouch laterally to ensure safe cholecystectomy . For this purpose, some surgeons use transabdominal 20 nylon sutures attached to keith needles for fixation and assist gallbladder retraction and some other used kirschner wire hook which is introduced through subcostal area to pull gallbladder in an upright direction during the operation . Yet, all these techniques consist puncture of the gallbladder with sharp needles especially if it is distended and carry the risk of bile leakage and contamination afterwards . They also have limited ability to mobilize whenever needed . To achieve gallbladder retraction and cephalad traction, this minigrasper is used not only in performing sufficient traction of the gallbladder in an upright position, but also it can allow good mobilization of the gallbladder in order to dissect calot's triangle safely . Use of this instrument has led no perforation and leakage of bile in the patients . Through a very small incision, 2 mm in length, without using any port, it has been introduced directly into the abdomen under camera control and made the dissection trouble - free as the manipulation of the gallbladder could be done easily to every direction needed . No gas leakage has been noted around the instrument after direct introduction into the abdomen in all cases . The most remarking effect of using this technique was its influence on the operation time . Previous reports using port systems or transabdominal sutures declare their mean operating time to be approximately at least 30 min to 70 min . Our procedure even on the first patient took only 40 min to conclude without any complications . We believe learning curve would be low and it would be possible to significantly decline operation periods in following patients . In conclusion, with the help of this instrument the operation period may be lessened, less scarring is achieved and surgical procedure is done more easily and safely without any additional costs.
Wilms tumor is the most common renal tumor in children, peaking at 23 years of age . Though uniformly fatal in previous decades, the prognosis for most patients has greatly improved due to advances in chemotherapy and surgical treatment . Most cases are unilateral and treated by resection at presentation, which allows for diagnostic pathology and bulk of disease removal . Surgery is then followed by several weeks of adjuvant chemotherapy, with radiotherapy used mainly for cases of tumor rupture or peritoneal seeding . Though primary kidney resection has long been the cornerstone of effective treatment for wilms tumors, this approach has recently been reevaluated in the context of synchronous bilateral disease . Bilateral wilms tumor accounts for only 5% of cases but is associated with higher morbidity and mortality . Frequently one kidney will have a much larger tumor burden, as in the present case, and surgery would consist of complete nephrectomy for one side and partial nephrectomy for the other . However, as many as 15% of stage v wilms tumor patients treated in this manner eventually develop chronic renal failure by age 20 compared with 1% of unilateral wilms tumor patients, prompting efforts to surgically spare as much normal renal tissue as possible . Children's oncology group trial aren0534calls for pre - operative neoadjuvant chemotherapy to be given in order to shrink tumors to facilitate partial nephrectomies to maximize preservation of normal renal tissue . Here, we describe a young girl with bilateral wilms tumor who had marked tumor response after 12 weeks of chemotherapy . Her case illustrates that tumor lysis precautions should be considered in children with a large amount of disease who undergo neoadjuvant treatment and that pathologic interpretation of the tumor is possible even after many cycles of chemotherapy . A normally developed 5-year - old girl presented to the emergency department with a 1 month history of worsening abdominal pain that localized to the right upper quadrant with intermittent vomiting and fevers over the week prior to presentation . On physical examination, the child was febrile to 37.6c with abdominal distension . A tender right - sided abdominal mass was found extending from the costal margin to the pelvis and beyond midline . Abdominal ct imaging identified a large mass arising from the right kidney with at least one smaller lesion in the left kidney (fig . Serum wbc and electrolytes were normal, with a serum creatinine of 0.40 mg / dl and a bun of 7 mg / dl . Figure 1:computed tomography of the patient's abdominal disease at presentation (a), after 6 weeks of chemotherapy per cog aren0534 (b) and post - surgically (c). (a) axial abdominal ct image at presentation showing large right - sided renal mass (23 13 15.8 cm; large triangle) and small contralateral renal lesion (small triangle), (b) axial abdominal ct after 6 weeks of chemotherapy showing marked response of the right - sided renal tumor (8.6 4.1 5.3 cm) to therapy . Left - sided renal renal tumor remained approximately stable in size (8 mm) throughout neoadjuvant therapy . (c) post - surgical abdominal ct reflecting right radical nephrectomy and wedge resection of left kidney . Computed tomography of the patient's abdominal disease at presentation (a), after 6 weeks of chemotherapy per cog aren0534 (b) and post - surgically (c). (a) axial abdominal ct image at presentation showing large right - sided renal mass (23 13 15.8 cm; large triangle) and small contralateral renal lesion (small triangle), (b) axial abdominal ct after 6 weeks of chemotherapy showing marked response of the right - sided renal tumor (8.6 4.1 5.3 cm) to therapy . Left - sided renal renal tumor remained approximately stable in size (8 mm) throughout neoadjuvant therapy . (c) post - surgical abdominal ct reflecting right radical nephrectomy and wedge resection of left kidney . The patient was given a presumptive diagnosis of bilateral (stage v) wilms tumor . The family consented for her to participate in the children's oncology group trial aren0534 which called for pre - operative chemotherapy in order to facilitate nephron - sparing surgery at the time of tumor resection . A central venous catheter was placed, and neoadjuvant chemotherapy with vincristine, dactinomycin and doxorubicin was initiated . Within hours of starting her therapy, we found that lactate dehydrogenase and uric acid were both elevated (1134 u / l and 6.3 mg / dl, respectively; fig . 2). The patient's hyperuricemia was felt to be consistent with tumor lysis syndrome, and allopurinol and alkalinized fluids were administered . The hyperuricemia soon resolved, and the patient maintained normal serum creatinine, potassium and phosphorus levels throughout her hospital stay . A follow - up ct scan after 6 weeks of therapy showed a marked response to chemotherapy of the right - sided lesion (fig . We decided to administer another 6 weeks of chemotherapy to improve her candidacy for right partial nephrectomy instead of whole nephrectomy . However, despite further tumor shrinkage, total nephrectomy eventually had to be performed for the right kidney because no clear surgical plane could be identified at the time of resection (fig . Pathologic interpretation of the right- and left - sided tumors confirmed wilms tumor in each and a focus of anaplastic disease on the right (fig . 10.8 gy of radiation to the right flank was administered because of the pathologic finding of focal anaplasia, in accordance with the aren0534 protocol . Presently the patient is well, with good renal function and in remission, now roughly 6 months after her end of therapy . Figure 2:tumor lysis metabolic abnormalities reflecting serum uric acid and creatinine levels in the context of the therapeutic timeline . Figure 3:pathologic images from right - sided lesion after 12 weeks of neoadjuvant chemotherapy . Note that the architecture of the tumor is still well preserved, confirming a diagnosis of wilms tumor . (a) lower - power power image (100) showing the area of anaplasia (black triangle) immediately adjacent to an area of scarring and fibrosis (white triangle), (b) a region of non - anaplastic glandular wilms tumor (400), (c) anaplastic focus of disease (white triangle; 400). Tumor lysis metabolic abnormalities reflecting serum uric acid and creatinine levels in the context of the therapeutic timeline . Note that the architecture of the tumor is still well preserved, confirming a diagnosis of wilms tumor . (a) lower - power power image (100) showing the area of anaplasia (black triangle) immediately adjacent to an area of scarring and fibrosis (white triangle), (b) a region of non - anaplastic glandular wilms tumor (400), (c) anaplastic focus of disease (white triangle; 400). Wilms tumor is responsible for roughly 6% of all childhood cancers and 95% of renal tumors in the pediatric population . Survivability from this malignancy now approaches 90% with proper management, including surgery, chemotherapy and radiation . Historically, most wilms tumors have been resected at diagnosis, with adjuvant chemotherapy to manage micrometastatic disease . Even with bilateral disease, chemotherapy would typically be administered only after the large primary tumor had been removed . However, current high - risk wilms tumor protocols, as apply to patients with bilateral disease, are designed to reduce long - term risk of renal failure by shrinking tumors with chemotherapy to enable nephron - sparing sub - total nephrectomy . Clinical trials by siop (international society of paediatric oncology) and other consortia have found that pre - nephrectomy chemotherapy may decrease the stage of disease and risk of tumor rupture during local control; however, this approach brings risks of tumor lysis syndrome, veno - occlusive disease and potential interference with tumor staging and pathologic diagnosis . In the present case, initial chemotherapeutic management of wilms tumor was complicated by hyperuricemia, presumably caused by tumor lysis syndrome in which metabolites are released from large numbers of cancer cells dying simultaneously in response to chemotherapy . Classically, tumor lysis syndrome occurs in the setting of initial treatment of hematologic malignancies such as burkitt's lymphoma and leukemia, presumably because of the large burden of disease and the exquisite chemosensitivity of such malignancies . Laboratory findings of tumor lysis syndrome include hyperuricemia, hyperkalemia and hyperphosphatemia with secondary hypocalcemia . We reason that our patient developed hyperuricemia because of her large tumor burden, the chemosensitivity of her disease and perhaps because of the presence of tumors in her kidneys that may have physically interfered with normal glomerular filtration . Many cases of tumor lysis can be managed conservatively with ample hydration, alkalization and xanthine oxidase inhibition by allopurinol . More severe or refractory cases may require administration of uric acid oxidase (rasburicase), a recombinant enzyme that degrades uric acid, or hemodialysis . Our case illustrates that wilms tumor, being a chemosensitive embryonal malignancy of childhood, can also be associated with tumor lysis syndrome . In the past, most patients with bilateral wilms tumors were treated up - front with surgery and therefore had much lower burdens of disease when adjuvant chemotherapy . This case illustrates that if surgery is postponed, tumor lysis precautions should be considered when chemotherapy is given to patients with large wilms tumors . This case also demonstrates that pathologic findings are preserved even after several weeks of chemotherapy.
In the united states, insufficient participation in leisure time physical activity constitutes a major threat to public health . Recent estimates suggest that 25% of americans do not engage in any physical activity at all . Even those engaging in physical activity are usually not doing so at recommended levels . In order to promote and maintain health, the american college of sports medicine (acsm) recommends a minimum of 30 minutes of moderate intensity aerobic physical activity five days a week or a minimum of 20 minutes of vigorous intensity aerobic physical activity three days a week . Despite these widely disseminated guidelines, the centers for disease control (cdc) report that americans have made no substantial progress towards achieving recommended levels of physical activity with the proportion of 1829 year olds meeting guidelines hovering around 35% and the proportion of adults 65 and older meeting guidelines at about 20% . These numbers are troubling, as aerobic exercise has been convincingly linked to the prevention of myriad negative health outcomes, including several forms of cancer . Numerous studies conducted over the past two decades have explored the association between physical activity participation and cancer prevention, consistently implicating strong or probable evidence for reduced risk of colon, breast, and endometrial cancers when physical activity recommendations are followed [36]. Likely mechanisms through which physical activity is believed to have an influence on cancer prevention include reduction in adiposity and changes to levels of circulating metabolic hormones and growth factors (e.g., estrogen, testosterone, and insulin - like growth factors) [79] as well as influences on dna methylation [10, 11]. In respect to prostate cancer, because physical activity activates gut motility, gastrointestinal transit time for food wastes is lessened and thus, exposure to carcinogens is attenuated . There is also research to suggest that immune function changes may mediate the relationship between physical activity and cancer development . The promising body of literature regarding the relationship between physical activity and cancer has led the national cancer institute (nci) to regard behavioral primary prevention of cancer (e.g., physical activity) as a top priority . Unfortunately, interventions designed to change behavior are typically met with only modest success, even when grounded in empirically supported theory, and behavioral adherence is reported to be a principal challenge faced by exercise promotion programs . Indeed, only 50% of individuals who adopt an exercise program stay with it for more than six months [14, 15]. Researchers devoted to the goal of improved physical activity participation have suggested that one likely determinant of physical activity behavior is the way in which individuals subjectively experience exercise . In previous work, we organized genetic, physiological, subjective, and motivational factors that may contribute to the initiation and maintenance of physical activity into a conceptual transdisciplinary framework [16, 17]. This framework has received support among both active and inactive samples, and provides the basis for the selection of phenotypes in the current study . Briefly, we proposed that genetic factors influence how an individual physiologically (e.g., body - temperature regulation) and subjectively (e.g., affective response, perceived exertion) responds to the experience of exercise . Physiological response influences how one subjectively responds to the experience of exercise (e.g., increased lactate during exercise may increase perceived exertion) and these subjective responses influence motivation to exercise (e.g., exercise self - efficacy, exercise intentions). Moreover, exercise behavior itself influences both how a person physiologically responds to the experience of exercise and gene expression, thereby recapitulating the framework . Importantly, this framework is meant to be dynamic such that the factors selected to represent physiological response, subjective response, and/or motivation can vary depending on the goals of each individual research study . The relationship between physiological changes induced by aerobic exercise (e.g., regulation of body temperature, heart rate, or blood pressure during exercise) and subjective responses to aerobic exercise (e.g., changes in affect during or immediately after exercise, ratings of perceived exertion or pain during an exercise bout) is one that has a clear influence on individual differences in exercise behavior . Bryan and colleagues found that physiological factors such as heart rate were related to mood response to exercise, and that mood response was a significant correlate of both motivation to exercise in the future and of current exercise behavior . Additionally, subjective experiences during exercise may be influenced by interpretations of exercise - induced physiological responses . For instance, increases in lactate levels during aerobic exercise may be perceived as painful to varying degrees across individuals, and this perceived pain will in turn influence subjective exercise experiences and potentially impact motivation to engage in exercise behavior in the future . Understanding potential influences on subjective response to exercise is especially important, given that affective responses to acute exercise have been found to predict long - term exercise behavior [19, 20]. Although the heritability of exercise participation in adults has been shown in twin studies to be approximately 50% (with peak heritability of 85%, occurring at age 19 - 20) [21, 22], there is a surprising lack of research regarding the role played by genetic factors for determining physiological and affective responses to exercise . These responses may serve as promising intermediate phenotypes for the linkage of genes to broader exercise participation phenotypes . Also important explained by de geus and de moor as the genetic variance causing differential responses to exercise training, given that the effects of exercise on health and fitness gains appear not to be uniform across individuals [24, 25]. One type of gene - by - exercise interaction that is relevant to the present study is the role of exercise in reducing the phenotypic effects of some detrimental genetic variants . For example, phares et al ., showed that sedentary individuals who possess two particular polymorphisms of the adr gene have unfavorable body composition . However, these individuals experience greater loss in percent body fat after 24 weeks of aerobic training in comparison with all other genotypes . It follows that weight loss interventions for individuals with this particular genotype would likely be successful if they focused on aerobic training . Thus, identifying particular genetic markers that are related to exercise behavior and physiological and affective responses to exercise may have clear implications for matching individuals to tailored exercise intervention programs . The goal of the current study is to determine whether genotypes predicted subjective physiological and affective responses to a 30-minute bout of aerobic exercise among sedentary individuals . Based on the literature and on our prior analysis of the relationships among a range of exercise response phenotypes (see for analysis and detailed information on the rationale for selection of phenotypes), the variables from the physiological responses to exercise domain included in our analysis were temperature, heart rate, systolic blood pressure, lactate, and norepinephrine, all measured as change scores from immediately prior to a bout of exercise to 30-minutes into the bout (just before the end of the bout). Genetic associations with vo2 max were also examined, as cardiovascular fitness is highly heritable [2730], and evidence exists for a strong genetic influence on athletic performance . Additionally, genetically influenced cardiovascular fitness traits play a role in determining individual experience of exercise intensity and perception of exertion during exercise . The variables selected from the subjective experience of exercise domain were affect (i.e., positive affect and affective valence), perceived pain, and rate of perceived exertion (rpe), which were also change scores measured from prior to the bout to just before the end of the bout . We chose the specific genetic factors for our analyses a priori based on evidence from the literature that they were linked to processes related to physiological and subjective responses during physical activity, general health and fitness traits, or because of evidence that they moderate responses to exercise interventions . A single nucleotide polymorphism (snp) in the fat mass and obesity - associated protein gene (fto; rs9930506) has been associated with obesity traits such as increased bmi and weight and susceptibility to obesity . Additionally, physical activity may slow down weight gain associated with the fto risk - allele . In addition, the -opioid receptor gene (oprm1) may be associated with pain sensitivity such that individuals possessing the rare g allele have an increased pressure pain threshold . Interestingly, this study also found gender differences in pain threshold among individuals with the g allele when heat pain was tested, such that women with this allele have lowered pain thresholds, and men exhibit higher pain thresholds . Snps located within in the slit2 gene(rs1379659), fam5c gene (rs1935881), kcnb2 gene (rs10505543), and rs10498091 (an snp associated with left ventricle mass) have all been found to be associated with echocardiography traits (e.g., left ventricle diastolic dimension, diameter, and systolic dimension) in a genome - wide association study . Another genome - wide analysis implicated creb1 in the prediction of submaximal exercise heart rate in response to exercise training [38, 39]. Thus, each of these snps was investigated in the current study in order to determine potential relationships with phenotypes related to physiological and affective response to an acute bout of aerobic exercise . Participants included in the present analysis were a subset of 238 individuals from a larger intervention study (costride) [17, 40] in which participants were randomly assigned to the stride exercise intervention (costride) or a health - and - wellness contact control condition (hw). Participants were men and women (ages 1845) who reported less than 90 minutes on average of at least moderate - intensity physical activity per week for the past three months . Individuals were excluded if they smoked cigarettes, were on a restricted diet, were taking psychotropic medications, were receiving treatment for any psychiatric disorder, were diabetic, had a history of cardiovascular or respiratory disease, had the flu or illness within the last month, or were pregnant (if female). All participants were required to be willing to be randomized to an intervention condition, to give informed consent, to be able to engage in moderate - intensity physical activity, to have a body mass index (bmi) between 18 and 37.5, and to have a regular menstrual cycle (if female). All participants were recruited from the denver - metro area and the university of colorado boulder community . The data reported herein are from assessments conducted prior to randomization, and the analysis and questions addressed are unique to this investigation . As described in detail below, we used the illumina human 1 m duov3 dna analysis beadchip to genotype the dna samples . The bead chips accommodate 4 samples each, and we ran a total of 50 bead chips . Thus, this experiment allowed for the genotyping of 200 individuals total . Due to limitations in funding, we were unable to genotype the remaining 38 individuals in the sample . Thus, individuals with the most complete baseline data (baseline dna sample, self - report questionnaire assessments, vo2 max fitness assessment, and submaximal exercise session) were selected to be genotyped out of the full sample . Statistical tests revealed no significant differences on demographic variables between participants who were included in genotyping procedures and those who were not included in genotyping procedures (details available from the first author). This reduced sample (n = 200) was comprised primarily of females (n = 160) and most participants identified as white (n = 137), followed by hispanic / latino (n = 22), asian american (n = 22), african american (n = 9), native american (n = 5), and mixed ethnicity (n = 5), the average age of participants at baseline was 28.68 (sd= 7.86) years old and mean body mass index (bmi; weight in kg / height in m) was 25.18 (sd = 4.72). On average participants reported an average of 28.14 minutes of voluntary physical activity in the past week (sd= 50.95), and reached an average vo2 max peak of 34.06 ml / kg / min (sd= 8.11). Prior to randomization to intervention condition and after giving informed consent, participants completed three sessions: (1) an orientation (baseline) session in which self - report questionnaire assessments were completed, (2) a vo2 max cardiovascular fitness assessment, and (3) a submaximal exercise session . Prior to exercise sessions, each participant was instructed to eat a meal comprised of both carbohydrates and protein and to consume at least 300 calories two hours before coming into the lab (e.g., if a participant is scheduled to come into the lab at 12:00 p.m. a researcher instructed him / her to eat the 300 calorie meal at 10:00 a.m. and no later). Participants were also instructed to drink at least 17 oz . Of water two hours prior to coming into the lab . Participants were instructed not to exercise on their own prior to the laboratory session, and not to consume alcohol during the 24 hours prior to testing . Further details regarding recruitment, selection of measures, and study procedures are available elsewhere . Consistent with established procedures, maximal oxygen capacity (vo2 max) was assessed during a balke protocol (a graded, incremental exercise test) on a motorized treadmill . Vo2 max was assessed with online computer - assisted open - circuit spirometry using the medgraphics cardi02/cp system . Prior to the fitness test, saliva samples (5 ml) were collected for dna extraction and measurements of height and weight were taken for calculation of bmi . Approximately one week after the fitness test, participants completed a standardized, short 30-minute bout of physical activity on the treadmill at 65% of their previously established vo2 max, calculated during the fitness test (vo2 max test session). Prior to beginning activity, an intravenous catheter was inserted by a nurse to collect blood samples during the bout . Intensity was maintained by measuring oxygen uptake and expired co2 for two to three minutes at the beginning of exercise and at 10 and 20 minutes during exercise . Lactate concentration and catecholamine levels (epinephrine and norepinephrine) were collected via blood samples immediately before activity began (11.5 ml), and 10 (5.5 ml) and 30 (11.5 ml) minutes into activity . Tympanic temperature was measured by taking an average of 2 - 3 temperature readings at each measurement . Readings of temperature, blood pressure, and heart rate were taken before activity, at 10 minutes, 20 minutes, and 30 minutes (directly before completion) during activity . Subjective experiences during exercise were assessed at six points during the submaximal session: five minutes prior to activity, immediately before activity began, and 10, 20, and 30 minutes into activity (directly before completion of the session). The present study focuses only on change scores created from subtracting the values obtained immediately before the exercise bout began from the values obtained 30 minutes into the bout . For the time points that occurred 10, 20, and 30 minutes into the exercise bout, participants were assessed while they were exercising the bout was not interrupted to make these assessments . While participants continued their session on the treadmill, a research assistant held up cards with the questionnaire items displayed on them . Participants indicated the number that they felt reflected their current subjective states, and their responses were manually recorded . Physiological measures were obtained at these time points using the iv catheter that was inserted prior to the bout . Positive affect was assessed using 3 items from the 12-item physical activity affect scale (paas). Participants rated their current state for each item using a 5-point scale (0 = do not feel to 4 = feel very strongly). The adjectives assessed by the 3-item positive affect subscale were enthusiastic, energetic, and upbeat (= .81). Affective valence during exercise was assessed using the 11-point single - item feeling scale (fs), which ranges from 5 = very bad to + 5 = very good . Perceived exertion was assessed using the 15-point single - item rating of perceived exertion (rpe) that ranges from 6 to 20 (6 = no exertion at all, 20 = maximal exertion). Perceived pain was assessed using a single - item 12-point borg category ratio-10 scale (cr10) (0 = no pain at all, 10 = extremely intense pain). Samples were genotyped on the illumina infinitum assay platform using the human 1 m duov3 dna analysis beadchip (illumina, inc ., san diego, ca, usa) following the manufacturer's protocol at the university of colorado, boulder . In this assay, the dna is then resuspended in hybridization buffer and applied to the bead chip array for an overnight incubation . The amplified and fragmented dna samples anneal to locus - specific 50-mers (covalently linked to one of over 1,000,000 bead types) during the hybridization step . The samples then undergo single - base extension and staining, followed by more washing . The arrays are allowed to dry, and then scanned using the illumina iscan system . Genotype calls were made using illumina's genomestudio software in conjunction with the genome studio genotyping module . Additionally, we excluded snps with a minor allele frequency (maf) of <10% and snps that showed significant deviation from hardy - weinberg equilibrium (p <1 10). Following these quality control checks, although we used a genome - wide approach to genotyping, we only tested a total of 14 snps which were selected for analysis in this study based upon their potential association with aerobic exercise response phenotypes suggested by prior studies (see table 2 for hardy - weinberg p values and minor allele frequencies for each snp tested). Our search was conducted primarily using pubmed, and was focused on snps that were directly associated with specific phenotypes of interest and to traits that may be associated with those phenotypes . Analyses were run using the snp and variation suite for genetic analysis (svs) (version 7.5.6, golden helix inc ., bozeman, mt).the 14 snps selected for inclusion based on our search were tested for associations with the phenotypes using a correlational trend test assuming additive effects of allele dosages for each snp (i.e., homozygous for the minor allele = 0; heterozygous = 1; homozygous for the major allele = 2). These analyses focused on correlations between particular snps suggested by the relevant literature and exercise response phenotypes drawn from our previous work and the existant exercise literature . Due to the fact that both exercise phenotypes and candidate snps were selected a priori based on the literature as well as our transdisciplinary framework, critical alpha for all tests was maintained at the .05 level for all analyses . Additionally, given that the aim of this study was to examine changes in physiological and subjective responses to exercise over the course of the 30-minute exercise bout, it was not necessary to compare subjects cross - sectionally at the baseline or 30-minute time points . Rather, all phenotype values were determined using a change score created by subtracting each subject's baseline values from the values obtained by that subject 30 minutes after the exercise bout began . In order to determine whether allele frequencies for all snps examined in this study were significantly different across racial / ethnic groups, additionally, major and minor alleles, as well as minor allele frequencies (mafs) for caucasians, african americans, asians, and hispanic / latino participants are reported in table 3 . Significant differences in genotype across racial / ethnic groups were found for three snps, rs1935881 (10,200) = 19.25, p = .037, rs1799971 (10,200) = 38.13, p <.001, and rs8044769 (10,200) = 21.54, p = .018 . For rs1935881, the maf is lower among asians . For rs1799971, an maf of 0 was found in african americans . For rs8044769, results of associations between snps and exercise response phenotypes presented below are uncorrected (no pca correction applied). A total of 10 different phenotypes were examined for association with genetic variants in this study . Given that many of these phenotypes may have common underlying physiological bases, we tested for associations between these phenotypes . In the following results, all phenotypes tested and reported (except for vo2 max) refer to a change score created by subtracting preexercise values from the values obtained 30 minutes into the exercise bout . Vo2 max was significantly correlated with change in lactate (r = .177, <.05), heart - rate (r = .434, p <.01), systolic blood pressure (r = .193, p <.01), and rate of perceived exertion (r = .157, p lactate change was correlated with temperature change (r = .214, p <.01), heart rate change (r = .429, p <.01), systolic blood pressure change (r = .208, p <.01), change in affective valence (as measured by the feeling scale) (r = .173, p <.05), and pain change (r = .215, p <.05). Norepinephrine change was significantly correlated with positive affect change (r = .174, p <.05) and pain change (r = .165, p <.05). Temperature change was significantly correlated with heart - rate change (r = .172, p <.05) and affective valence change (r = .149, p <.05). Heart rate change was significantly correlated with systolic blood pressure change (r = .185, p <.05) and rate of perceived exertion change (r = .231, p <.05). Rate of perceived exertion change was significantly correlated with affective valence change (r = .163, p <.05) and pain change (r = .316, p <.01). Finally, positive affect change was significantly correlated with affective valence (r = .455, p <.05). The creb1 snps rs2360969 and rs2253206 were associated with temperature change during exercise (rs2360969, r = .17, p = .02; rs2253206, r = .17, p = .02) indicating that for rs2360969, individuals with the t allele had greater changes in temperature over the course of the exercise, and for rs2253206, individuals with the a allele had greater changes in temperature during exercise . These same snps were also significantly associated with vo2 max (rs2253206, r = .17, p = .01; rs2360969, r = .14 p = .049), such that for rs2253206, individuals with the g allele had higher vo2 max, and for rs2360969, individuals with the c allele had higher vo2 max . The oprm1 snp rs1799971 was significantly associated with lactate change during exercise (r = .17, p = .02), norepinephrine change during exercise (r = .16, p = .03), and change in rpe during exercise (r = .14, p = .048), indicating that individuals with the rare g allele had greater changes in lactate, norepinephrine, and rate of perceived exertion change over the course of exercise . The fto snp rs8044769 was related to change in positive affect during exercise (r = .16, p = .03), and individuals with the c allele had greater change in positive affect over the course of the exercise . The fto snp rs3751812 was associated with positive affect change during exercise (r = .14, p = .04), such that individuals with the t allele experienced greater changes in positive affect . The fto snp rs9941349 was significantly related to change in systolic blood pressure during exercise (r = .15, p = .04), and individuals with the t allele experienced greater increases in systolic blood pressure during exercise . The fto snp rs7201850 was significantly related to change in systolic blood pressure during exercise (r = .17, p = .027), with individuals possessing the t allele experiencing greater increases in systolic blood pressure over the course of the exercise bout . The slit2 snp rs1379659 was associated with norepinephrine change during exercise (r = .18, p = .01), with individuals with the g allele experiencing greater changes in norepinephrine during exercise . Finally, the fam5c snp rs1935881 was associated with change in norepinephrine during exercise (r = .16, p = .03). Individuals with the g allele had greater changes in norepinephrine over the course of the exercise bout (all associations initially reported in the manuscript changed only slightly after applying the pca correction . Pca corrected p - values for genotype - phenotype associations are as follows: rs1799971 and norepinephrine change (p = .104), rs1799971 and rpe change, (p = .038), rs8044769 and positive affect change (p = .038), rs3751812 and positive affect (p = .036), rs1935881 and norepinephrine change (p = .059), rs1379659 and norepinephrine change (p = .010), rs9941349 and systolic blood pressure change (p = .053), rs7201850 and systolic blood pressure change (p = .04998), rs2360969 and temperature change (p = .031), rs2253206 and temperature change (p = .066), rs1799971 and lactate change (p = .015), rs2253206 and vo2 max (p = .035), and rs2360969 and vo2 max (p = .032)). Due to the fact that several of the variants that were associated with a particular phenotype were in the same gene, it is likely that these snps are in high - linkage disequilibrium with one another . These snp sets within single genes are rs3751812 and rs8044769 in fto, both significantly associated with positive affect change, rs2253206 and rs2360969 in creb1, both significantly associated with temperature change as well as vo2 max, and rs7201850 and rs9941349, both in fto, both significantly associated with systolic blood pressure change . To examine whether these snps were in ld, we ran correlations on each set of 2 snps in the same gene that were associated with the same phenotype . The correlation between rs2360969 and rs2253206 was .805 (p <.01), the correlation between rs3751812 and rs8044769 was .676 (p <.01), and the correlation between rs7201850 and rs9941349 was .938 (p <.01). In order to further explore the direction of the relationship of genotype on exercise response, we graphed the adjusted means for each genotype of three snps which demonstrated particularly robust relationships with exercise response phenotypes . We graphed the relationship between rs2360969 and temperature 30 minutes into the exercise bout, between rs1799971 and rpe 30 minutes into the exercise bout, and between rs8044769 and positive affect score 30 minutes into the exercise bout . As can be seen in figure 1, individuals with the tt genotype of rs2360969 showed the highest temperature after 30 minutes of aerobic exercise, controlling for baseline temperature . In figure 2, we show that individuals with the ag / gg genotypes on rs1799971 show greater rpe after 30 minutes of aerobic exercise than individuals with the aa genotype, controlling for baseline rpe . In figure 3, we show that individuals with the cc genotype in rs8044769 show the highest ratings of positive affect after 30 minutes of aerobic exercise, controlling for baseline positive affect . The present study replicated prior findings suggesting that snps in the creb1, fto, oprm1, slit2, and fam5c genes are all related to phenotypes encompassing various responses to exercise . Our study tested conceptually relevant phenotypes that to date had not been explored in this way inanyother exercise research . Given that the physiological response to aerobic exercise involves a complex interplay of metabolic, cardiovascular, musculoskeletal, ventilator, and hormonal functions, these genes and snps are likely to explain only a small portion of the variability in individual differences in response to aerobic exercise . Subjective responses to exercise may be yet more complex, involving sociocultural factors, effects of previous exercise experiences, and anticipated consequences / rewards of exercise . Additionally, our findings suggest that individuals performing equivalent bouts of aerobic exercise may have vastly different subjective perceptions of this exercise (overall experiences which can range from negative to positive), and that these perceptions may be influenced by genotype . Giving sedentary individuals information about their propensity to respond to exercise in a particular way could provide useful insight, allowing these individuals to temper their expectations of what aerobic exercise should feel like for them or allowing intervention designers to incorporate external reinforcement contingencies (e.g., social interaction) for individuals who are less likely to experience intrinsic rewards from exercise . Despite several inherent limitations, the present study's findings linking genetic variants to exercise responses among sedentary individuals presents promising initial evidence associating genes and exercise behavior . However, it is unlikely that variation at a single genetic locus could fully explain variation in physiological and subjective responses to exercise more possibly, there are many genetic variables influencing this phenotype, each of which contributes only bya small fraction of the observed variation . When combined into a genetic composite, these loci would likely correlate more strongly with phenotypic response . So, although the correlations between genotype and exercise response found in this study are not large, they represent a necessary first step in forming genetic composite scores that are likely to be more highly correlated and significantly predictive of exercise responses . In summary, linking snps to specific physiological and psychological mechanisms that contribute to exercise response will assist in informing individually tailored exercise programs, as well as deepen our understanding of the relationship between genetics, physiology, and psychology underlying health behaviors associated with cancer prevention . Our study showed that for rs3751812, the presence of a t allele increased change in positive affect during exercise . This finding is somewhat at odds with previous work suggesting that tt individuals have higher bmi on average . Rs3751812 was found to have a strong association with bmi in african - derived populations, with the tt genotype predicting increased bmi . However, the relationship between bmi and positive affective response to exercise is unclear because the hassanein study did not include information about exercise behavior of participants . It is possible that rs3751812 individuals are predisposed to have higher bmi, but if they engage in aerobic exercise, they are likely to have a more positive affective response . This is one example of how knowledge about the effect of a particular genotype could be used to prescribe tailored interventions for overweight individuals with the tt genotype, exercise could be recommended as a more effective weight loss tool, given that these individuals have a more positive affective response to exercise . We also found that for rs8044769, the tt individuals had greater changes in positive affect during exercise . In a hispanic american sample, rs8044769 was found to be weakly associated with waist - to - hip ratio, and the c allele showed an association with variation in bmi . Prior research suggests that the c allele of rs8044769 is associated with greater variation in bmi . This snp seems to be related to body fat mass, predisposition to obesity, and response to aerobic exercise yet the nature of this relationship requires further exploration . Creb1 is a key component of long - term cardiac memory formation (specific t - wave patterns on an electrocardiogram), as well as long - term memory formation in the brain [54, 55]. Our results indicate that for the creb1 snp rs2253206, individuals with the a allele (ag genotypes, and to a greater extent aa genotypes) have a greater change in temperature during exercise . If greater temperature change while exercising translates into a more unpleasant subjective exercise experience, then our findings suggest that the aa individuals (and to a lesser extent ag individuals) may have less pleasant subjective experiences of exercise than gg individuals . Rs2253206 was shown to be strongly associated with heart rate (hr) change in response to a 20-week endurance training program, with gg and ag genotypes and showing 57% and 20% better change in hr than the aa participants . Our results make sense in the context of the rankinen findings, as the aa individuals may have more unpleasant exercise experiences due to increased temperature, which could influence their ability to exercise effectively (and thus decrease the heart rate improvements they can obtain from an exercise intervention). We also found that this snp was related to vo2 max (an indicator of cardiovascular fitness), such that the gg individuals had greater vo2 max than ag individuals, who had greater vo2 max than aa individuals . These results also coincide with our findings and the findings from previous research, as gg individuals may be more fit to begin with, and also more capable of gaining increased fitness through training, due to the fact that they experience exercise as less painful . Additionally, we found that for rs2360969, tt individuals experienced greater change in temperature than did ct and cc individuals . Rs2360969 has also been shown to be related to heart rate response endurance training [38, 39], however, these studies did not state direction of effect for this snp . In our analysis, rs1799971 (the a118 g polymorphism) was related to rpe, as well as to lactate change during exercise and norepinephrine change during exercise . For all three of these phenotypes, individuals with the rare g allele showed greater change during the exercise bout . Previous research on this snp has found that individuals with the g allele (genotypes of either ag or gg) demonstrated higher pressure pain thresholds than individuals with the aa genotype . This study also found that when heat pain was tested, a sex by genotype interaction emerged, such that the g allele was associated with lower pain ratings among men but higher pain ratings among women . The a118 g variant has greater binding affinity for -endorphin (an exogenous opioid that activates the mu opioid receptor), which is one possible mechanism by which this snp could influence pain sensitivity . The relationship between rs1799971 and subjective responses to pain may extend to the pain and exertion experienced during aerobic exercise . Given that our sample was 79.5% female, our findings of greater lactate, norepinephrine and rpe change over the course of exercise for the gg / ag group is in the same direction as the findings for females in the fillingim study . These results lend further support to the idea that individuals (and perhaps particularly women) with the ag / gg genotype have lowered pain threshold, and the present study suggests increases in lactate and norepinephrine as possible physiological explanations, at least in the context of aerobic exercise - induced pain . Prior studies have shown that rs1379659 in fam5c is associated with echocardiographic traits, and specifically left ventricular systolic dimension . The results of our study suggest that it is also associated with change in norepinephrine in response to exercise . To date, research has not examined the relationship between fam5c and aerobic exercise response . Given the connection between this gene and cardiac function, examining the potential relationship between fam5c and aerobic exercise would provide a logical next step for research in this area . Previous research has demonstrated an association between the slit2 snp rs1935881 and echocardiographic traits, specifically left ventricular diastolic dimension . The results of this study suggest that it is also related to norepinephrine change during exercise . Further research is needed to elucidate more specific relationships between slit2 and response to aerobic exercise . The genes discussed above represent potential candidates for further explanation in terms of their relationship to exercise response phenotypes . More than a decade's worth of research on the psychophysiological responses associated with exercise has demonstrated that the subjective experience of exercise, how sensations are remembered, anticipated, and interpreted, is closely tied to subsequent exercise behavior [14, 19, 20, 47, 57]. A better understanding of the genetic basis for subjective responses to aerobic exercise may have the potential to lead to more effective and sophisticated intervention designs . Eventually, these advances in the basic science of exercise response could lead to the implementation of interventions tailored on the level of individual genetic variants . Primary prevention of cancer through behavioral intervention is now a top priority of the nci . This approach is intuitive given that approximately 30% of total cancer deaths are related to energy imbalance (e.g., excessive adiposity) [58, 59]. Physical inactivity is not the only contributing factor to energy imbalance, but it is a major contributing factor as trends clearly show that the least physically active regions of the country are also the most obese . The hopeful perspective on behavioral intervention for physical activity is that even small increases in the total amount of participation accumulated per week stands to lead to meaningful differences in cancer risk . For example, found evidence for a 38% reduction in risk for breast cancer with every additional 60 minutes of physical activity engaged in per week . The link between physical activity participation and reduced risk for cancer, especially of the colon, breast, and endometrium is convincing, but also dependent upon good adherence [36, 61]. For this reason, it is imperative that researchers continue to search for ways to improve the likelihood of adherence to behavioral interventions . One way to achieve this goal may be through increasing the amount of focus that is placed on subjective response phenotypes and their underlying genetic variants . Developing a better understanding of the link between genes, exercise - relevant physiological mechanisms, and the resulting exercise - response phenotypes is a first step towards tailoring individualized exercise programs that would likely increase adherence and lead to improved health outcomes and decreased rates of cancer and other diseases . As with all research that involves genetic analyses, we cannot rule out the possibility that other genetic factors, including rare or common snps, insertions, deletions, or copy number variants, could play a role in determining the physiological responses to exercise that were measured in this study . The phenotypes investigated in this study are likely to be polygenic traits, such that numerous genes and snps other than those examined in the present study may all contribute to these exercise response phenotypes . In contrast, the extent of the pleiotropic effects of the genes and snps investigated in this study are unknown . Thus, it is possible that the polymorphisms that influence exercise response may also be more strongly associated with other, possibly unrelated phenotypes that led to our findings . Another limitation of note is the present study's lack of power to detect moderation effects of demographic variables . It is possible that variables such as age or ethnicity could moderate the associations between genetic variation and response to exercise . Additionally, the results of this investigation are based on one single, standardized, bout of moderate - intensity exercise . For this reason, our results cannot be generalized to subjective exercise experiences that occur under less regulated circumstances (i.e., when type of activity, intensity, and duration are individually determined). Despite this limitation, there are many examples from the literature in which subjective responses to exercise are measured and analyzed based on a single bout of standardized exercise (e.g., [19, 20, 6265]) and therefore, our procedures and analyses are in concert with the approach previously established by the field . Importantly, the purpose of the present investigation was to understand how genetic variants are associated with particular subjective responses to exercise when the parameters of the exercise experience are standardized across all individuals . In the present study, this level of standardization was achieved by having all participants perform the same activity (treadmill walking), for the same duration (30 minutes), at the same intensity (65% of each individual participant's previously established vo2 max). Further, efforts were made to standardize variables external to the exercise bout as well (i.e., instructions detailing recommended calorie and water consumption prior to the bout described in section 2). It remains to be seen whether the snps and genes reported in this study to be related to exercise response phenotypes would show an association to these same phenotypes in other studies examining different types, duration, and intensity of exercise sessions . However, as noted, the size of the associations changed negligibly after a pca correction, suggesting that the population substructure did not play a major role . Overall, replication is needed in order to confirm findings from the present study, and to better understand the functional significance of these genes and snps in relation to physiological and subjective responses to aerobic exercise . The purpose of this paper was to explore the genetic underpinnings of individual physiological and subjective responses to aerobic exercise . One strength of this study was its focus on a sedentary population, a group that has been rarely tested in terms of associations between genetics and exercise phenotypes . The relationship between particular genetic variants and responses to exercise has important implications for the prevention of cancer via increasing exercise behavior in sedentary populations . Future studies designed to test genetic influences on a wide range of exercise response phenotypes would help to advance this goal, potentially leading to a panel of markers important for characterizing the physiological and subjective response to exercise . Moreover, giving feedback to sedentary individuals regarding the genetic basis for their strengths and weaknesses in fitness / exercise / sports activities could be a potentially useful motivational tool for increasing exercise behavior [13, 66]. In sum, expanding our understanding of the association between genetics and exercise response phenotypes has a myriad of implications for helping to increase exercise behavior in sedentary individuals, an outcome which is crucially important for the reduction of morbidity and mortality associated with cancer.
Dental implants are now a reliable solution for the functional and esthetic rehabilitation of partially and completely edentulous patients; this has been demonstrated by long - term clinical trials, with survival rates of greater than 95% [13]. In order to achieve long - term survival, osseointegration of the dental implant needs to occur; that is, a direct connection must be established between the bone and the implant surface, without the interposition of fibrous tissue; once established, this close bond must be maintained over time, resulting in a clinically asymptomatic fixation of the implant under functional load . Osseointegration is a complex phenomenon and depends on many factors; some are related to the implant (material, macroscopic design, and implant surface), others to the surgical - prosthetic protocol (surgical technique, loading conditions, and time), and others to the patient (quantity / quality of bone at the receiving site and the host response) [4, 5]. Although survival rates of dental implants are now high, there still remains a seemingly unavoidable number of failures: either cases in which correctly placed implants do not integrate with the bone or cases of peri - implant tissue infection [6, 7]. To be specific, failure to osseointegrate and peri - implantitis are the most frequent causes of early implant failure [3, 6, 7]. Such events occur during the early stages of healing (within 2 - 3 months of implantation) and therefore before the implant is functionally loaded with the prosthetic restoration; these failures are unevenly distributed within the general population and tend to occur in some subjects in particular . In these individuals early failures occur even when optimal materials are used, surgical protocols are strictly followed, and the quantity / quality of bone at the recipient site is sufficient [68]. All these observations would suggest the existence of specific patient - related risk factors; this prompts an investigation into the regulatory mechanisms controlling bone metabolism, bone remodelling, and bone turnover [9, 10]. It is a fat - soluble vitamin which promotes the absorption of calcium in the intestine and regulates calcium and phosphate homoeostasis in the tissues and it is a fundamental element in the mineralization of bones and teeth [1113]. It also acts as a hormone and is vital for the health of the blood vessels and the brain [14, 15]. It has been demonstrated that vitamin d plays a crucial role in the health of the cardiovascular tract, the immune system, and the respiratory tract [18, 19]. Vitamin d in an inactive form (cholecalciferol or vitamin d3) is ingested or produced in the skin on exposure to sunlight [11, 12]. This inactive form undergoes double hydroxylation in the liver and the kidneys and is thereby transformed into its active form, known as either calcitriol or 1,25-dihydroxyvitamin d3 [11, 12]. This active form exerts its action on various tissues by binding to the vitamin d receptors and regulating the transcription of specific target genes [1223]. Serum levels of vitamin d in the 25(oh)d form are the most accurate way of determining vitamin d status: a subject with <10 ng / ml is considered to be vitamin d deficient; one with 1030 ng / ml is considered to have low levels of vitamin d. the optimal blood level of vitamin d is a value> 30 ng / ml [12, 13]. Vitamin d deficiency is high in the general population: in italy, for example, it is estimated that about 80% of people can be deficient, particularly in the northern regions where exposure to the sun is lower . This deficiency increases with age and encompasses the majority of the elderly population of italy who are not taking vitamin d supplements . Until a few years ago, the guidelines estimated that the daily intake of vitamin d required to maintain adequate blood levels was 200 iu (5 mcg) in adults aged between 19 and 50, 400 iu (10 mcg) in adults aged between 51 and 69, and at least 600 iu (15 mcg) in those over 70 [12, 13]. These guidelines have now been revised upwards and it is currently believed that the amount of vitamin d which should be taken daily is 2000 iu (50 mcg) and up to 4000 (100 mcg) in the case of, for example, pregnant women [12, 13]. There is now substantial literature on the negative effects of low levels of vitamin d, especially in severely compromised patients: vitamin d deficiency seems to be associated with increased mortality, cardiovascular events, and reduced functioning of the immune and musculoskeletal systems [1519, 2123]. On the other hand, normalizing levels of vitamin d can lead to substantial benefits for critically ill patients, with effects on the muscles, the respiratory system, the heart, and the immune system [18, 21, 23]. Despite the importance of vitamin d and its effects on bone metabolism [11, 12] few studies have, to date, investigated the effects of its depletion on the osseointegration of dental implants [9, 2435]: almost all these studies have been done on animal models [2432] and very few on humans [3335]. The purpose of this retrospective study was therefore to investigate any possible correlation between low blood levels of vitamin d and early implant failure (failure occurring in the four months prior to the full restoration of the implant, because of a lack of osseointegration or because of infection). All patients who had been treated with morse - taper connection dental implants (leone implant system, florence, italy) [3, 8] inserted to support fixed prosthetic restorations in one single dental centre (gravedona, como, italy), in the period between january 2003 and december 2015, were evaluated for possible enrollment into this retrospective study . Patients were enrolled into the study if they were over 18 years of age, had good oral and general health, and had not undergone bone regenerative therapy prior to implant placement . The exclusion criteria were incomplete medical records, the presence of specific systemic diseases (uncontrolled diabetes mellitus, immunodeficient states, and bleeding disorders), and the abuse of alcohol and drugs; patients undergoing radiotherapy and chemotherapy and those who were pregnant were also excluded . All the data used for the study were obtained from the medical records of the patients enrolled . The patient data was evaluated; this included gender (male or female), age at time of surgery, history of chronic periodontal disease, smoking habits, and serum vitamin d levels . Vitamin d levels were taken from blood tests, which had been requested two weeks prior to surgery . The medical records also contained a range of information as regards the implant or implants, that is, their site (maxilla or mandible), location (incisor, canine, premolar, and molar), the length and diameter of the implant, the type of prosthetic restoration, and the loading conditions . These included their cause (lack of osseointegration in the absence of infection, infection of the peri - implant tissues or peri - implantitis, or implant failure due to progressive bone loss caused by to prosthetic overload). It also included their classification: early failure, occurring in the early healing period, that is, the four months after implant placement, prior to the placement of restoration and loading, or late failure, occurring after loading . There were also details of any possible biological complications (peri - implant mucositis and peri - implantitis) and/or prosthetic complications (mechanical and/or technical). All implants were inserted under the same strict protocol by the same specialist (c. m.) who had 25 years' experience in implant dentistry, in the period between january 2003 and december 2015 . The implants were inserted after raising a full thickness mucoperiosteal flap; the implant site preparation and implant placement were performed in compliance with modern surgical protocols and in accordance with the manufacturer's instructions . After placement, cover screw was positioned and the implants were submerged . Immediately after positioning, patients were prescribed antibiotic coverage with 2 g of amoxicillin (or 600 mg of clindamycin in patients allergic to penicillins) for 6 days . Patients were given detailed instructions on oral hygiene and were prescribed chlorhexidine 0.12% mouth rinse twice a day for 6 days . The implants were left to heal submerged for a total period of 4 months, to allow undisturbed healing and achieve osseointegration . After 4 months of undisturbed healing, the patient was recalled for the implant to be uncovered . Two weeks later, the sutures were removed and an impression was taken for the manufacture of the temporary restoration . The temporary restoration was maintained in situ for 3 months, in order to monitor the response of the implant, as well as the peri - implant tissues, to masticatory load; at the end of this period, the temporary restoration was replaced with the final restoration . The final restorations were metal porcelain, cemented with a zinc oxide - eugenol cement . A periapical radiograph was taken to check on the sealing of the restoration . All patients were included in a follow - up protocol with an annual check - up at one of the scheduled professional oral hygiene sessions . Early implant failure, occurring within 4 months after implant placement and therefore prior to placement of the prosthetic restoration and the functional load of the implant, was the primary outcome studied . Early implant failures were divided into two different categories: (a) early failures due to lack of osseointegration and subsequent implant mobility, in the absence of clinical signs of infection; (b) early failures due to infection of the bone tissue around the implant, with inflammation (peri - implantitis) of peri - implant tissues and the presence of fistula, pain, swelling, pus and/or exudate, pocket depth> 6 mm with bleeding, and marginal bone resorption> 2.5 mm . All the data retrieved from the individual medical records were recorded on a generic spreadsheet (excel, microsoft office, redmond, ma, usa) which was used for the descriptive, qualitative, and quantitative analyses . The mean, standard deviation, median, and confidence intervals were calculated for the quantitative variables (e.g., patients' age and vitamin d levels in serum). A patient - based technique was used to calculate implant survival . In this analysis, the event was implant failure: thus in patients receiving more than one implant, the occurrence of even a single implant failure led to the patient being classified as a failure . The influence of different variables on implant survival was taken into consideration: gender (male or female), age at time of surgery (three age groups were examined: <40, 4060 years, and> 60 years), smoking habits (regardless of the actual number of cigarettes smoked), a history of chronic periodontitis, and serum levels of vitamin d. in the analysis of serum levels of vitamin d, three classes of patients were considered: severely deficient patients (serum vitamin d <10 ng / ml), patients with low levels (serum vitamin d between 10 and 30 ng / ml), and patients with adequate levels (serum vitamin d> 30 ng / ml). The influence of each of these variables on implant survival was calculated using the chi square test . The overall implant survival, the survival within the different groups, and the analysis of the influence of the different variables on survival were all made using dedicated statistical analysis software (spss 17.0, spss inc ., of the 915 patients originally evaluated for enrollment in this study, 93 presented with conditions corresponding to the exclusion criteria and were therefore excluded from the assessment . By contrast, 822 patients (mean age 57.3 14.2 years; median age 58; range 1890; and 95% ci, 56.358.2), receiving 1625 implants, did not have any of the conditions contained in the exclusion criteria and were therefore enrolled into this retrospective study . The distribution of patients by groups, with relative incidence of failures, was reported in table 1 . In total, 27 early failures were recorded (19 due to failure of osseointegration and 8 due to peri - implant tissue infection), with an overall incidence of 3.2% . No differences were observed in the incidence of early failures between males and females (p = 0.97) nor according to age at time of surgery (p = 0.98). Although the percentage of early failures in smokers was slightly higher than that detected in nonsmokers, there was no statistically significant difference (p = 0.56) between these two groups of patients . The same was true for patients with a history of periodontal disease; they displayed a slightly higher incidence of early failures than patients who had not been affected by periodontitis, but this difference was not significant (p = 0.73). The average serum level of vitamin d in the general population was 29.9 ng / ml (12.1; median 29; range 573; and 95% ci, 29.130.7). In patients in whom early implant failure occurred, the average serum level of vitamin d was 25.5 (13.2; median 24; range 855; and 95% ci, 20.630.4). Statistical analysis reported a rather low incidence of early failures (2.2%) in patients with blood vitamin d levels> 30 ng / ml . The incidence of early failure was almost double in patients with insufficient serum levels of vitamin d (1030 ng / ml) and became even higher (9.0%) in patients with serious vitamin d deficiency . Although the statistical analysis revealed a trend toward an increased incidence of failure in patients with severe vitamin d deficiency, the analysis did not reveal a statistically significant difference (p = 0.15) in the incidence of early implant failure in these three groups of patients . Similar results (p = 0.14) were obtained comparing the incidence of failures in the group of severely deficient patients (2/22: 9.0%) with the incidence of failures in all other patients (25/800: 3.1%). Finally, the statistical analysis did not reveal a significant difference (p = 0.13) when comparing the incidence of failures in the group of patients with serum vitamin d levels> 30 ng / ml (9/394: 2.2%) with the incidence of failures in all other patients (18/428: 4.2%). A relatively small number of experimental studies has attempted to investigate the effects of vitamin d on the osseointegration of dental implants [2432]. The majority of these studies would appear to indicate a positive effect of vitamin d on osseointegration, but it is not yet entirely clear whether supplementation would promote the healing of peri - implant bone tissue clinically [24, 3335]. A recent review of the literature on animal studies has shown that vitamin d supplementation can stimulate new bone formation and increase the contact between the bone and the surface of titanium implants . Specifically, kelly et al . Demonstrated that vitamin d deficiency could significantly compromise the establishment of osseointegration of ti6al4v implants in rats . In an experimental study on ovariectomized rats, the authors demonstrated that vitamin d deficiency could impair the formation of peri - implant bone; the normalization of blood levels via supplementation of vitamin d stimulated new bone formation . Similar results were reported by zhou et al ., who found an increase in osseointegration in osteoporotic rats given vitamin d supplements, and wu et al ., who demonstrated an increase in the percentage of contact between bone and implant in diabetic rats given vitamin d supplements . Finally, liu et al . Reported that the administration of vitamin d could increase the fixation of dental implants in mice suffering from chronic kidney disease . A further possibility for study, in order to understand the effects of the administration of vitamin d on bone healing of the peri - implant tissues, is that of coating the implant surface with vitamin d [3032]. Evaluated the effect of the topical application of vitamin d to the surface of implants inserted in postextraction sockets in dogs, with histological and histomorphometric analyses of tissues removed at 12 weeks . Topical application of vitamin d increased the percentage of bone to implant contact of 10% . Similarly promising results were reported by cho et al . In a histological and histomorphometric study on rabbits, where the coating of anodized implant surfaces with a solution of poly(d, l - lactide - co - glycolide) plga and 1,25-dihydroxyvitamin d3 (1,25-(oh)2d3) stimulated the apposition of new bone on fixtures . Finally, in a further experimental work in rabbits, implants with a surface coated in 1,25-(oh)2d3 have shown an improved tendency to osseointegrate compared to noncoated implants; however, this difference was not statistically significant . Unfortunately, very few clinical studies have so far investigated the effects of vitamin d deficiency on osseointegration and on bone regeneration in dentistry [3335]. This is probably due to the fact that there are many factors which can determine the success or failure of dental implants; the attention of clinicians has been mostly focused on drawing up surgical and prosthetic protocols and identifying new materials and implant surfaces to improve osseointegration, rather than on the analysis of patient - related risk factors [69]. In a recent clinical work, alvim - pereira et al . Found no relationship between polymorphism of the vitamin d receptor and implant failure . In a randomized, investigated the effects of supplementation with a combination of vitamin d3 (5000 iu) and calcium (600 mg) on the formation of new bone following maxillary sinus lift . Ten patients were assigned to the test group and given vitamin d and calcium; ten other patients were assigned to the control group and received only calcium . Six to eight months after surgery for bone regeneration, bone samples were taken for histological analysis during implant placement . Although supplementation with vitamin d3 would have increased the serum levels of vitamin d with potentially positive effects on bone remodelling at the cellular level, no statistically significant difference was demonstrated between the two groups at the histological level . The results of our study would appear to suggest that a severe deficiency of vitamin d in the blood might be related to an increase in the incidence of early implant failure . In fact, the incidence of early implant failure was rather low (2.2%) in patients with normalized levels of vitamin d in the blood (> 30 ng / ml), rose to almost double (3.9%) in patients with insufficient serum levels (1030 ng / ml), and were rather high (9.0%) in patients characterized by severe deficiency states . However, despite the tendency to an increased incidence of early failure in patients characterized by deficiency states, the differences between the three groups of patients were not statistically significant (p = 0.15). Our study also confirms that the serum values of vitamin d in the local population are rather low: we found that the proportion of patients with insufficient levels was 49.4% and that the percentage with a severe deficiency was 2.7% . This is not surprising, as most of the patients treated came from northern italy and southern switzerland, regions where exposure to sunlight is somewhat reduced for long periods of the year . In the light of this, the administration of vitamin d in the weeks prior to placement of a dental implant could be useful, particularly in patients with severe deficiency states; in these patients, vitamin d supplementation should be maintained for the whole life, in order to guarantee a good remodelling of the bone around the implant . This study has the distinction of being one of the first clinical studies carried out on a large number of patients to investigate the possibility of an association between low blood levels of vitamin d and the incidence of early failure in implantology . By restricting the analysis to early failures, occurring in the first period of healing and therefore prior to placement of the prosthetic restoration, we were able to focus our research and avoid a range of factors (linked to the restoration itself and the prosthetic load) which could have confused the issue in the study . It is, in fact, well known that implant survival and thus osseointegration depend on a large number of factors (related to the surgical and prosthetic protocol, the materials used, and lastly the patient) [4, 5] and it can be difficult to identify which of them might be determining the success or failure of the treatment . In order to avoid this and to limit the confounding factors, the same materials were used for all the patients in this study (the same implant system for all the patients) [1, 3, 8]. In addition, the same surgical protocol was used, involving submerged healing in the absence of prosthetic loading . Thus the only possible confounding factors were the different quantity and quality of bone at the implant receiving sites and the patients' responses: these are unavoidable factors . However, some of those categories of patients most at risk of implant failure (patients undergoing bone regeneration to create the conditions for the positioning of the implant fixtures or those with particular medical conditions which might increase the risk of treatment failure) were excluded in the present study . It is a retrospective work, in which the number of patients having a severe deficiency of vitamin d in the blood was low (only 22); thus the presence of even just one less failure in this group would have led to quite different results . It is possible that some residual confounding may have biased the association between vitamin d and implant failures that we observed . For instance, this study did not investigate the influence of other patient - related factors (e.g., the bone quality) which can affect implant survival in the period immediately following implant placement . In addition, if subjects with low levels of vitamin d were also likely to receive more than 1 implant, their risk of being classified as failures may increase . However, no patient in this study experienced more than 1 failure, and the probability of implant failure was not higher (1.5% versus 2.1%) in presence of another implant . Therefore, randomized, controlled clinical trials are needed to confirm the presence of an association between low serum levels of vitamin d and an increase in the incidence of early failure in implantology . It would be appropriate to assess whether supplementation of vitamin d in the weeks before the operation could lead to a reduction in early failures, whether due to lack of osseointegration or implant infection . Further scientific studies with an appropriate design and a more rigorous statistical analysis will therefore be required in order to thoroughly investigate this issue . Until now, very few studies, and those mainly on animal models, have involved assessing the influence of blood levels of vitamin d levels on the osseointegration of dental implants . Although most of these studies have shown that the administration of vitamin d can improve the healing of the peri - implant bone tissue, it is not yet clear whether vitamin d supplements can promote the osseointegration of dental implants . Our retrospective clinical study aimed to investigate if there is a link between low levels of vitamin d in the blood and an increased risk of early implant failures . Although the incidence of early implant failures was higher in patients with low serum levels of vitamin d, our study failed in proving an effective link between low levels of vitamin d in the blood and an increased risk of early implant failure . Further higher level studies (prospective controlled trials or, even better, randomized controlled clinical trials) with a more rigorous statistical analysis are therefore needed to investigate this issue . If an association between low serum levels of vitamin d and higher risk of early implant failure could be demonstrated, the clinician could give a set dose of vitamin d in the weeks before surgery, in order to normalize serum levels and obtain a positive effect on the healing process.
Root canal treatment is an effective, less invasive and ideal treatment modality for pulpally involved tooth and salvaging it from extraction . Cleaning, shaping and three dimensional obturation of root canal system are essential steps in root canal treatment . The bacteria impervious seal that is essential for success is provided by the root canal sealer and obturation material . Introduced mineral trioxide aggregate (mta), a tricalcium silicate cement as a perforation repair material . Being biocompatible and bioactive it gained popularity for pulp capping, pulpotomy, apexification and as root end filling material . Recently, it is finding application as a root canal sealer and as obturation material . When used as a root canal sealer, mta has the ability to regenerate periodontal ligament and form cementum in the root canal space and accessory canals, thus closing the leeway spaces that can result in the treatment failure . More recently, sealers based on mta have been introduced and found to have good sealing ability and higher push - out bond strengths . In addition, sealers based on mta demonstrated apatite - like deposits in contact with physiological solutions and a biocompatibility similar to mta . A sealer of good working consistency could be developed by mixing mta with adequate quantities of water - soluble polymer . Epoxy resin sealers like ah plus was found to bond better to the core obturation material and root dentine . They have the advantages such as less shrinkage, high radio opacity, low solubility, better periapical repair and biocompatibility . Studies have evaluated the success of root canal treatment, the incidence of pain and healing ability and many other aspects of the root canal sealers both in vivo and in vitro . Evaluation of post - operative pain with visual analogue scale (vas), periapical healing with periapical index (pai) are widely used methods and morphometric evaluation with vixwin pro digital image analysis software is a new, reliable and accurate technique . Zinc oxide eugenol is an efficient and proven root canal sealer while epoxy resin has equivalent properties to compete with it . This study was done to compare the clinical and radiological outcome of mta or epoxy resin as a root canal sealers compared to zinc oxide eugenol sealer using gutta - percha as the obturating material in teeth with periapical radiolucency . This study was conducted in patients in an age group of 18 - 50 years with teeth indicated for root canal treatment . Teeth with calcified canals, retreatment cases, pregnant patients, systemic diseases and lactating mothers were excluded . The 45 teeth indicated for root canal treatment were allotted into three groups with 15 teeth in each group . The cases were assigned into any one of the following groups: group 1: zinc oxide eugenol (tubliseal) as root canal sealer (n = 15)group 2: epoxy resin (ah plus) as root canal sealer (n = 15)group 3: mta (proroot mta) as root canal sealer (n = 15). Group 1: zinc oxide eugenol (tubliseal) as root canal sealer (n = 15) group 2: epoxy resin (ah plus) as root canal sealer (n = 15) group 3: mta (proroot mta) as root canal sealer (n = 15). Caries was excavated and if necessary pre - endodontic management was done with composite resin . Access cavity was prepared with access preparation kit (dentsply maillefer, ballaigues, switzerland). After irrigation with 2.5% sodium hypochlorite (naocl) (prime dental products, thane, india), a k - file of appropriate size was introduced in the root canal and working length was verified with propex ii apex locator (dentsply maillefer, ballaigues, switzerland). This was confirmed by taking a radiograph using parallel cone technique with the help of a film positioning device (endoray ii, densply rinn . Cleaning and shaping was done with k - files (sybron endo, orange, ca) and protaper rotary system (dentsply maillefer, ballaigues, switzerland) for all the teeth . 2.5% naocl, ethylenediaminetetraacetic acid (anabond stedman, kanchipuram, india) and normal saline (baxter, alathur, india) were used as irrigants . After cleaning and shaping, the canals were dried and medicated with calcium hydroxide (endo cal, m dent, bkk, thailand) mixed with normal saline . After 1 week, patients were recalled and the intracanal medicament was removed and evaluated . Once the patient was free of pain, discomfort and canals were dry, the teeth were obturated according to their groups . For group 1: zinc oxide eugenol (tubliseal, kerr / sybron, romulus, mi) was used as root canal sealer . The apical extent of the master cone was confirmed with radiograph and the canals were dried . The root canal was coated with the sealer using lentulospirals (densply canada, woodbridge, canada) in a slow speed handpiece (nsk, tochigi, japan). Obturation was performed with gutta - percha cones and sealer by lateral compaction technique . For group 2: epoxy resin (ah plus, dentsply detrey, konstanz, germany) was used as a sealer . Mta (proroot mta, densply tulsa, johnson city) was used as root canal sealer in group 3 . To improve the handling properties of mta and to get a sealer like consistency, the powder was mixed with propylene glycol in a mixing pad . Mta sealer was coated in canal walls using lentulo spirals in a slow speed handpiece and obturated as in groups 1 and 2 . Permanent restorations were done with composite resin (filtek z 350, 3 m espe) and full coverage restoration if necessary after obturation . Follow - up evaluations were done after an interval of 1 week and after 6 months . Before commencing the evaluation for pain, every patient was explained about the usage of vas using the following criteria: immediately after obturation and placement of coronal seal every patient was asked to mark the pain intensity using a 10 cm vas . All subjects were recalled after 1 week of post - obturation for evaluation of pain and clinically examined . Immediate post - obturation radiograph (base line data) was evaluated for pai score and recorded in the evaluation sheet using the criteria described by orstavik . At the 6 month follow - up visit, again a radiograph was taken using parallel cone technique and the pai score was assessed [figure 1]. Sample radiographs: (a) pre - operative, (b) immediate post - operative and (c) after 6 months for the groups respectively immediately after obturation, a digital radiograph was taken (base line data) with gendex rvg unit for morphometric analysis (gendex dental systems, hatfiled, pa, usa). The image was then subjected to morphometric area measurement with the area measurement tool in the vixwin pro digital image analysis software (version 1.3, gendex dental systems, hatfiled, pa, usa) by outlining the radiolucency . The area measured was recorded in the evaluation sheet . At the 6 month, again the same procedure was repeated and the area was measured using the same criteria as mentioned before [figure 2]. Area measurement by outlining the radiolucency using vixwin pro digital image analysis software wilcoxon's signed rank test was used for comparison of pain and pai score within the groups at different intervals . Kruskall - wallis analysis of variance test followed by mann - whitney u test was used for comparison of pain and pai scores between groups at different intervals . Wilcoxon's signed rank test was used for the comparison of area among the three groups at base line and at 6 months . Before commencing the evaluation for pain, every patient was explained about the usage of vas using the following criteria: immediately after obturation and placement of coronal seal every patient was asked to mark the pain intensity using a 10 cm vas . All subjects were recalled after 1 week of post - obturation for evaluation of pain and clinically examined . Immediate post - obturation radiograph (base line data) was evaluated for pai score and recorded in the evaluation sheet using the criteria described by orstavik . At the 6 month follow - up visit, again a radiograph was taken using parallel cone technique and the pai score was assessed [figure 1]. Sample radiographs: (a) pre - operative, (b) immediate post - operative and (c) after 6 months for the groups respectively immediately after obturation, a digital radiograph was taken (base line data) with gendex rvg unit for morphometric analysis (gendex dental systems, hatfiled, pa, usa). The image was then subjected to morphometric area measurement with the area measurement tool in the vixwin pro digital image analysis software (version 1.3, gendex dental systems, hatfiled, pa, usa) by outlining the radiolucency . The area measured was recorded in the evaluation sheet . At the 6 month, again the same procedure was repeated and the area was measured using the same criteria as mentioned before [figure 2]. Wilcoxon's signed rank test was used for comparison of pain and pai score within the groups at different intervals . Kruskall - wallis analysis of variance test followed by mann - whitney u test was used for comparison of pain and pai scores between groups at different intervals . Wilcoxon's signed rank test was used for the comparison of area among the three groups at base line and at 6 months . Pain comparison was performed intergroup and intragroup at three specific time periods . The standard deviation (sd) of vas score immediately after obturation for the groups are 8.46 (11.26), 10.46 (14.86) and 7.63 (8.46) respectively . Group 2 was found to have more vas score (p = 0.19) than other two groups but was statistically not significant (p = 0.19). When the pain was compared within the group at 1 week and at 6 months, there was no difference . Vas score after 6 months were 1.63, 1.06 and 0.33 respectively and there was no statistically significant difference [tables 1 and 2]. Comparison of pain between groups measured by vas at baseline, 1 week and at 6 months comparison of pain within group measured by vas at baseline, 1 week and at 6 months there was a highly significant (hs) difference in the pai score was found on comparing the base line and at 6 months . However, no difference among the groups was found at any interval [table 3]. Comparison of periapical index score within groups at baseline and at 6 months the sd of area measured immediately after obturation for the groups were 8.14 (8.40), 9.57 (9.20) and 4.32 (5.20) respectively and area after 6 months were 5.4 (5.7), 6.80 (8.11) and 4.14 (3.93) respectively . There was a hs difference in the area measurement comparing the base line and at 6 months, but no difference among the groups at any interval [table 4]. Pain comparison was performed intergroup and intragroup at three specific time periods . The standard deviation (sd) of vas score immediately after obturation for the groups are 8.46 (11.26), 10.46 (14.86) and 7.63 (8.46) respectively . Group 2 was found to have more vas score (p = 0.19) than other two groups but was statistically not significant (p = 0.19). When the pain was compared within the group at 1 week and at 6 months, there was no difference . Vas score after 6 months were 1.63, 1.06 and 0.33 respectively and there was no statistically significant difference [tables 1 and 2]. Comparison of pain between groups measured by vas at baseline, 1 week and at 6 months comparison of pain within group measured by vas at baseline, 1 week and at 6 months there was a highly significant (hs) difference in the pai score was found on comparing the base line and at 6 months . However, no difference among the groups was found at any interval [table 3]. The sd of area measured immediately after obturation for the groups were 8.14 (8.40), 9.57 (9.20) and 4.32 (5.20) respectively and area after 6 months were 5.4 (5.7), 6.80 (8.11) and 4.14 (3.93) respectively . There was a hs difference in the area measurement comparing the base line and at 6 months, but no difference among the groups at any interval [table 4]. Invasion of microorganisms into the pulp is responsible for the pathogenesis and necrosis of the vital tissue . Elimination of infection from the root canal system followed by its maintenance was found to induce healing . Root canal sealers along with obturation material will provide a bacteria proof seal of the root canal system, preventing the leeway space and communications between the intracanal and extracanal environments . The present study was designed to compare the clinical and radiological outcome of three different root canal sealers . No difference in the age, sex, tooth type, tooth distribution, root morphology, pai score and area measurement at baseline suggest that the groups were equally distributed [table 5]. Baseline values of vas score, pai scores and area measurement zinc oxide eugenol was used as a positive control in this study . This is because it has a history of long - term clinical usage with definitive success rate . It has good handling properties, sealing ability, minimal tissue toxicity, less water solubility, antimicrobial property periapical repair and good radio opacity . Ah plus is an epoxy resin based sealer that is widely used because of its compatibility with resin based materials used for obturation and post - endodontic restorations . Because of better flow and long setting time, ah plus sealer can penetrates deeper into the surface micro irregularities as well inside the lateral root canals . These properties lead to greater intertwining of the sealer with dentin structure, which, together with the cohesion among the cement molecules provides greater adhesiveness and resistance to dislodgment . Mta mixed with propylene glycol was used as a root canal sealer in this study . Mta sets hard and its setting time is long enough (165 5 min) to be used as a sealer . Calcium sulfate sealers based on mta has demonstrated apatite - like deposits in contact with physiological solutions and a biocompatibility similar to mta . Pain was evaluated at 3 time periods - immediately after obturation, 1 week after obturation and 6 months after obturation . On comparison of pain between groups, there was no statistically significant difference in pain experienced by the subjects in any of the time intervals evaluated . Clinical difference in pain intensity was observed by patients with epoxy resin sealer experiencing more pain, but was not statistically significant . The results of this study are in accordance with results obtained by alacam in his study . He found no statistical significant difference between the presence and type of post - operative discomfort and the type of sealer utilized . Comparison with in the group at different interval has provided a hs difference immediately after the procedure and after 1 week or after 6 months . According to a meta - analysis by nixdorf et al ., the incidence of persistent tooth pain after endodontic therapy is 5.3% . The radiological outcome of root canal treatment using three different sealers was evaluated with pai . When compared with in the group, there is a highly statistically significant difference in the pai score at baseline and after 6 months . This dictates that healing of the periapical lesions may take longer time period than 6 months . But, according to a study by jean camps, there was a noticeable change in the periapical lesion after comparing the radiographs taken immediately after treatment, after 3 months and 6 months of endodontic therapy . However, there was no increase in pai score in any of the groups . This implies that all the three sealers used were highly efficient in inducing healing of the periapical tissues . Morphometric area measurement was done using vixwin pro digital image analyzing software (gendex dental systems). The periapical lesions were circumscribed by drawing the outline and the software provided the measured area in cm . There was no difference in the baseline data . When compared at different time periods within the same group there was a decrease in the area measured in all the groups after a period of 6 months . There are no previous clinical studies evaluating area measurement using vixwin pro digital image analysis software as criteria for success of endodontic therapy . So, comparison of results with previous studies in the literature is not possible, but ideally it can be judged that, pai records the periapical status of a tooth based on previous reference radiographs, which is more subjective and differs between the examiners . Area measurement makes it more objective, better comparison between pre - operative and follow - up evaluation with less inter examiner bias . In this study, mta was mixed with propylene glycol to get a sealer consistency . However in the future studies, an ideal powder liquid ratio with the vehicle should be established . Though radiographically detectable difference in periapical lesions can be appreciated in 6 months as used in this study, further longer period evaluation should be carried out in the future . Furthermore, the removal of material from the root canal system, if retreatment is indicated should also be evaluated . Being a biocompatible material and having the property to induce the deposition of cementum and periapical repair, mta can prove to be a better material than other sealers . The scope of this study was to evaluate the effectiveness of mta as a root canal sealer considering its radiological and clinical success rate, usage and feasibility . Further long - term clinical trials with more criteria should be conducted to conclude the superiority of mta among other root canal sealers . Among the three root canal sealers used in this study, no sealer was proven to be superior . Mta has performed equally well when compared with the zinc oxide eugenol and ah plus . Further long - term clinical trials with more criteria should be conducted to conclude the superiority among these materials.
Osteoarthritis (oa) is characterized by cartilage breakdown, synovial fibrosis, and osteophyte formation . The main clinical symptom of oa is chronic joint pain, which is typically treated using analgesic drugs, such as nonsteroidal anti - inflammatory drugs and corticosteroids [1, 2]. However, due to potential adverse effects [3, 4] and variable efficacy of these drugs for providing symptomatic pain relief, more effective analgesics are needed to improve oa treatment and patient care . In addition, because only a few effective disease - modifying drugs are available for oa [5, 6], a better understanding of the mechanisms that drive oa pain is required to guide drug development . Nerve growth factor (ngf) is a widely known pain mediator and plays a critical role in the modulation of oa pain [811]. The neutralization of ngf with tanezumab, an anti - ngf monoclonal antibody, has robust analgesic effects on oa pain [8, 10, 11]. Due to these effects, the regulation of ngf has been investigated in several in vitro and in vivo studies [12, 13]. Evidence from these studies suggests that the activity of ngf is mediated by inflammatory cytokines . For example, in an experimental arthritic mouse model, il-1, but not tnf-, increased ngf levels in knee joints . However, the mechanism regulating ngf expression in synovial tissue (st) remains unclear . St contains macrophage- and fibroblast - like cells in the lining layer [1416]. The treatment of cultured synovial oa fibroblasts with il-1 and tnf- induces the production of ngf . More recent studies have shown that macrophages produce a number of inflammatory cytokines, including il-1, il-6, and tnf-, which contribute to oa progression and associated joint pain [1721]. Despite these findings, the regulation of ngf expression in synovial macrophages is not fully understood . Str / ort mice are a well - characterized oa model that spontaneously develop oa with a progression resembling that of humans [2224]. Synovial hyperplasia is also observed in str / ort mice, and we previously showed that the cd11b+ macrophage population in st str / ort mice is higher than that found in c57bl/6j mice [17, 18]. In addition, we reported that compared to synovial fibroblasts (cd11b), synovial macrophages produce high levels of il-1 and tnf- in str / ort mice . Here, we characterized the expression profiles of several inflammatory cytokines and ngf in the st of str / ort mice . In addition, the regulation of ngf expression by inflammatory cytokines in synovial macrophages and fibroblasts was also examined . Nine - month - old male str / ort (average body weight, 40.6 4.1 g) and c57bl/6j (control; average body weight, 32.8 1.2 g) mice (charles river laboratories, inc ., specific pathogen - free colonies of each mouse line were maintained at nippon charles river laboratories (kanagawa, japan) in a semibarrier system with a controlled environment (temperature: 23 2c; humidity: 55% 10%; lighting: 12 h light / dark cycle). All experimental protocols were approved by the kitasato university school of medicine animal care committee . C57bl/6j and str / ort mice were sacrificed by the intramuscular injection of a mixture of medetomidine, midazolam, and butorphanol tartrate . After removing skin with a scalpel, st was harvested, and total rna was then extracted from the harvested tissue using trizol (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions . The extracted total rna was used as a template for first - strand cdna synthesis using superscript iii rt (invitrogen) in pcr reaction mixtures consisting of 2 l cdna, specific primer set (0.2 m final concentration), and 12.5 l sybr premix ex taq (takara, kyoto, japan) in a final volume of 25 l . The primers for il6 and ngf were designed using primer blast software and were synthesized by hokkaido system science co., ltd . The other primers used in this study were designed based on previously published primer sequences . The sequences of the pcr primer pairs used in this study are listed in table 1 . Quantitative pcr was performed using a cfx-96 real - time pcr detection system (bio - rad, hercules, ca, usa). The pcr cycles consisted of an initial denaturation step at 95c for 1 min, followed by 40 cycles of 95c for 5 s and 60c for 30 s. mrna expression was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna . The gene expression levels in the st of str / ort mice were compared with those in the st of c57bl/6j mice (str / ort / c57bl6j). In addition, the gene expression levels in cd11b+ cells were compared with those in cd11b cells (cd11b+/cd11b). St samples were collected from both knees of five str / ort mice, and mononuclear cells were then isolated from the collected tissue by digestion with type i collagenase for 2 h at 37c . The obtained mononuclear cells were suspended in 500 l phosphate - buffered saline (pbs) containing biotinylated anti - cd11b antibody . Following a 30 min incubation at 4c, the cells were washed with pbs, mixed with streptavidin - labelled magnetic particles (bd imag streptavidin particles plus - dm; bd biosciences, tokyo, japan), and incubated for 30 min on ice in an imag separation system (bd biosciences). Warmed (37c) -minimum essential medium (mem) was added to the cell suspension to collect unbound (cd11b - negative) cells, and an additional 3 ml -mem was added to collect cd11b - positive cells after removing the tub from the magnetic support . The cd11b - positive and cd11b - negative cells were collected by centrifugation at 300 g for 10 min, and the obtained cells were cultured in -mem in six - well plates . Tnf-, il-1, il-6, and ngf expression in the cells was analyzed by reverse transcription - polymerase chain reaction (rt pcr). St - derived mononuclear cells, including synovial macrophages and fibroblasts, which were isolated as described above, were cultured in -mem in six - well plates . After a 1-week incubation at 37c in a 5% co2 incubator, synovial fibroblasts were incubated with either mouse recombinant tnf- (25 ng / ml), il-1 (50 ng / ml), or il-6 (100 ng / ml) (biolegend, san diego, ca, usa) for 24 h. cells that were not treated with any cytokines were used as controls . To examine the effect of tnf- on synovial macrophages and fibroblasts, cd11b - positive and cd11b - negative cells were collected from st, as described above, and were then cultured in -mem in six - well plates . After a 1-week incubation at 37c in a 5% co2 incubator, cd11b - positive and negative cells were incubated with mouse recombinant tnf- (25 ng / ml) or il-1 (50 ng / ml) for 24 h. cells that were not treated with tnf- were used as controls . The treated and control cells were harvested for total rna isolation, as described above, and ngf expression was analyzed by rt pcr . Ten l3 and l4 drg samples were harvested from the 9-month - old c57bl/6j and str / ort mice and were then immersed in a buffered paraformaldehyde fixative at 4c overnight . The samples were further incubated in pbs containing 20% sucrose for 24 h at 4c . After freezing in liquid nitrogen, each specimen was sectioned at 10 m thickness on a cryostat (leica microsystems, cm3050s, wetzlar, germany) and was then treated for 90 min at room temperature with a blocking solution of pbs containing 0.05% tween-20 and 1% skim milk . The sectioned drg specimens were stained with rabbit antibodies against calcitonin gene - related peptide (cgrp; 1: 1000; immunostar, hudson, wi, usa) and transient receptor potential vanilloid 1 (trpv1; 1: 500; calbiochem, san diego, ca, usa) by incubation for 18 h at 4c . The drg sections were then incubated with alexa 488-conjugated goat anti - rabbit igg (for cgrp immunoreactivity, 1: 1000; molecular probes) and alexa 488-conjugated goat anti - rabbit igg (for trpv1 immunoreactivity, 1: 1000; molecular probes). The immunostained sections were visualized using a fluorescence microscope (axiovert 200, zeiss, jena, germany) in a treatment - blinded manner . The numbers of cgrp- and trpv - positive cells were counted, and the proportion of these cells to the total number of nucleated cells in drg was calculated for each drg sample . Differences between c57bl/6j and str / ort mice were examined using the t - test . All statistical analyses were performed with spss software (version 11.0; spss, inc ., chicago, il, usa). A p value of <0.05 was considered statistically significant . Real - time pcr analysis of the genes encoding tnf-, il-1, il-6, and ngf showed that the expression levels of these genes were significantly elevated in the st of str / ort mice compared to those in control c57bl/6j mice (figures 1(a)1(d)). Activated macrophages reportedly produce higher levels of inflammatory cytokines, including tnf-, il-1, and il-6, than tissue - resident macrophages [18, 26, 27]. To determine whether macrophages in st also produce inflammatory cytokines, expression of the genes encoding tnf-, il-1, and il-6 in cd11b - positive cells isolated from the st of str / ort mice was examined by real - time pcr . Tnf-, il-1, and il-6 gene expression in cd11b - positive cell fractions was higher than that in cd11b - negative cell fractions (figures 2(a)2(c)). In contrast, no differences in the gene expression of ngf were detected between the two examined cell fractions (figure 2(d)). Real - time pcr analysis revealed that the gene expression of ngf increased significantly in isolated synovial cells in the presence of exogenously added tnf- and il-1 compared to untreated control cells (figure 3). In contrast, the gene expression of ngf was not affected in il-6-treated synovial cells (figure 3). Ngf expression was also significantly increased in the presence of exogenously added tnf- in isolated populations of synovial fibroblasts and macrophages compared to untreated control cells (figure 4(a)). Ngf expression was also significantly increased in the presence of exogenously added il-1 in isolated populations of synovial fibroblasts, but not in synovial macrophages (figure 4(b)). To investigate pain - related sensory innervation by ngf, the proportion of trpv1- and cgrp - positive cells in the drg of str / ort mice was investigated by immunohistochemistry . The number of trpv1- (figures 5(a), 5(b), and 5(e)) and cgrp - positive cells (figures 5(b), 5(d), and 5(f)) in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice . In the present study investigating the mechanisms underlying the regulation of ngf and development of oa pain, higher expression of the genes encoding tnf-, il-1, il-6, and ngf was observed in the st of an oa str / ort mouse model compared to that in c57bl/6j mice . In addition, ngf expression was specifically detected in the cd11b - positive and cd11b - negative cell fractions isolated from st, whereas higher tnf-, il-1, and il-6 gene expression was observed in the cd11b - positive cell fractions . Notably, the treatment of cultured synovial cells with tnf- and il-1 stimulated ngf expression and tnf- also stimulated ngf expression in cd11b - positive and cd11b - negative cell fractions . The number of trpv1- and cgrp - positive cells in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice . Taken together, these findings suggest that tnf- regulates ngf expression in both synovial fibroblasts and macrophages and that il-1 regulates ngf expression in synovial fibroblasts and that these factors may contribute to the development of pain in oa patients by innervation of the peripheral nervous system . Ngf is upregulated in human synovial fibroblasts, suggesting that this factor plays an important role in oa pathology [12, 13, 28]. Ngf elevated inflammatory knee joint and tnf- and il-1 stimulate synovial fibroblasts to produce ngf . Reported that ngf expression is upregulated in the knee joint of a carrageenan - treated inflammatory arthritis model and is further stimulated by the intraarticular injection of tnf- and il-1 . Further, tnf- and il-1 treatment of cultured synovial fibroblasts derived from a human oa patient also increased ngf production . However, because these studies consisted of analyses of whole knee joints or cultured synovial fibroblasts exogenously stimulated with tnf- and il-1, the cell populations responsible for regulating ngf and producing tnf- and il-1 in st were not conclusively determined . Here, elevated expression of the inflammatory cytokines tnf-, il-1, and il-6 was observed in the st of str / ort mice, and tnf- and il-1 were increased in synovial macrophages isolated from this oa model . In addition, ngf expression was stimulated by tnf- and il-1 in the synovial fibroblast fraction . Taken together, these findings suggest that tnf- and il-1 stimulate ngf expression in synovial fibroblasts . A recent immunohistochemical analysis revealed that ngf not only is localized to fibroblasts, but is also produced by a specific population of macrophages in human st . However, the factor regulating ngf in macrophages was not determined . In the present study, isolated synovial macrophages and fibroblasts from str / ort mice expressed ngf at similar levels to each other . Ngf expression in the macrophage fraction was also stimulated by tnf-. Taken together, these findings indicate that tnf- stimulates ngf expression in both synovial fibroblasts and macrophages . Ngf plays a key role in the generation of acute and chronic pain and peripheral sensitization [3033]. Recent evidence suggests that the trpv1 ion channel and cgrp are involved in the development of ngf - induced persistent mechanical and thermal hypersensitivity in rats [30, 31]. Ngf acutely modulates trpv1 activity [32, 33] and also stimulates cgrp expression in drg neurons in vitro and in vivo . Consistent with this speculation, inhibition of ngf by treatment with tanezumab was recently shown to have high analgesic efficacy in oa knees compared with placebo . In the present study, cgrp- and trpv1-positive cells, which are localized in drg neurons in monoiodoacetate- (mia-) treated oa animals [35, 36], were increased in the drg of str / ort compared to c57bl/6j mice, suggesting that the elevation of synovial ngf in str / ort mice contributes to peripheral sensitization . However, several studies have shown that human oa chondrocytes also produce ngf by inflammatory cytokines [37, 38]. Further investigations are needed to clarify the contribution of chondrocytes derived ngf on peripheral sensitization . First, the mechanism underlying the development of oa pain that was proposed based on the present results was based on cross - sectional analysis and therefore no data on the effects of the observed changes on the progression of oa are available . Second, although ngf was found to be elevated in the st of oa mice, it remains to be determined if the elevation of synovial ngf levels contributes to peripheral sensitization . Finally, present study did not confirm that the protein levels of these cytokines correlated with the expression levels . In conclusion, the present results support the hypothesis that tnf- regulates ngf expression in synovial fibroblasts and macrophages and il-1 regulates ngf expression in synovial fibroblasts in oa mice . Tnf- and il-1 may therefore regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain.
Hepatitis b virus (hbv) is a major health problem, with approximately 400 million chronically infected people worldwide, and 1560% of the normal population in many african countries may be positive for one or more of the serological markers of hepatitis b virus infection . These chronically infected patients not only are at an increased stage of developing liver cirrhosis and hepatocellular carcinoma, but also serve as a potential reservoir of infection . The major structural protein of virus envelope, hepatitis b surface antigen (hbsag), is universally considered as a diagnostic marker of hbv infection . The absence of hbsag in the serum and the presence of antibodies to core antigen (anti - hbc) usually indicate resolved infection . Occult hbv infection (obi) usually has a serological evidence of previous hbv infection that has been described in a few cases . Hiv coinfection has been reported to modify the natural history of hbv with potential consequences on morbidity and mortality . Data on obi in art untreated hiv patients is limited from a vast number of african countries like nigeria, where prevalence of hbv monoinfection, mode of transmission, viral genotype, and mutational pattern varies considerably in different parts of the country . No previous study from nigeria on prevalence on obi among any groups has been carried out . This is particularly important as exposure to hbv is common among hiv - infected cases because of shared routes of transmission . Notably, there is considerable variation in prevalence of hiv / hbv coinfection according to geographic regions and exposure risk . Successful implementation of art leads to immune reconstitution that can potentially result in immune mediated liver injury in the setting of hbv coinfection . Some studies have reported an association between obi and elevated transaminase; therefore identification of obi is of importance . Of the 1,200 hiv - infected patients enrolled in the haart clinic of the specialist hospital, ikole, ekiti state, nigeria, from october, 2012, to april, 2013, we identified 980 hbsag negative patients (art - nave subjects). Among them, 188 were selected for the study by a simple random method . Informed consent was obtained from the patients, and the institutional committee approved the study protocol . All samples were tested for hbsag, anti - hbs, anti - hbc, anti - hcv, and anti - hiv using elisa (drg diagnostics, marburg, germany). All anti - hbc positive samples were retested for hbsag as well as for anti - hbc, and only repeat positive samples were included in the study . Dna was extracted from all the serum samples using qiaamp dna blood mini kit (qiagen gmbh, hilden, germany) following the manufacturers' instructions . Briefly, samples (200 l) were incubated with protease and lysis buffer . After incubation, there were two washing steps, and the nucleic acids were eluted in a volume of 50 l of elution buffer . The presence of hbv dna was examined in all samples using a routine diagnostic pcr . Primer pairs were designed from the highly conserved overlapping regions of the s and p genes of the hbv genome . A nested pcr was performed: outer primer pairs were hbpr134 (sense) 5-tgctgctatgcctcatcttc-3 and hbpr135 (antisense) 5-cagagacaaaagaaaattgg-3 and the inner primer pairs were hbpr75 (sense) 5-caaggttatgttgcccgtttgtcc-3 and hbpr94 (antisense) 5-ggtataaagggactcacgatg-3. Pcr amplifications were carried out in 25 l reaction volumes with 5 ng of genomic dna, 10x pcr buffer (20 mm tris - hcl ph 8.4, 50 mm kcl; qiagen), 2 mm of dntps, 50 ng of each primer, and 1 u ampli taq gold dna polymerase (applied biosystems) on a ptc 200 cycler (peltier thermal cycler watertown, massachusetts, usa). Thermal cycling parameters were initial denaturation at 94c for 2 min, followed by 35 cycles of 30 sec at 94c denaturation, 30 sec at 52c annealing temperature, and 45 sec at 72c extension, followed by a final extension of 5 min at 72c . Thermal cycling parameters remained the same as in the first pcr round except for the number of cycles that is increased to 40 cycles of amplification . Each pcr product (5 l) was analysed by electrophoresis in 2% agarose gels . A positive control (hbv plasmid dna) and a negative control of the master mix only were integrated to each run to validate the pcr products that yielded a 340 bp fragment . Quantification of hbv dna was performed with quantitative real - time pcr using a previously described procedure in a geneamp 7300 sequence analyzer (applied biosystems, perkin - elmer, foster city, ca). Hbv - plasmid dna was used to generate a standard curve following a serial 10-fold dilution . Mean age and all the numerical data were analysed using student's t - test . The chi - square test and fisher's exact test were used to compare categorical data . For the purpose of our study, the demographic, biochemical, and virological parameters of the study group are summarized in table 1 . The mean age was 35 (range: 367) years . The majority (45%) had multiple sexual partners and 25% of the subjects had a history of concomitant alcohol use . Overall, 29/96 (29.2%) of patients were reactive for anti - hbc, an indication of prior exposure to hbv dna, and majority 6/8 (75%) of the patients were female (table 2). Thus, in the total study population, 21/188 (11.2%) of patients were identified as obi and 62.5% of the obi patients had cd4 count less than 200 cells / mm . Averagely the hbv viral load was <50 copies / ml in the obi samples examined by quantitative pcr . Serum levels of ast and alt were higher among patients with obi in comparison to anti - hbc positive hbv dna negative individuals, but the difference failed to reach standard significance (p = 0.13 and p = 0.07), respectively . The comparison of different demographic, biochemical, and virological factors between hbv dna positive and negative cases was illustrated in table 3 . The distribution of the study participants as per the 1993 revised classification system for hiv infection and expanded surveillance case definition for aids among adolescents and adults was as shown in table 4 . The present study represents a comprehensive cross - sectional analysis of prevalence of obi in an art nave hiv positive cohort comprising various risk groups . Most previous studies looking at the clinical effects of obi in hiv include a large number of patients on anti - hbv drugs as a component of art . This study describes the risk factors associated with obi, frequently of anti - hbc positivity and its possible values as a serological marker for identifying hiv - infected patients who benefit from hbv dna assay . We found the prevalence of occult hbv to be 11.2% among a random selected group of hiv - infected patients . The prevalence of obi in hiv positive individuals varies worldwide between 0 and 90%, depending on the geographic regions, risk factors, and the exposure involved . In the present study, the prevalence of anti - hbc (29.2%; 28 of 96) and obi (28.6%; 8 of 28) among the art nave hiv positive cohort was higher compared to previous report on blood donors from studies done in areas of india, areas which reported 21.3% obi among the hbsag negative anti - hbe positive donors . Within nigeria, hbv and hcv coinfection among hcv - infected patients most of the previous studies on hbv / hiv co - infection are aimed at detecting hbv prevalence in the hiv population are based on hbsag positivity (prevalence 9.9 to 11%), but reports on obi are scarce . A previous study on intravenous drug users in northeastern india detected a prevalence of 15.9% . Among the obi cases, the rate of anti - hbs was lower (2/8; 25%), which may be due to the fact that hiv - infected patients are prone to lose anti - hbs immunity at a higher frequency than the general population . Previous reports suggested that the lower hbv replication was associated with milder hepatic damage . Among the subjects with obi, elevated alt or ast however, our study is cross - sectional; therefore evaluation of long term clinical significance of obi should be better addressed by follow - up studies . The low level of viral load obtained in this study buttress the findings in another study that showed that showed that almost all obi cases are infected with replication incompetent hbv, revealing a strong suppression of overall replication activity and gene expression, thereby resulting in a significant reduced viral load . Hiv patients are screened for concomitant chronic hepatitis b using hbsag elisa, and it is not considered cost - effective to perform hbv dna testing for all hiv patients in our resource - poor setting . Our study tried to identify possible clinical and serological markers which could guide dna testing in these patients . Obi is reported to be common among hcv infection, but we found its prevalence to be low among our study group . However, anti - hcv was not tested among the other hiv positive samples attending the specialist hospital, ikole, ekiti state, nigeria . Furthermore, none of the risk factors were found to be statistically significant markers of obi and cannot be used as an independent marker for identifying patients who should benefit from hbv dna estimation . However, as one third of the anti - hbc positive negative patients were positive for hbv dna (8 of 28), it is recommended that hiv positive patients with hbsag negative / anti - hbc positive patterns should be tested for the presence of hbv dna irrespective of their anti - hbs status . Nevirapine is commonly included in the first line art regimens at most treatment centers in nigeria . Our study identified only 21 subjects with obi, and all the samples were collected from a single center indicating that results might differ in setting with significant different demographic characteristics . A main implication of the presently viable data is therefore further emphasizing the need for efficient hbv vaccination programs . Overall, the present study highlights the need for screening hbv before the initiation of any haart containing anti - hbv regimens in hbv / hiv coinfected patients . It necessitates the use of nat for effective laboratory diagnosis of occult hbv infections in hiv positive patients, especially in developing countries where these assays are not widely available.
Epilepsy is generally deemed intractable or refractory to treatment if the use of two or more appropriately chosen antiepileptic drugs (aeds) fails to adequately control seizures . The aeds in question must be at the maximum tolerated doses, at a sufficient therapeutic level and not be discontinued due to side effects . A less commonly used definition of intractability requires monthly seizures for a period of 18 months.1 despite optimal medical management with appropriate aeds, 20%30% of adults and 10%40% of children diagnosed with epilepsy continue to suffer from seizures.28 furthermore, after the failure in efficacy of two aeds, the chance of achieving seizure freedom by introducing subsequent drug regimens has been shown to be less than 10%.2,812 intractable epilepsy is a significant problem for the quality of life (qol) of the individual patients . Persistent seizures heavily impact on the patients behavior, cognitive ability and psychosocial well - being . It has been shown that this condition can lead to poor academic achievements, increased probability of unemployment, low self - esteem, anxiety and depression, social isolation and ultimately to decreased quality of life.13,14 in addition, the unremitting seizures can pose a significant financial burden, with patients suffering from poor seizure control reported to represent approximately 25% of epilepsy cases but accounting for greater than 85% of epilepsy associated costs, mainly due to repeated hospital admissions and extensive investigations.15 further to the financial burden, patients with refractory seizures have higher mortality rate than patients whose seizures are well - controlled, who have mortality rates comparable to the general population.16 this is likely to be due to differences in the underlying etiology rather than the effect of seizures per se . Patients with refractory seizures have higher rates of structural abnormalities or inborn errors of metabolism (iem). In addition, sudden unexpected death in epilepsy (sudep) is forty times more common in patients who continue to suffer from seizures compared to patients who are seizure free.17 the number of new aeds has significantly increased in the last 20 years, effectively doubling the number of anticonvulsants available for physicians to prescribe . Whilst the efficacy of new molecules in controlling seizures has not dramatically increased, safety and tolerability profiles are significantly better than for the older aeds . Many of the new drugs have novel mechanisms of action and exhibit fewer drug - interactions . When considering the most appropriate choice of treatment this is of particular importance in children with refractory epilepsy, a large proportion of whom present specific age - dependent epilepsy syndromes . Accurate identification of the epilepsy syndrome is the foundation for the choice of an appropriate treatment and increases the likelihood of seizure remission.18 in some instances, identifying etiologies can radically alter the potential treatment options, for example metabolic treatments such as the ketogenic diet for epilepsies resulting from iem.19 surgery may be the most appropriate treatment option in some cases, especially where strong evidence exists for its efficacy, such as for the anterior temporal lobectomy in drug - resistant temporal lobe epilepsy.20 however, the most appropriate timing for surgery remains debated . When surgery is not a suitable treatment option, neurostimulation therapies such as vagus nerve stimulation (vns) may be considered . This review will outline the evidence for the use of different treatment options for patients with intractable seizures . Whilst the focus will be on the pharmacological options, we will also review the extent and quality of evidence supporting the efficacy of surgical, metabolic, neurostimulatory and herbal interventions in the context of the associated side - effects, safety and patient preferences . Before considering any changes to pharmacological treatment in a patient with refractory seizures, confirmation that the events are epileptic in origin is essential . It is estimated that up to 30% of patients seen in specialist epilepsy clinics present with non - epileptic attacks alone or in combination with epileptic seizures.21 in these patients, the prescription of aeds is often inappropriate and can lead to significant side effects, in addition to the financial burden to the health services . In many patients with refractory seizures, video - electroencephalography (video - eeg) monitoring is essential to characterize the events and reach diagnostic conclusions with a high level of confidence . A better understanding of the underlying etiology can inform treatment decisions and lead to optimal seizure control over a shorter time period . This is also the case for patients who suffer from a specific epilepsy syndrome, for whom the importance of correct classification at an early stage should also be emphasized . The correct identification and classification of seizures and epilepsy types allows evidence - based choice of syndrome - specific or etiology - specific treatment intervention, as is the case for valproate treatment as a first line agent in juvenile myoclonic epilepsy.18 this stepwise approach to correct diagnosis and subsequent treatments options is outlined in figure 1 . Patients with refractory seizures have, by definition, tried at least two appropriate aeds and their seizures have failed to be adequately controlled . At this point the physician faces the decision to either add a further aed to the current treatment or to pursue sequential monotherapy . If sequential monotherapy is chosen, the drugs that are failing to control seizures are slowly tapered off and replaced with different aeds which can potentially improve seizure control . Sequential monotherapy was the main treatment paradigm from the 1970s through to the 1990s, when sodium channel blocking aeds were the only treatment option . Combining drugs with the same mechanism of action lead to a slightly better seizure control, but at the expense of a marked increase in adverse drug interactions and pharmacodynamic amplification of side effects.22 this practice changed in the 1990s, when new aeds were introduced with novel mechanisms of action . These drugs exhibited fewer pharmacokinetic interactions, exhibited fewer side effects when combined and raised the possibility of synergistic drug combinations . Clinical practice over the past 20 years has been characterized by rational polytherapy: numerous drug combinations have been considered and a wealth of evidence on which combinations are most effective and where drug interactions are most likely to occur has accumulated.23 when contemplating polypharmacy for a patient with epilepsy, it is important to consider the concept of total drug load in addition to the number of drugs being prescribed . Although general agreement on the definition has not been reached, drug load is usually defined as the total amount of drug exposure for a treatment regimen, and has been quantified as the prescribed daily dose of aed(s) divided by the defined daily dose for the aed(s) as outlined by the world health organization . Drug load has been demonstrated to correlate with the number of adverse events better than the number of aeds being prescribed.24 it has been argued that for the use of multiple aeds to be rational, the combination of two drugs must provide supra - additive therapeutic efficacy, whereby the combined efficacy of the two drugs is greater than when the drugs are given alone . Alternatively, combining aeds may be more advantageous than prescribing monotherapy at standard doses if they demonstrate simple additive efficacy but less than additive side effects . However, pharmacokinetic drug interactions often contraindicate specific drug combinations . Some of the traditional aeds, including phenobarbital, phenytoin and carbamazepine are metabolized in the liver and are potent inducers of the cytochrome p450 (cyp450) system . These drugs act by blocking voltage - dependent sodium channels preventing the repetitive and uncontrolled neuronal discharges seen in epilepsy . Phenobarbital, in addition, enhances gaba mediated inhibition, which further acts to prevent the repetitive neuronal firing.25 in the developed world, phenobarbital is now prescribed rarely due to its sedative side effect profile . However due to its relatively low cost it is regularly prescribed in developing countries . All of the drugs described above induce hepatic enzymes and decrease the serum concentration of additional aeds that may be prescribed . As a consequence of this effect, each drug may impede the other, by preventing effective serum concentrations being reached.22 it is therefore advised that the combination of an enzyme - inducing aed with another aed which is metabolized in the liver requires particular caution . Interactions with other medications should also be considered when prescribing hepatic enzyme inducing drugs, particularly in patients taking exogenous contraceptive hormones, immunosuppressant therapy, a number of corticosteroids and some antineoplastic chemotherapeutic agents, all of which can be the subject of enzyme - induction . Physicians should also be aware of the additional adverse effects that enzyme - inducing drugs can produce, including secondary hyperparathyroidism, reduced serum estosterone concentrations, menstrual irregularities and elevated cholesterol levels, which may impact on a patient s cardiovascular disease risk . Consequently, it has been suggested that the older, enzyme - inducing aeds should not be considered as first - line treatment options for patients with refractory seizures.26 among older aeds, valproate is characterized by a number of mechanisms of action, including sodium channel blockade and potentiation of gaba mediated inhibition . It is also metabolized by the liver, but has no enzyme - inducer properties . The newer aeds, including gabapentin, levetiracetam, pregabalin, vigabatrin, lamotrigine, oxcarbazepine, topiramate and zonisamide, are characterized by a more favorable tolerability profile, as their hepatic enzyme induction properties are minimal or absent . Many of these drugs have novel or unknown mechanisms of action and demonstrate similar levels of efficacy and fewer side effects, less adverse pharmacokinetic drug interactions and are tolerated better by patients.25 when compared to carbamazepine in double - blind randomized control trials for treatment of partial onset seizures, gabapentin, lamotrigine and oxcarbazepine were found to have an similar efficacy but better levels of tolerability.2729 until recently, direct comparison studies in treatment - refractory epilepsy populations have been sparse . A recent systematic review and meta - analysis of studies on patients with refractory partial epilepsy suggested that topiramate and levetiracetam have a greater efficacy in controlling seizure frequency than the other new aeds.30 the same study found that gabapentin is likely to be less effective than other new aeds . In terms of tolerability, oxcarbazepine and topiramate were associated with significantly more withdrawals and therefore more poorly tolerated than the other new aeds . Gabapentin and levetiracetam, on the other hand, were associated with significantly fewer withdrawals and were therefore better tolerated . It should be noted that these conclusions were based on indirect comparisons and should be treated with caution . Although the newer aeds exhibit fewer side effects than traditional aeds, the potential for significant cognitive and behavioral side effects should be considered when prescribing . Drugs that have a mechanism of action involving the potentiation of gaba mediated inhibition such as vigabatrin, tiagabine, topiramate and gabapentin have been associated with sedation and negative effects on mood . Some patients who are prescribed these aeds are at risk of developing depression as a side effect, especially patients with a past psychiatric history.31 topiramate has been associated with unfavorable cognitive effects, including impaired concentration, psychomotor slowing, language regression, comprehension problems and memory deficits.32 other new aeds which act by inhibiting the glutamatergic excitatory neurotransmission have been associated with an taking advantage of these properties, lamotrigine may be considered in patients with concomitant depression, apathy and hypersomnia.31 in addition, psychotic symptoms have been reported as adverse events following aed administration, especially with topiramate and levetiracetam, although the extent that the medication plays in producing these symptoms remains uncertain.33 it is recommended that baseline mood and cognitive ability is taken into account when deciding which aed to prescribe in refractory patients, in order to minimize any impact that prescribing may have on mood and cognition . In addition to the above evidence, clinical studies on polytherapy are informative when considering using a combination of aeds . One study on 47 patients with cognitive impairment showed that seizures were controlled more effectively by a combination of phenobarbital and phenytoin or phenobarbital and carbamazepine, than by phenytoin and carbamazepine combination.34 this suggested that a combination of two sodium - channel blocking aeds does not improve seizure control significantly . Other studies indicate that a combination of carbamazepine and valproate or carbamazepine and vigabatrin is more effective in seizure control than a combination of carbamazepine and phenytoin, although these studies were not controlled for drug concentration.35,36 the aed combination for which there is the most convincing evidence is lamotrigine and valproate . A large study aiming to assess the efficacy of lamotrigine monotherapy also evaluated the effect on seizure control whilst taking lamotrigine as add - on to the first aed (carbamazepine, phenytoin or valproate). This study showed that patients who were taking lamotrigine and valproate experienced an 83% reduction in seizures, whilst those taking the lamotrigine and carbamazepine or lamotrigine and phenytoin combinations experienced only a 43% and 34% reduction in seizures, respectively.37 these findings should be interpreted with some caution, however, as different doses of lamotrigine were used in each combination . For resective surgery to be a viable option, the patient with treatment - refractory epilepsy should ideally have a single epileptogenic focus in a non - eloquent cortical region (ie, not involved in key language, memory or motor processes). It should be noted that exceptional cases do exist, where larger resections such as hemispherectomies are acceptable if the seizures are severe and the benefit gained from such surgery can be justified.38 major surgery such as corpus callosotomy can be considered as last treatment option in palliative care for patients with intractable seizures . This invasive intervention is usually aimed at preventing secondary generalization in patients who are regularly experiencing loss of consciousness and a high frequency of seizure - related injuries . Most epileptologists consider surgical management as second line therapy for patients with intractable seizures.39 this is especially true for patients with drug - resistant temporal lobe epilepsy (tle), for whom temporal lobectomy can represent an effective treatment option . A randomized control trial found that 58% of patients randomly allocated to surgery for temporal lobe resection did not present with any seizures which impaired consciousness after one year, compared to 8% in the treatment arm receiving the optimum medication regimen (p <0.001).20 a multi - center study which followed 339 patients with epilepsy for 2 years post - operatively showed that temporal lobe resection is the most effective type of resective surgery in this patient population: 68% of the patients who received temporal lobectomies experienced seizure remission for 2 years, compared to 50% of the patients who underwent extratemporal resections.40 another non - randomized controlled study which followed up patients with refractory seizures, assigned either to surgical treatment or continued drug therapy, found that 44.6% of 242 patients with pharmacoresistant tle who received surgery were seizure - free for 12 months, compared to 4.3% of those who received aeds only (p <0.001).41 it should be emphasized, however, that not all patients with tle have the same probability of achieving seizure freedom through temporal lobe resection . A meta - analysis of 83 studies including 7,343 patients undergoing epilepsy surgery showed that the proportion of patients experiencing seizure freedom for at least 5 years post - operatively was highest in those who underwent temporal lobe resection (66%), even though a substantial proportion of patients undergoing occipital, parietal or frontal lobe resections demonstrated post - operative seizure freedom (46%, 46% and 27% respectively).42 it should be noted that post - operative seizure freedom for the first few years following surgery might not persist long - term . Isolated relapses can still occur many years after the procedure, with a probability of seizure relapse of 4% per year for the 5 years following surgery.43 overall, patients with structural abnormalities apparent on pre - operative imaging studies, such as hippocampal sclerosis or foreign tissue lesions are more likely to experience post - operative seizure freedom compared to patients with no obvious lesion.4446 when considering surgery for refractory epilepsy, there are a number of risks that need to be taken into account alongside the potential benefits . The surgery itself can cause a substantial period of disability, with patients typically having to take 48 weeks off work or school.47 the most common medical complications are deep vein thrombosis, infection and transient endocrine abnormalities . Psychiatric complications are reported post - resection in 25% of the patients, including transient dysphoria, depression and occasionally mania.48 although less commonly reported, permanent neurological and neuropsychological complications can result from resective surgery . These differ depending on the site of resection, and tend to be more common and disabling if the site is close to areas of the cortex involved in key cognitive functions . Furthermore, the procedure has the possibility to create new lesions which may become epileptogenic and trigger subsequent seizures.47 on the other hand, patients are often withdrawn from aeds following surgery, and therefore relieved from many of the adverse effects of these medications . Surgical procedures are generally considered after the failure of two appropriate drug regimens, although the number of unsuccessful trials with aeds, alone or in combination, before considering surgery remains a matter of debate.47,49 when contemplating surgery, it is important to assess the likelihood of seizure remission using further drug regimens compared to surgical resection, whilst considering the risks associated with such procedures . Although current evidence suggests that temporal lobe surgery is the most effective treatment option after the failure of two aeds, the same cannot be said for other surgical procedures, such as frontal lobe resection, whereby the overall benefit from surgery may not be greater than trying alternative drug regimens . In these cases, the risks for each treatment option should be weighed up on a case - by - case basis until more definitive data is available to guide therapeutic decisions . Patients with intractable epilepsy who are not suitable for epilepsy surgery or those who continue to experience seizures post - operatively may benefit from neurostimulation . This treatment option for patients with intractable epilepsy involves electrical stimulation of the nervous system using surgically implanted devices, or non - invasive stimulation as is the case for repetitive transcranial magnetic stimulation (rtms). Vagal nerve stimulation (vns) therapy is the only neurostimulation method to have received approval by the usa food and drug administration, whilst both vns and deep brain stimulation (dbs) are approved for use in patients with refractory epilepsy in the uk . In vns therapy, intermittent electrical stimulation is delivered to the left vagus nerve, which has ascending fibers with widespread connections to the limbic, autonomic and reticular brain regions . The reduced thalamic blood flow that is observed in vns has been proposed to underlie the mechanism for seizure reduction.50 in addition to a number of retrospective studies, efficacy for this treatment option has been demonstrated in two randomized, placebo - controlled, double - blind trials, which reported a median seizure reduction of 24.5%28.0% in the group receiving high level vns compared to just 6.1%15.0% in patients receiving low level vns (p = 0.01 and p = 0.04, respectively).51,52 the risks associated with implantation of the vns device are relatively low, with a 3%5% chance of infection.5355 vocal cord dysfunction, throat discomfort, change in voice quality and sleep apnea can present post - operatively, and such consequences should be discussed with the patient when considering this treatment . An additional factor that should be considered when planning vns therapy is that patients are usually unable to undergo magnetic resonance imaging (mri) after implantation, since the changing magnetic fields may induce electrical currents in the device.56 dbs involves electrical stimulation of specific subcortical nuclei, which have widespread neural connections . The anterior nucleus of the thalamus is often the target of dbs due to its widespread projections to the limbic system . In a randomized controlled trial of 109 patients with refractory epilepsy who had dbs electrodes implanted in the anterior nucleus of the thalamus there was a 29% greater reduction in seizures for 54 patients who had the stimulator switched on compared to 55 patients who had their stimulation turned off after a blinded period of 3 months (p = 0.002).57 infection, hemorrhage and stimulation - induced seizures were amongst the most frequently reported complications . In the same study, impaired memory and higher levels of depression were observed in the group receiving the active treatment . Dbs has been recently licensed in the uk, and nice guidelines suggest that this method should be used in highly selected cases, due to the lack of efficacy data and the associated risks.58 the centromedian nucleus is also a potential location for dbs, since it is part of the reticulo - thalamo - cortical system which is involved in mediating cortical excitability and wakefulness . One double - blind crossover trial reported a 30% reduction in generalized tonic - tonic seizures when the device was turned on compared to when it was off, although this change was not significant, possibly due to the small sample size of 7 patients.59 further evidence for centromedian nucleus stimulation efficacy can be found in a longitudinal study which reported good efficacy for generalized tonic - clonic seizures, atypical absences and tonic seizures, however caution should be taken in interpreting these findings because of the lack of control groups.60 unlike other methods of neurostimulation, responsive neurostimulation (rns) does not deliver electrical stimulation at specific frequencies throughout the day . Instead, the rns device is composed of a combined recorder and stimulator device, which detects clinically relevant epileptiform discharges and delivers an appropriate electrical stimuli in response . A recent multicenter double - blind randomized controlled trial in adults with intractable partial seizures demonstrated a reduction in seizure frequency of 37.9% in the treatment arm compared to 17.3% in the control group (p = 0.012).61 the fourth and only non - invasive neurostimulation technique, low frequency rtms, is thought to suppress cortical excitability either via inducing long term depression or alternatively by enhancing gabaergic inhibition.62 although evidence for its efficacy is limited, a number of small - scale studies suggest antiepileptic properties (see fregni and pascual - leone for a comprehensive review).63 over the past century, the efficacy of dietary therapies for intractable seizures has increasingly been recognized . Currently there are four main types of metabolic therapy in use for the reduction of seizures . Three of these have a high fat content and low carbohydrate content the classic ketogenic diet, the medium chain triglyceride (mct) diet and the modified atkins diet (mad)and the fourth controls the carbohydrate quality, the low glycemic index treatment (lgit). These treatments are usually considered in patients with intractable epilepsy who have failed to respond to several aeds and are not suitable candidates for surgery . The classic ketogenic diet is a high fat and low carbohydrate diet that uses long chain fatty acids (lcfas) as its main source of fat . Typically the patient must consume 34 grams of fat for every 1 gram of carbohydrate plus protein . As a result, between 86% and 90% of the calories from this diet are provided from lipids.64 the mct diet provides more ketones per calorie consumed than in the classic ketogenic diet, which uses mainly lcfas . In mct, more carbohydrates and proteins can be consumed for every gram of fat . The mad recommends increased consumption of foods high in fat whilst restricting the amount of carbohydrates . Unlike the classic ketogenic and mct diets, there is no limit on the amount of protein consumed or the total number of calories per day.65 the typical mad uses a ratio of 1 gram of fat for every gram of carbohydrate plus protein, and consequently is less restrictive than the other two high fat diets . The lgit restricts carbohydrate intake to food items with a glycemic index of less than 50 at 4060 grams per day, in order to prevent large fluctuations in blood glucose concentrations, which are thought to exacerbate seizures.66 there is a large body of evidence demonstrating the efficacy of the metabolism - based therapies for refractory seizures, with the majority focusing on the classic ketogenic diet . Two meta - analyses of observational studies showed that 15.6%15.8% of patients on the diet can achieve seizure freedom whilst 33.0%55.8% of the patients can have a> 50% reduction in number of seizures.67 one of the only randomized controlled trials to investigate the efficacy of the classic ketogenic diet followed children with intractable or focal - onset epilepsy who were randomized to a ketogenic diet or continuation of a normal diet for a 3-month period . Of the patients who received the ketogenic diet, 38% had a seizure reduction of> 50% . This was significantly greater than the control group, where only 6% had> 50% seizure reduction (p <0.001).68 the results of a follow - up study comparing the effectiveness of the lcfa versus the mct diet suggest there is no increased benefit using either of the two diets for seizure reduction.69 despite the lack of prospective randomized control trials, the mad has also demonstrated substantial efficacy in controlling seizures . Of the patients who have been studied on the mad, 45% have demonstrated 50%90% seizure reduction and 28% have> 90% seizure reduction . Similar results have been reported in two retrospective studies of the lgit, with 38%73% of patients achieving> 50% seizure reduction.70,71 the best candidates for the mad tend to be adults and adolescents, for whom compliance on the lcfa and mct diets is an issue.64 this is because the food consumed on the mad is similar to that being consumed by peers . The same can be said about the lgit, which in addition can be considered for patients who find the other 3 high - fat diets unpalatable . There is a body of evidence suggesting that metabolism - based therapy can be considered earlier in the treatment plan for patients with specific epilepsy syndromes . One notable example is west syndrome (infantile spasms), where the ketogenic diet is successfully used as a first line agent.72 the same diet is also thought to be especially effective in unspecified symptomatic generalized epilepsy, multifocal epilepsy, juvenile absence epilepsy, continuous spike and slow wave of sleep, myoclonic - astatic epilepsy (mae or doose syndrome), severe myoclonic epilepsy of infancy (dravet syndrome), and seizures in patients with tuberous sclerosis complex and landau - kleffner syndrome.68,73,74 ketogenic diets are also recommended for patients suffering from glucose transporter defects and pyruvate dehydrogenase deficiency, who are unable to utilize glucose for brain metabolism.75,76 the most common side effects of dietary treatments are constipation, acidosis, temporary hypercholesterolemia, kidney stones and hunger.64 growth restriction has also been observed in children.77 the ketogenic diet is contraindicated in patients who are unable to use ketone bodies as an energy source or those who require high levels of glucose . Such conditions include pyruvate decarboxylase deficiency, primary carnitine deficiency, fatty acid oxidation abnormalities, the porphyrias and some mitochondrial disorders.78 generally, as with aeds, it is suggested that after adhering to dietary therapy for a period of 2 years, the treatment should slowly be tapered off . Many patients who become seizure free whilst adhering to the diets remain seizure free off the diet . This has been demonstrated in one study, which reported the risk of seizures recurring when stopping the diet at only 20%.79 it has been hypothesized that the dietary therapies have a long - term, disease modifying effects, although the observed phenomenon could just be explained by the natural history of the seizure disorder.64 in addition to showing high levels of efficacy, similar to many aeds, the dietary interventions are generally quite tolerable, can have associated positive effects such as weight loss,80 and demonstrate effectiveness in a relatively short time period . Whilst aeds may take several weeks to be titrated up to a therapeutic dose and vns may take months or years to show an optimal effect,81 the effects of a ketogenic diet can be seen within a few days . Furthermore, the diets are cost effective compared to many aeds.64 in practice, a median of 5 aeds are prescribed before considering a metabolic treatment such as the ketogenic diet.82 furthermore, 60% of child neurologists only use a ketogenic diet as a last resort treatment.83 it is therefore suggested that the metabolic treatments should be considered earlier in the course of treatment, especially in patients with refractory seizures who are not suitable for or willing to undergo resective surgery . Herbal treatments are generally viewed as alternative and complementary forms of medicine by both patients and physicians . There is, however, a small body of evidence demonstrating the effectiveness of some herbs at controlling seizures . Out of all the herbal treatments, cannabinoids have the largest body of evidence supporting their use as anticonvulsants . One randomized, double - blind controlled trial reported 50% of patients with secondary generalized epilepsy receiving cannabidiol becoming virtually seizure - free for the duration of the study . However, only 15 patients were included and to our knowledge no follow - up study has been performed.84 the use of cannabis for epilepsy has been legalized in canada and licensed in 14 states within the united states, although it is prohibited in most european countries.85huperzine a. has been shown to be effective in animal models of epilepsy, and a clinical trial to assess its effectiveness has been planned.86,87 there is also laboratory data supporting the efficacy of kava (piper methysticum) and mistletoe (viscum sp), but no clinical evidence exists to support their use . A number of other herbal treatments that are commonly used to treat seizures have either been shown to have no effect on the severity of seizures, or in fact demonstrate proconvulsant properties and exacerbate the condition . In addition, much evidence is anecdotal, or when clinical studies have been performed, these often display poor methodology and do not have sufficient levels of statistical power to make firm conclusions from . Further, good quality evidence in the form of controlled trials is required to justify the use of herbal treatments in a clinical setting (see pearl et al for a comprehensive review).88 for resective surgery to be a viable option, the patient with treatment - refractory epilepsy should ideally have a single epileptogenic focus in a non - eloquent cortical region (ie, not involved in key language, memory or motor processes). It should be noted that exceptional cases do exist, where larger resections such as hemispherectomies are acceptable if the seizures are severe and the benefit gained from such surgery can be justified.38 major surgery such as corpus callosotomy can be considered as last treatment option in palliative care for patients with intractable seizures . This invasive intervention is usually aimed at preventing secondary generalization in patients who are regularly experiencing loss of consciousness and a high frequency of seizure - related injuries . Most epileptologists consider surgical management as second line therapy for patients with intractable seizures.39 this is especially true for patients with drug - resistant temporal lobe epilepsy (tle), for whom temporal lobectomy can represent an effective treatment option . A randomized control trial found that 58% of patients randomly allocated to surgery for temporal lobe resection did not present with any seizures which impaired consciousness after one year, compared to 8% in the treatment arm receiving the optimum medication regimen (p <0.001).20 a multi - center study which followed 339 patients with epilepsy for 2 years post - operatively showed that temporal lobe resection is the most effective type of resective surgery in this patient population: 68% of the patients who received temporal lobectomies experienced seizure remission for 2 years, compared to 50% of the patients who underwent extratemporal resections.40 another non - randomized controlled study which followed up patients with refractory seizures, assigned either to surgical treatment or continued drug therapy, found that 44.6% of 242 patients with pharmacoresistant tle who received surgery were seizure - free for 12 months, compared to 4.3% of those who received aeds only (p <0.001).41 it should be emphasized, however, that not all patients with tle have the same probability of achieving seizure freedom through temporal lobe resection . A meta - analysis of 83 studies including 7,343 patients undergoing epilepsy surgery showed that the proportion of patients experiencing seizure freedom for at least 5 years post - operatively was highest in those who underwent temporal lobe resection (66%), even though a substantial proportion of patients undergoing occipital, parietal or frontal lobe resections demonstrated post - operative seizure freedom (46%, 46% and 27% respectively).42 it should be noted that post - operative seizure freedom for the first few years following surgery might not persist long - term . Isolated relapses can still occur many years after the procedure, with a probability of seizure relapse of 4% per year for the 5 years following surgery.43 overall, patients with structural abnormalities apparent on pre - operative imaging studies, such as hippocampal sclerosis or foreign tissue lesions are more likely to experience post - operative seizure freedom compared to patients with no obvious lesion.4446 when considering surgery for refractory epilepsy, there are a number of risks that need to be taken into account alongside the potential benefits . The surgery itself can cause a substantial period of disability, with patients typically having to take 48 weeks off work or school.47 the most common medical complications are deep vein thrombosis, infection and transient endocrine abnormalities . Psychiatric complications are reported post - resection in 25% of the patients, including transient dysphoria, depression and occasionally mania.48 although less commonly reported, permanent neurological and neuropsychological complications can result from resective surgery . These differ depending on the site of resection, and tend to be more common and disabling if the site is close to areas of the cortex involved in key cognitive functions . Furthermore, the procedure has the possibility to create new lesions which may become epileptogenic and trigger subsequent seizures.47 on the other hand, patients are often withdrawn from aeds following surgery, and therefore relieved from many of the adverse effects of these medications . Surgical procedures are generally considered after the failure of two appropriate drug regimens, although the number of unsuccessful trials with aeds, alone or in combination, before considering surgery remains a matter of debate.47,49 when contemplating surgery, it is important to assess the likelihood of seizure remission using further drug regimens compared to surgical resection, whilst considering the risks associated with such procedures . Although current evidence suggests that temporal lobe surgery is the most effective treatment option after the failure of two aeds, the same cannot be said for other surgical procedures, such as frontal lobe resection, whereby the overall benefit from surgery may not be greater than trying alternative drug regimens . In these cases, the risks for each treatment option should be weighed up on a case - by - case basis until more definitive data is available to guide therapeutic decisions . Patients with intractable epilepsy who are not suitable for epilepsy surgery or those who continue to experience seizures post - operatively may benefit from neurostimulation . This treatment option for patients with intractable epilepsy involves electrical stimulation of the nervous system using surgically implanted devices, or non - invasive stimulation as is the case for repetitive transcranial magnetic stimulation (rtms). Vagal nerve stimulation (vns) therapy is the only neurostimulation method to have received approval by the usa food and drug administration, whilst both vns and deep brain stimulation (dbs) are approved for use in patients with refractory epilepsy in the uk . In vns therapy, intermittent electrical stimulation is delivered to the left vagus nerve, which has ascending fibers with widespread connections to the limbic, autonomic and reticular brain regions . The reduced thalamic blood flow that is observed in vns has been proposed to underlie the mechanism for seizure reduction.50 in addition to a number of retrospective studies, efficacy for this treatment option has been demonstrated in two randomized, placebo - controlled, double - blind trials, which reported a median seizure reduction of 24.5%28.0% in the group receiving high level vns compared to just 6.1%15.0% in patients receiving low level vns (p = 0.01 and p = 0.04, respectively).51,52 the risks associated with implantation of the vns device are relatively low, with a 3%5% chance of infection.5355 vocal cord dysfunction, throat discomfort, change in voice quality and sleep apnea can present post - operatively, and such consequences should be discussed with the patient when considering this treatment . An additional factor that should be considered when planning vns therapy is that patients are usually unable to undergo magnetic resonance imaging (mri) after implantation, since the changing magnetic fields may induce electrical currents in the device.56 dbs involves electrical stimulation of specific subcortical nuclei, which have widespread neural connections . The anterior nucleus of the thalamus is often the target of dbs due to its widespread projections to the limbic system . In a randomized controlled trial of 109 patients with refractory epilepsy who had dbs electrodes implanted in the anterior nucleus of the thalamus there was a 29% greater reduction in seizures for 54 patients who had the stimulator switched on compared to 55 patients who had their stimulation turned off after a blinded period of 3 months (p = 0.002).57 infection, hemorrhage and stimulation - induced seizures were amongst the most frequently reported complications . In the same study, impaired memory and higher levels of depression were observed in the group receiving the active treatment . Dbs has been recently licensed in the uk, and nice guidelines suggest that this method should be used in highly selected cases, due to the lack of efficacy data and the associated risks.58 the centromedian nucleus is also a potential location for dbs, since it is part of the reticulo - thalamo - cortical system which is involved in mediating cortical excitability and wakefulness . One double - blind crossover trial reported a 30% reduction in generalized tonic - tonic seizures when the device was turned on compared to when it was off, although this change was not significant, possibly due to the small sample size of 7 patients.59 further evidence for centromedian nucleus stimulation efficacy can be found in a longitudinal study which reported good efficacy for generalized tonic - clonic seizures, atypical absences and tonic seizures, however caution should be taken in interpreting these findings because of the lack of control groups.60 unlike other methods of neurostimulation, responsive neurostimulation (rns) does not deliver electrical stimulation at specific frequencies throughout the day . Instead, the rns device is composed of a combined recorder and stimulator device, which detects clinically relevant epileptiform discharges and delivers an appropriate electrical stimuli in response . A recent multicenter double - blind randomized controlled trial in adults with intractable partial seizures demonstrated a reduction in seizure frequency of 37.9% in the treatment arm compared to 17.3% in the control group (p = 0.012).61 the fourth and only non - invasive neurostimulation technique, low frequency rtms, is thought to suppress cortical excitability either via inducing long term depression or alternatively by enhancing gabaergic inhibition.62 although evidence for its efficacy is limited, a number of small - scale studies suggest antiepileptic properties (see fregni and pascual - leone for a comprehensive review).63 over the past century, the efficacy of dietary therapies for intractable seizures has increasingly been recognized . Currently there are four main types of metabolic therapy in use for the reduction of seizures . Three of these have a high fat content and low carbohydrate content the classic ketogenic diet, the medium chain triglyceride (mct) diet and the modified atkins diet (mad)and the fourth controls the carbohydrate quality, the low glycemic index treatment (lgit). These treatments are usually considered in patients with intractable epilepsy who have failed to respond to several aeds and are not suitable candidates for surgery . The classic ketogenic diet is a high fat and low carbohydrate diet that uses long chain fatty acids (lcfas) as its main source of fat . Typically the patient must consume 34 grams of fat for every 1 gram of carbohydrate plus protein . As a result, between 86% and 90% of the calories from this diet are provided from lipids.64 the mct diet provides more ketones per calorie consumed than in the classic ketogenic diet, which uses mainly lcfas . In mct the mad recommends increased consumption of foods high in fat whilst restricting the amount of carbohydrates . Unlike the classic ketogenic and mct diets, there is no limit on the amount of protein consumed or the total number of calories per day.65 the typical mad uses a ratio of 1 gram of fat for every gram of carbohydrate plus protein, and consequently is less restrictive than the other two high fat diets . The lgit restricts carbohydrate intake to food items with a glycemic index of less than 50 at 4060 grams per day, in order to prevent large fluctuations in blood glucose concentrations, which are thought to exacerbate seizures.66 there is a large body of evidence demonstrating the efficacy of the metabolism - based therapies for refractory seizures, with the majority focusing on the classic ketogenic diet . Two meta - analyses of observational studies showed that 15.6%15.8% of patients on the diet can achieve seizure freedom whilst 33.0%55.8% of the patients can have a> 50% reduction in number of seizures.67 one of the only randomized controlled trials to investigate the efficacy of the classic ketogenic diet followed children with intractable or focal - onset epilepsy who were randomized to a ketogenic diet or continuation of a normal diet for a 3-month period . Of the patients who received the ketogenic diet, 38% had a seizure reduction of> 50% . This was significantly greater than the control group, where only 6% had> 50% seizure reduction (p <0.001).68 the results of a follow - up study comparing the effectiveness of the lcfa versus the mct diet suggest there is no increased benefit using either of the two diets for seizure reduction.69 despite the lack of prospective randomized control trials, the mad has also demonstrated substantial efficacy in controlling seizures . Of the patients who have been studied on the mad, 45% have demonstrated 50%90% seizure reduction and 28% have> 90% seizure reduction . Similar results have been reported in two retrospective studies of the lgit, with 38%73% of patients achieving> 50% seizure reduction.70,71 the best candidates for the mad tend to be adults and adolescents, for whom compliance on the lcfa and mct diets is an issue.64 this is because the food consumed on the mad is similar to that being consumed by peers . The same can be said about the lgit, which in addition can be considered for patients who find the other 3 high - fat diets unpalatable . There is a body of evidence suggesting that metabolism - based therapy can be considered earlier in the treatment plan for patients with specific epilepsy syndromes . One notable example is west syndrome (infantile spasms), where the ketogenic diet is successfully used as a first line agent.72 the same diet is also thought to be especially effective in unspecified symptomatic generalized epilepsy, multifocal epilepsy, juvenile absence epilepsy, continuous spike and slow wave of sleep, myoclonic - astatic epilepsy (mae or doose syndrome), severe myoclonic epilepsy of infancy (dravet syndrome), and seizures in patients with tuberous sclerosis complex and landau - kleffner syndrome.68,73,74 ketogenic diets are also recommended for patients suffering from glucose transporter defects and pyruvate dehydrogenase deficiency, who are unable to utilize glucose for brain metabolism.75,76 the most common side effects of dietary treatments are constipation, acidosis, temporary hypercholesterolemia, kidney stones and hunger.64 growth restriction has also been observed in children.77 the ketogenic diet is contraindicated in patients who are unable to use ketone bodies as an energy source or those who require high levels of glucose . Such conditions include pyruvate decarboxylase deficiency, primary carnitine deficiency, fatty acid oxidation abnormalities, the porphyrias and some mitochondrial disorders.78 generally, as with aeds, it is suggested that after adhering to dietary therapy for a period of 2 years, the treatment should slowly be tapered off . Many patients who become seizure free whilst adhering to the diets remain seizure free off the diet . This has been demonstrated in one study, which reported the risk of seizures recurring when stopping the diet at only 20%.79 it has been hypothesized that the dietary therapies have a long - term, disease modifying effects, although the observed phenomenon could just be explained by the natural history of the seizure disorder.64 in addition to showing high levels of efficacy, similar to many aeds, the dietary interventions are generally quite tolerable, can have associated positive effects such as weight loss,80 and demonstrate effectiveness in a relatively short time period . Whilst aeds may take several weeks to be titrated up to a therapeutic dose and vns may take months or years to show an optimal effect,81 the effects of a ketogenic diet can be seen within a few days . Furthermore, the diets are cost effective compared to many aeds.64 in practice, a median of 5 aeds are prescribed before considering a metabolic treatment such as the ketogenic diet.82 furthermore, 60% of child neurologists only use a ketogenic diet as a last resort treatment.83 it is therefore suggested that the metabolic treatments should be considered earlier in the course of treatment, especially in patients with refractory seizures who are not suitable for or willing to undergo resective surgery . Herbal treatments are generally viewed as alternative and complementary forms of medicine by both patients and physicians . There is, however, a small body of evidence demonstrating the effectiveness of some herbs at controlling seizures . Out of all the herbal treatments, cannabinoids have the largest body of evidence supporting their use as anticonvulsants . One randomized, double - blind controlled trial reported 50% of patients with secondary generalized epilepsy receiving cannabidiol becoming virtually seizure - free for the duration of the study . However, only 15 patients were included and to our knowledge no follow - up study has been performed.84 the use of cannabis for epilepsy has been legalized in canada and licensed in 14 states within the united states, although it is prohibited in most european countries.85huperzine a. has been shown to be effective in animal models of epilepsy, and a clinical trial to assess its effectiveness has been planned.86,87 there is also laboratory data supporting the efficacy of kava (piper methysticum) and mistletoe (viscum sp), but no clinical evidence exists to support their use . A number of other herbal treatments that are commonly used to treat seizures have either been shown to have no effect on the severity of seizures, or in fact demonstrate proconvulsant properties and exacerbate the condition . In addition, much evidence is anecdotal, or when clinical studies have been performed, these often display poor methodology and do not have sufficient levels of statistical power to make firm conclusions from . Further, good quality evidence in the form of controlled trials is required to justify the use of herbal treatments in a clinical setting (see pearl et al for a comprehensive review).88 patients with treatment refractory seizures continue to represent a large proportion of patients diagnosed with epilepsy . The fact that recurrent seizures and associated side effects from aeds significantly impact on patient s qol means that treatment decisions should be made in light of the best evidence for improved seizure control, enhanced qol and the greatest tolerability profile . Confirmation that the events are epileptogenic in origin and early identification of an underlying etiology or specific epilepsy syndrome is essential to guide appropriate treatment for which syndrome or etiology specific efficacy exists . This may be in the form of monotherapy, polytherapy or alternative treatment options including surgery, neurostimulation, metabolic treatments and herbal remedies . Despite the fact that decisions to alter a treatment regimen may be influenced by local availability and funding, physicians should balance the evidence for efficacy in seizure reduction with the associated risks, side effects and patient preference.
In august of 2005, hurricane katrina devastated the new orleans area with high wind, heavy rainfall, and a storm surge of about 7 m which caused the collapse of the levee system surrounding the city . Approximately 80% of the city was flooded to varying depths for many weeks before the us army corps of engineers was able to implement temporary levee repairs and install emergency pumping capacity . In the aftermath of the flood event, the infrastructures of the city along with residences and commercial buildings were grossly contaminated with sediments deposited by the floodwaters and subsequently by microbial overgrowth supported by the residual moisture, high humidity, and elevated temperatures in the area . After floodwaters had receded, various surveys were conducted for measurement of indicators of microbial contamination in air, dust, and damaged building materials, including total and culturable mold spores, fungal fragments, mycotoxins, 13--d - glucan, and bacterial endotoxin . Generally, observed levels of microbial contaminants in these surveys were elevated, often extremely so, and were relatable to the depth and duration of flooding, and indoor levels were typically higher than those in the surrounding outdoor environment [15]. Subsequent to the posthurricane flooding event, there has been extensive rebuilding in the new orleans area . Residents who personally performed repairs of their properties as well as various skilled and unskilled laborers working in the construction and building maintenance trades were at risk for inhalation exposures to dust containing microbial and other agents during demolition, removal, and repair of flood - damaged and contaminated infrastructure and building materials [68]. Exposures to microbial contaminants in agriculture, waste management, and in water - damaged and moldy buildings have been linked to various upper and lower respiratory illnesses and adverse effects including rhinitis, hayfever, toxic pneumonitis (tp), hypersensitivity pneumonitis (hp), and respiratory infections including pneumonia and exacerbation or initiation of asthma [912]. The potential for respiratory illness arising from inhalation exposure to bioaerosols and microbial contaminants during restoration activities in the post - hurricane katrina environment was of particular concern . As a part of a 5-year longitudinal study investigating the risk of respiratory illness associated with work in and around flood - damaged structures in post - hurricane katrina new orleans, baseline findings from initial cross - sectional survey are reported . The study cohort consisted of 791 adults residing or working in the greater new orleans metropolitan area . Study participants were recruited from several sources: (1) employees of three large institutions in the city of new orleans, two of which are academic and the third is a branch of local government (n = 488 total). All three institutions experienced heavy flood damage to their buildings and facilities and utilized their regular staff as well as contract labor to perform restoration work . Recruiting from the academic institutions focused primarily on workers from departments normally engaged in maintenance, custodial, and facilities services . Support personnel (clerical, managerial, etc .) From the targeted departments were included in the recruitment . (2) members of a local union hall for the skilled and unskilled building trades (n = 63). (3) private building contractors and self - employed tradesmen (n = 95). (4) other residents of the new orleans area (n = 145), many of whom performed restoration work on their own properties . Overall, 54% of the study cohort reported a skilled or unskilled trade as their primary occupation, including carpentry (n = 50), electrician (n = 27), plumbing (n = 12), paint / drywall (n = 21), hvac (n = 12), groundskeeping (n = 20), general construction (n = 102), general maintenance (n = 36), operating / building engineering (n = 18), and being mechanic / machinist (n = 15). An additional 15% of study participants worked in custodial or janitorial services (n = 115). Testing was conducted in a mobile laboratory van outfitted with spirometry and interview work stations and ancillary equipment . The mobile laboratory van was moved to the work locations or union hall of the study participants for the duration of their respective testing period, generally 2 to 3 weeks, and to the parking lots of several large building supply stores, in order to allow private contractors and self - employed construction tradesmen to participate . Spirometry testing procedures and equipment have been previously described and comply with both the original and updated american thoracic society spirometric test criteria . Spirograms were collected with a sensormedics model 1022 dry rolling seal spirometer interfaced to a laptop computer running omi spirometry software version 5.05.9 (occupational marketing, inc ., all spirometric testing was conducted by the same individual who is a member of the research staff and is a certified pulmonary function technician; in addition, all spirometric test results were quality assured and interpreted by senior study investigators . Predicted lung function parameters and lower limit of normal (lln) lung function values for forced expiratory volume in one second (fev1), forced vital capacity (fvc), and fev1/fvc ratio were computed from predictive equations developed by hankinson et al . . Separate predictive equations were used for caucasians, african americans, and latinos . Predicted values for study participants of asian heritage were calculated using the equations for caucasians . In addition to race, the predicted values were based on age, gender, and height . The lln values were calculated by subtracting 1.645 see from the predicted values, where see was the standard error of the estimate and 1.645 is the 95th percentile of a standard normal distribution . Those participants with chronic obstructive pulmonary disease (copd) were identified according to the gold criteria, that is, fev1/fvc% predicted less than 70% and fev1% predicted less than 80%; however, only prebronchodilator lung function values were available and thus may not have adequately differentiated asthma (with reversible obstruction) from copd . Ever asthma on questionnaire were therefore excluded from the analyses of copd prevalence as a function of exposure . A demographic, medical, smoking, and occupational questionnaire it was based on a modified version of the standardized questionnaire reported by burrows et al ., which accounts for a variety of putative and established risk factors and potential confounders for the development of airways disease including asthma, allergic disease, historical confounding exposures, serious childhood respiratory illness, cigarette smoking history, environmental tobacco smoke, and age, gender, and race . Additional questions were designed to capture the development of specific symptoms after hurricane katrina that might be associated with living and working in the post - katrina environment . These included post - hurricane katrina onset of asthma, sinus symptoms, pneumonia, and transient fever and cough absent infection, with the latter used as an indicator of possible hypersensitive (hp) or toxic (tp) reaction . Asthma was defined dichotomously and required a positive response to both of the following questions: have you ever had asthma or attacks of shortness of breath with wheezing in the chest when not having a cold? Followed by the response to the question how old were you when your asthma started? Was used in conjunction with the participant's date of birth to determine whether asthma onset was after september 30, 2005 (post - katrina new onset asthma). Dyspnea was also defined dichotomously and required a positive response to the question do you have shortness of breath when hurrying on level ground? The interview also included queries on pre- and post - katrina work and occupation, and detailed information was gathered on time spent after hurricane katrina performing five specific types of hurricane / flood remediation work: demolition and ripout, trash removal, landscape restoration, sewer line repair, and mold remediation . Participation in any of these work activities, herein identified as restoration work, was assumed to result in occupational or vocational exposure to flood - related contaminants . Participants were asked to report the number of hours spent in each of the five restoration work activities, for each year since the hurricane up to the point of interview, and the type and relative frequency of any respiratory protective equipment that may have been used during the work . Restoration of personal property was included in the total time spent in restoration work along with any from the subject's regular employment . The study protocol was approved by the authors' institutional review board and all study participants provided a written informed consent . For the current hayfever compared to current trouble with pollen, grass, or fur, analysis indicated that the parallel question significantly enhanced the positive response rate for these symptoms (22% claiming current hayfever versus 39% claiming sensitivity to pollen, grass, or animal fur; p <0.0001). Similar but nonsignificant results were observed for ever and current asthma versus attacks of dyspnea and for chronic bronchitis versus copd . The unadjusted prevalence rate ratios for each symptom or condition for those doing any restoration work versus those not doing any restoration work were calculated within smoking categories based on 2 2 contingency tables . The prevalence rate ratio was defined as prr = p1/p2, where p1 = a / n1 and p2 = c / n2 represent the sample proportion of exposed (n1) and unexposed (n2) individuals with disease . If a and b represent the number of exposed subjects who do and do not have disease, respectively, and c and d represent the number of unexposed subjects who do and do not have disease, respectively, the asymptotic 95% confidence interval for prevalence rate ratio is calculated using the following standard logarithmic transformation: ln(prr)1.96b / an1+d / cn2 . Exponential transformations on the confidence limits of this log transformed interval provided the asymptotic 95% confidence intervals for prevalence rate ratio . Multiple logistic regression analyses were used to compute adjusted prevalence odds ratios for each symptom or condition per 100 hours of restoration work as well as to compute asymptotic 95% confidence intervals for prevalence odds ratios . Due to significant interactions between gender and total hours of restoration work, logistic regression analyses were performed separately by gender and adjusted for age (because of a significant correlation with prevalence of pneumonia) and smoking categories . All interactions between age, smoking category, use of respiratory protective equipment (ever versus never), and total hours of restoration work were considered and were not significant . Multiple linear regression related% p fev1, fvc, and fev1/fvc to restoration work hours, use of respiratory protection, gender, asthma classification, and smoking category . All possible interactions were considered and were not significant, and no significant exposure associations were detected . The majority of the study cohort was african american and male (table 1). Current smokers comprised 28% of the cohort, whereas 18% were ex - smokers and 54% had never smoked . 3.7% of the study cohort reported having pneumonia after hurricane katrina and almost half reported newly developing sinus symptoms . Among those reporting never having had asthma prior to hurricane katrina (n = 539), about 4.5% reported new onset asthma . Episodes of transient fever and cough occurring after hurricane katrina were reported by about 29% of the study cohort . Multiple episodes were also common in this reporting group: the median number of episodes was 3, and 10% of the group reported having 12 or more such occurrences . Overall, lung function parameters were somewhat depressed in the cohort (tables 1 and 4) and correlated with cigarette smoking and presence of current asthma symptoms . Percent predicted (% p) fev1 averaged 93.4% (sd: 16.0) for current smokers, while ex- and never smokers had a mean level of 96.0% (sd: 15.4), p = 0.037 by t - test . The proportions of the cohort falling below lln for fev1 and fvc were also somewhat elevated (5% being the expected proportion based on the definition of lln), particularly for the current and ex - smokers, as expected . Participants reporting current asthma symptoms had a mean% p fev1 of 89.2% (sd: 21.8) and% p fvc of 91.8% (sd: 16.8), while asthmatics without current symptoms had a mean% p fev1 of 95.8% (sd: 18.0) and% p fvc of 96.7% (sd: 15.2). Participants who never had asthma had mean% p fev1 of 96.1% (sd: 13.6) and% p fvc of 96.4% (sd: 13.6). Almost 75% of the study participants reported having performed some restoration work activity after hurricane katrina (n = 587), and details on the actual time spent in these activities were self - reported by 474 or 81% of this group (table 2). Demolition / ripout was the most commonly reported restoration work activity, followed by landscape restoration and trash / debris removal . The distributions of time spent in these activities were highly skewed because many study subjects worked as much as 16 hours per day, seven days per week, for extended durations after hurricane katrina . The majority of the study subjects also reported time spent in more than one type of restoration work activity . For the total combined hours spent in any of the specific restoration work activities, the mean and median values reported by 474 subjects with complete data were 1646 and 620 hours, respectively . Among those who reported performing restoration work, 80.1% reported some use of respiratory protective equipment (i.e., filtering facepiece or air - purifying cartridge respirator). 202 study subjects reported no time spent in restoration work . The prevalence rates for post - hurricane katrina episodes of transient fever and cough and new onset sinusitis were significantly elevated for those reporting any restoration work (prr: 1.7 and 1.3, resp . ; table 3). The prevalence rate ratios were statistically significant for ex- and never smokers but not current smokers . The prevalence rate ratios for post - hurricane katrina new onset asthma were elevated for the overall cohort (prr = 2.2) and especially for ex- and never smokers (prr = 2.7) but were not statistically significant (p = 0.09); the overall lack of significance may have been due in part to the low incidence (29 cases) and the reduced size of the base population; that is, only those who never had asthma prior to hurricane katrina . Statistically significant elevations in prevalence rate ratios for those having done any restoration work were not observed for pneumonia, dyspnea, copd, and being below lln for any of the lung function parameters . Figure 1 illustrates the prevalence of respiratory symptoms and conditions with quartiles of reported time spent in post - hurricane katrina restoration work activities (proportions for new onset asthma and pneumonia are multiplied by 10 in the figure for purposes of scaling). The unadjusted proportions for several post - hurricane katrina symptoms and conditions, including transient fever and cough, dyspnea, new onset sinus symptoms, new onset asthma, and pneumonia, show trends of increasing prevalence with time in restoration work . Statistically significant increases in prevalence odds ratio with restoration work time were observed only for transient fever and cough, for new onset sinus symptoms, and for dyspnea . When analyzed by logistic regression, the prevalence of fever and cough was statistically significantly associated with restoration work time, but only for men, with a prevalence odds ratio of 1.016 per 100 hours . Likewise, for new onset sinus symptoms in men, the prevalence odds ratio was 1.042 per 100 hours . In contrast, only women exhibited a statistically significant association between the prevalence of dyspnea and restoration work time, the odds ratio being 1.031 per 100 hours . The restoration work time - gender interactions observed in the logistic regression analysis of the prevalence odds ratios for these symptoms may be confounded with job type since women in the study tended to be in the custodial / janitorial occupations, whereas men were more likely to be in the building, construction, or maintenance trades . The exposures associated with restoration work done within these two broad categories of occupation are likely to be qualitatively and quantitatively different . The prevalence odds ratios for post - katrina new onset asthma and for pneumonia with restoration work time were elevated but were not statistically significant by logistic regression analysis . For new onset asthma the crude, unadjusted proportions in each of the restoration work time quartiles (figure 1) were all higher than for participants with no restoration work time (1.99.1% versus 1.7%). For pneumonia, the unadjusted prevalence among those with no restoration work was 3.0% whereas subjects in each of the first three quartiles of restoration work time exhibited higher prevalence rates (3.25.1%); however, those in the highest quartile of restoration work time had a pneumonia prevalence rate of only 2.5% . The group mean values for% p fev1, fvc, and fev1/fvc ratios generally were depressed but were within 5% of the normal except for smokers performing restoration work . Current smokers who did restoration work showed lower overall predicted lung function compared to smokers who did not; however, multiple linear regression analysis yielded no statistically significant correlations of any of the lung function parameters with restoration work time after adjustment for smoking, gender, asthma status, and use of respiratory protective equipment . The results of this study suggest moderate adverse impact on respiratory health from time spent in post - hurricane katrina flood restoration activities . Published reports and public health surveillance systems generally did not show increases in emergency room visits or hospitalizations resulting from exposures in the post - hurricane katrina environment, although there have been a few reports of increased risk for respiratory effects . In a survey of 525 new orleans firefighters, 79% had contact with floodwaters following hurricane katrina, and 38% reported new onset respiratory symptoms including sinus congestion, throat irritation, and cough . The prevalence rate ratio for those who had contact with floodwater versus those who did not was 1.9 and was statistically significant . First responders would have had significant exposures to flood sediments and associated contaminants, in addition to microbial agents . Inhalation exposure to aerosolized sediment collected in the aftermath of hurricane katrina was also shown to elicit significant pulmonary inflammation, increased airways resistance, and airway hyperreactivity in a mouse model . There were widespread anecdotal reports of persistent nonproductive cough, often with sore throat and rhinorrhea, in the population residing in new orleans in the fall of 2005 . An investigation of this phenomenon by the louisiana department of health and hospitals concluded that visits to medical facilities for respiratory complaints in the population of new orleans were not related to exposure to dust or molds at the residence or at work . It is likely that katrina cough was an irritant phenomenon resulting from a dry fall season with high levels of airborne particulate matter, coinciding with the start of the regular allergy and flu seasons [21, 24]. The prevalence of episodes of fever and cough in the present study population is clearly elevated for those who have done restoration work . However, given the overall strong correlation with restoration work time, the common reports of multiple and distinct episodes of fever and cough, and the inclusion of the febrile component in the symptom complex, tp is likely to be underlying many of these reports and appears to be a common adverse effect of restoration work exposures in the post - hurricane katrina environment . Unlike hypersensitivity pneumonitis, it is uncertain whether toxic pneumonitis and inhalation fevers result in significant lasting decrements in lung function, and functional parameters are expected to return to baseline upon recovery from an episode [25, 26]. This study did not identify any restoration work - related decrements in functional parameters, nor in the prevalence of being below lln . The world health organization, in its report on guidelines on indoor air quality related to damp indoor spaces and mold, concluded that the evidence is inadequate to identify an association between damp indoor environments or the presence of mold with risk of alterations in lung function . However, in a recent study of 6,443 individuals in the european community respiratory health survey, lung function measurements across 9 years showed statistically significant excess declines in fev1 of 2.25 ml / year and an additional 7.43 ml / year for women who reported dampness in the home and visible damp spots in the bedroom, respectively . Annual excess declines of such small magnitude are difficult to detect over a short time period and are unlikely to result in a detectable group difference in function, measured cross - sectionally, after only a few years in the post - hurricane katrina environment, as in this baseline study . However, our study population is being evaluated annually over the course of a 5-year period, and currently undetectable decrements in lung function may reach the level of significance when measured directly over this extended period of time . It is generally accepted that exposure to flood - related microbial contaminants can exacerbate existing asthma, and there is a mounting evidence that such exposures increase the risk of development of new asthma [11, 12]. In a recent extensive review and meta - analysis of the literature from 1980 to 2010, overall odds ratios of 1.49 (c.i . : 1.281.72) and 1.68 (c.i . : 1.481.90) were found for the associations of asthma and wheezing, respectively, in children living in homes with visible mold . In this study, there was an observable elevation in prevalence of new onset asthma after hurricane katrina which increased with increasing quartiles of restoration work time but was not statistically significant . The lack of significance may be due in part to a self - selection process occurring in the cohort, with some study subjects who developed new onset asthma in the wake of hurricane katrina avoiding or terminating further restoration work exposure because of personal health concerns . Furthermore, the reported restoration work time is at the time of the interview, not at the time of development of new onset asthma, which always preceded the interview . Some of the study subjects may have continued to engage in and increase their time in restoration work to varying extent after developing asthma, but there is no information on the magnitude of this confounding nor its effect on the analysis . This study has several additional limitations: as noted, health outcomes were assessed cross - sectionally, and there was a significant time element over which the baseline information was collected for the entire study population . Those subjects evaluated later in the study period could therefore have opportunity for greater amounts of time spent in restoration work with concomitant increase in risk for development of respiratory effects . As the longitudinal component of the study moves to completion, additional reports of development of respiratory symptoms and conditions are coming to light, and the ability to detect ongoing small excess decrements in lung function will also increase . Exposure to flood - related contaminants was assumed to be related to reported time spent performing restoration work . However, there is no information as to a particular individual's exposure intensity nor can it be assumed that all exposures associated with restoration work activities were qualitatively or quantitatively similar . Finally, the reliance on self - reporting of respiratory symptoms and conditions could have led to misclassification due to recall bias . This study provides further evidence that workers performing restoration work on flood - damaged structures are at risk of respiratory health impacts from exposure to microbial - contaminated dust and debris . Moderate adverse respiratory health effects including toxic pneumonitis and sinusitis were commonly reported in the study cohort, and the prevalence of new onset asthma among restoration workers was noticeably elevated . While it is unclear from this cross - sectional analysis whether restoration work exposures have adversely affected pulmonary function in the population, the functional parameters overall are depressed in the cohort . Ongoing longitudinal health surveillance of this study cohort, along with a quantitative exposure assessment, will examine whether there is an increased risk for long - term or irreversible effects on respiratory health and how the risks relate to the nature and magnitude of the exposures occurring during posthurricane flood restoration work.
Road traffic injuries are responsible for the death of 1.23 million people around the world annually (1). In terms of disability adjusted life year (daly ((1). Road traffic injuries ranked ninth in 1999, and are expected to rise to the third place by 2020 (2). These events account for 1 - 2% of gross national product (gdp) of different countries (3). In iran, cost of road traffic injuries is 2.19% of gdp that is significantly higher than the global average (4). More than 20% of deaths caused by traffic crashes belong to pedestrians . According to the estimates, about 273000 pedestrians around the world lost their lives due to traffic crashes in 2010 (5). In 2013, 22% of all fatalities as a result of road traffic crashes in the european union(eu) were fatalities of pedestrians (6). Studies have shown that most traffic crashes related to pedestrians, particularly in high - income countries, occur in interurban streets and roads (7). For example, in the european union countries, 70% of these crashes occur in cities, and this figure is 76% in the u.s . Pedestrians are associated with the highest contribution of deaths caused by traffic crashes in the world s densely populated cities . For instance, in mumbai and delhi, pedestrians accounted for 78% and 53% of traffic fatalities, respectively (9). According to the statistics of tehran traffic police, more than 6,000 traffic crashes causing injury and more than 100 traffic crashes causing death annually occur in tehran . In fact, more than 40% of deaths from traffic crashes in tehran occur for pedestrians (11). Given the importance of this issue, in the recent years, many studies have been conducted in different parts of the world, especially in densely populated cities, on the role of environmental and demographic factors in frequency, spatial distribution, and severity of traffic crashes related to pedestrians in urban areas, using different methods of spatial analysis and a variety of statistical models (12 - 21). In the recent years, due to the increase in automobile production, traffic load has dramatically increased in urban and suburban streets and roads of iran, while these transport infrastructures have not been developed qualitatively and quantitatively commensurate with car production . Since a major proportion of traffic crashes in iran occur due to environmental factors, especially the quality of roads and streets and as the capacity of streets and roads, studying the urban and interurban roads and streets, particularly in densely populated cities with heavy traffic such as tehran, in terms of the environmental factors involved in traffic crashes can be helpful to more accurately identify the environmental factors of such crashes and their contribution . The results of such studies can be used in better planning for reduction of this type of risk factors . Hence, this study aimed to determine the high - risk areas and spatially analyze the traffic crashes causing death to pedestrians in tehran in 2013 and 2014 . Tehran is the largest city and the capital of iran, with an area of more than 612 square kilometers (22). Tehran is divided into 22 districts, 370 parishes, and 560 traffic zones, with more than 535 km of highway and 445 arterial streets (24). Statistical analyses were performed by descriptive and inferential statistics in spss and arc - gis . Analytic surveys were conducted in the following steps: first, the exact location of traffic crashes causing death to pedestrians was extracted by reviewing the files of crashes in the database of tehran traffic police . Second, according to the universal transverse mercator (utm), geographical coordinates of the traffic crashes locations with the accuracy of one to three meters were recorded and saved in a computer . The utm system divides the earth into 60 zones, each 6 of longitude in width and uses a secant transverse mercator projection in each zone . The point of origin of each utm zone is the intersection of the equator and the zone's central meridian . Third, using arc - gis, different layers of geographic information were put on each other and mapping was extracted . Finally, global moran s index with euclidean distance method was used to study and analyze the distribution pattern of traffic crashes related to pedestrians in terms of being either cluster or scattered . Moran s i index is the most common index used for measuring the spatial autocorrelation to determine how close are the objects compared to other close objects . A strong autocorrelation occurs when the values of a variable that are spatially close to each other are correlated . In other words, if the spatial objects or the values of variables related to them are randomly distributed in space, there must apparently be no relationship between them . Moran s index studies the distribution pattern of these spatial objects by considering the values of the studied variable in terms of being cluster or scattered . If the value of moran's index is equal to + 1 or near + 1, it implies a complete cluster pattern or existence of autocorrelation . If this value is zero, it means that the pattern is random or multipolar accumulation . Finally, if moran's index is equal to -1 or near -1, it shows that the crashes follow a scattered pattern . Hot spots analysis: for this analysis, global getis - ord g index with euclidean distance method was calculated for each feature . Z - score of this index shows that to what extent the studies feature has been distributed cluster - like, which may be statistically significant . In this study, this index was used to display the areas with high, low or moderate frequency of traffic crashes associated with pedestrians . Getis - ord g index values were interpreted based on comparing the observed values and the expected values . If the observed values of getis - ord g index in an area are more than the expected values, that area is considered among the hot spots, and if the observed values are less than the expected values, that area is considered among the cold spots . One hundred ninety - eight incidents were studied; 92 of which, (46.4%) occurred in april 2013 to march 2014 and other 106 cases (63.6%) occurred in april 2014 to march 2015 . The highest and the lowest frequency of crashes belonged to january (26 cases) and june (10 cases). Figure 1 demonstrates the trend of the traffic crashes causing death to pedestrians in 2013 - 2015 in tehran . Distribution of fatal crashes associated with pedestrians in tehran by different months: 2013 - 2015 overall, 158 cases (79.8%) of crashes have occurred in tehran highways . Azadegan highway, which stretches from northwest to southeast of tehran, had the highest frequency of crashes (25 cases). During the study period, fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west, east and south of tehran . Figure 2 depicts the distribution of studied crashes in tehran and their position to highways, and urban to interurban bus terminals . Mapping of fatal crashes associated with pedestrians in tehran and their position relative to highways and urban and interurban passenger terminals: 2013 - 2015 among the 22 districts of tehran, districts located in the south, east, and northwest of tehran, as well as districts located in the center of the city, have shown the highest and the lowest frequency of traffic crashes, respectively . Figure 3 displays the distribution of studied crashes in the 22 districts of tehran in 2013 and 2014 . Mapping of fatal crashes associated with pedestrians in the 22 districts of tehran in 2013 - 2015 result revealed that the density of events in the areas with dominant transportation, industrial and military land use was higher than the areas with dominant residential land use (fig . 4). During the study period, fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west, east and south of tehran . Mapping of fatal crashes associated with pedestrians in tehran and their position relative to land use in 2013 - 2015 figure 5 depicts the distribution pattern of traffic crashes causing death to pedestrians in traffic zones . Moran s i index value showed that the studied crashes had a cluster - like distribution (p<0.001). Distribution pattern of fatal crashes associated with pedestrians in tehran by traffic areas in 2013 - 2015 the hot spots in tehran in terms of traffic crashes causing death to pedestrians included the western, southern, northern, partly eastern suburbs, and cold spots were mainly located in the central areas of tehran (fig . Getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes was statistically significant (p<0.001). Low and high - risk traffic areas in terms of frequency of fatal crashes associated with pedestrians in tehran in 2013 - 2015 the results of this study revealed that occurrence of the traffic crashes causing death among pedestrians in tehran during different months of 2013 and 2014 does not follow a specific and fixed trend . For instance, the highest and the lowest frequencies of studied crashes in 2013 have been recorded in january - february and october, respectively, while the highest and the lowest frequencies belonged to may and december, respectively . However, it can be generally concluded that the number of crashes in this period during the summer months (especially june and august) is fewer than the winter months (especially january and march). Since some studies have shown that the frequency of traffic crashes associated with pedestrians increases with increased pedestrian volume (10,25,26), these seasonal differences can be attributed to changes in pedestrian volume . Since some studies such as those conducted by cloutier, cottrill, green, miranda - moreno, mcarthur et.al found that the number of schools or students is associated with the frequency of traffic crashes associated with pedestrians in urban areas (17,26 - 29), school holidays and reduced traffic of students in june and august can be one of the reasons for the reduced frequency of such crashes during these months . Therefore, better organization of students traffic and promotion of the safety of the streets near schools can be considered as one of the strategies for reducing the deaths from traffic crashes related to pedestrians in tehran . This study showed that about 80% of the studied crashes have occurred in highways and freeways . Highways located at the main western and southern entrances and exits of tehran such azadegan highway (from northwest to southeast of tehran) and karaj special road, which connects karaj to 36 km west of tehran had the highest share of such crashes . These results are consistent with the findings of studies conducted by wier et.al in san francisco and mueler et al . In washington that showed that frequency of traffic crashes associated with pedestrians since traffic load in the western and southern highways of tehran is higher than other areas and there is an intense congestion of different means of transport in some of these areas, one reason for the high frequency of fatal crashes in these areas would be the high traffic load and congestion of different means of transportation . Therefore, the reasons of this problem need to be identified by conducting more studies . In addition, the necessary measures should be taken to reduce the rate of traffic crashes through organizing the passage of vehicles and pedestrians and separating the passage of light and heavy means of transportation in the western and southern highways of tehran . Distribution of studied crashes in terms of proximity to or remoteness from the west (azadi) and south (khazaneh) terminals showed that the density of traffic crash was relatively high at the end of the roads leading to azadi passenger terminal such as tehran - karaj freeway, karaj special road, afsarieh bridge, and the eastern part of azadegan highway . Thus, one of the reasons for the high frequency of traffic crashes causing death to pedestrians in south and west of tehran is the location of azadi and khazaneh passenger terminals in these areas . On the other hand, distribution of the studied crashes in terms of proximity to or remoteness from urban passenger terminals does not follow a specific pattern and the frequency of traffic crashes associated with pedestrians is very low near these passenger terminals, especially those far from interurban passenger terminals . Therefore, it can be concluded that the streets and roads around the urban passenger terminals are of low risk in terms of traffic crashes associated with pedestrians . Comparison of the 22 districts of tehran in terms of the frequency of fatal crashes involving pedestrians shows that the highest frequency of this type of crashes has occurred in district 2, which accounts for 13.6% of the total cases, while this district covers only 8% of the total area of tehran . Given that most crashes in district 2 have happened on highways, the relative high length of highways could be mentioned as one of the reasons for the higher frequency of traffic crashes associated with pedestrians in district 2 of tehran . Highways in this district, with a length of about 61 km, account for about 11% of all highways of tehran . Results of this study revealed that high - risk traffic areas in terms of crashes causing death to pedestrians during the studied years are mostly located in the west and south of tehran . High value of moran s i index indicated the distribution of crashes by the 560 traffic areas of tehran followed a cluster - like pattern . In other words, the location of occurrence of these crashes in tehran did not follow a random distribution . In addition, findings demonstrated that the hot spots of the studied crashes were mostly located in the west and south of tehran, while the cold spots were mainly focused in the central parts of the city . Since getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes is statistically significant, it can be stated that some certain factors were involved in non - random distribution traffic crashes causing death to pedestrians in tehran and high frequency of these crashes in high - risk areas . Due to the environmental and structural differences between the high - risk and low risk areas, which are mainly located in the center of tehran, environmental factors similar studies in other parts of the world, including the studies conducted by slaughter et.al in new york, anderson in london, taquechel et.al in atlanta, wang and kockelman in texas, siddiqui et.al in florida and kuhlmann et.al in colorado, have also shown that environmental factors are highly involved in non - random distribution of traffic crashes related to pedestrians (12,14,16,31 - 33). It seems that one of the factors determining the distribution of the studied crashes in tehran, which is the higher frequency of crashes in the suburban areas than the central part of the city, is the difference between drivers and pedestrians in terms of traffic culture . The drivers and pedestrians in central parts of the city have higher education levels and respect traffic regulations more than those in the suburban areas . Higher traffic load on the outskirts of the city compared to the downtown was another reason for this issue . Enforcement of regulation of traffic limits and alternative passage of vehicles with even or odd license plates in the central parts of tehran causes the central parts of the city to have a lighter traffic load than the margins of tehran . This was one of the few studies in iran that has used the files drawn by police experts for conducting an applied research to determine the high - risk areas in a metropolis . Using these files, we recorded the geographical coordinates of the location of crashes and we prepared a map to determine the distribution pattern of traffic crashes causing death to pedestrians and their position relative to other spatial objects . This study was conducted using only the data of crashes registered by tehran traffic police . Comparison of the statistics provided by the police and forensic medicine organization showed that the actual number of fatal crashes in tehran is more than those mentioned in the database of tehran traffic police and the information related to some crashes causing death does not exist in this database . Therefore, it is not possible to acquire information on their location of occurrence through the police files . In such studies, it is better that the relevancy of the death of the injured by traffic crashes to these crashes be defined based on the time of death after crash . This time in different countries varies between 6 days to one month, and in iran, this time has been defined to be one month . However, due to the lack of consistency between the data provided by tehran traffic police and forensic medicine organization, relevancy of the death of the injured by traffic crashes to these crashes in this study was determined based on the data extracted from data based on tehran traffic police . Various districts of tehran, excluding central districts, are considered high - risk areas . Therefore, the majority of traffic crashes causing death to pedestrians have occurred on highways located at the main entrance; that is, districts located in south, east and northwest of tehran . Significant cluster - like distribution of crashes by the 560 traffic zones of tehran and existence of hot spots in terms of the studied crashes in the western and southern parts of tehran indicates the decisive role of environmental factors in occurrence of traffic crashes causing death to pedestrians . Moreover, it is necessary to organize the pedestrians traffic in high - risk areas . This study was part of a thesis conducted in shahid beheshti university of medical sciences . We would like to sincerely thank all who helped us in this study, especially gen . Hosseini, the commander of tehran traffic police, personnel of crashes department of tehran traffic police, particularly lt . Soroush, headquarters of public health faculty and members of the epidemiology department of shahid beheshti university of medical sciences.
Since its first application in 1980, extracorporeal shock wave lithotripsy (swl) has become the preferred treatment method in many ureteric and kidney stone diseases . Advances in stone treatment in the endoscopic age, such as the ability to perform retrograde intrarenal surgery more frequently and almost independently of the size of the stone and percutaneous nephrolithotripsy (pnl) gaining less invasive features defined as mini and micro, can be listed as developments that have hindered the preference of swl . The wide use of swl is due to its higher efficacy in selected cases while its low morbidity rates is one of the most important advantages of this method, making it the first treatment choice in many cases today, despite the other treatment alternatives that are available . However, there are conditions that limit the use of the method and affect its success . Among the factors that affect the success and results of swl are; the type of lithotripter; stone - related factors such as the size, structure, number, and localization; the anatomy and the functioning of the kidney; and patient specific structural features [3, 4, 5]. Al ansari et al . Investigated the prognostic factors affecting the success of swl in 427 patients and they demonstrated that in cases with renal stones larger than 30 mm, the stone's size, localization, number, radiologic renal features and congenital renal anomalies were significant factors, while ureteral stent use, age, gender, and the nature of the stone (de novo or recurrent) had no effect . . Reached the same conclusions in their study of 2954 cases with renal stones that were smaller than 30 mm . In addition, factors such as the presence of additional interventions pre- and post - swl, complications, and costs can also affect the efficacy of the treatment [2, 3, 4, 7]. The routine use of ureteral stent prior to swl is not recommended in renal stone cases despite the lack of any defined criteria in the guidelines [2, 8]. While the use of ureteral stents can reduce post - swl complications such as obstruction and renal colic, it does not prevent steinstrasse formation and infectious complications, and does not increase stone - free rates [8, 9, 10]. Patient discomfort, pain in bladder, and issues related to urination that are associated with ureteral stent use can often be experienced . According to kirkali et al ., pre - swl ureteral stent use should be preferred only in solitary kidney patients . The goal of our retrospective study of over 1361 patients was to compare the stone - free rates, steinstrasse formation, treatment efficacy and complications between patients with renal pelvic stones with and without pre - swl stent and to contribute to medical literature based on real life experiences . This is a retrospective study conducted by scanning the medical data of 1378 patients treated with swl for renal pelvic stones at our clinic between 1995 and 2011 . Seventeen patients who had percutaneous nephrostomy tube placement prior to swl were excluded from the study . The median age of the 1361 patients included in the study was 40 (1 - 85) years . All patients had routine renal function tests, urinalysis and urine culture, coagulation tests, kidney - ureter - bladder x - ray (kub), intravenous pyelography (ivp), and ultrasonography (usg) before swl . An abdominal contrast - free computerized tomography (ct) patients with urinary system infections were treated with antibiotics according to their culture results prior to swl . Uncontrolled infections, coagulation dysfunctions, ureteropelvic junction obstruction, and pregnancy were considered contraindications for swl . The swl procedure was carried out via siemens lithostar lithotripter (siemens medizinische technik, erlangen, germany). The size of the stone was calculated in squared centimeters, by multiplying the widest width and length observed in kub . When multiple stones were observed, the sum of their sizes was calculated . In order to avoid statistical bias in this study, the patients were separated into 3 groups based on the size of the stone: 1 cm (group 1), 1.12 cm (group 2), and> 2 cm (group 3). Table 1 displays the patient characteristics, prior interventions for stones on the same side, stone characteristics, and treatment features of the 1361 patients . Specialized in swl and the energy and shock wave count for each patient were determined by the same physician . Prior to the procedure, an ureteral stent (percuflex plus 4.8 f x 26 cm, boston scientific, quincy, ma, usa) was placed in solitary kidney patients, patients with renal ectasia of grade 2, and patients with obstruction symptoms lasting a long period of time (> 1 month). Swl was performed on all patients in the supine position, under fluoroscopic control and as an outpatient procedure . Fourteen patients had the procedure under anesthesia where 0.10.2 mg / kg midazolam and 0.5 mg alfentanil were used for analgesic sedation . The treatment was initiated with 13 kilovolt (kv) and was increased with 0.3 kv increments up to the highest level that the patient could tolerate . The procedure was ended when full fragmentation of the stones was observed in the fluoroscopic control . The procedure was considered unsuccessful in cases where fragmentation was not achieved at the end of the 3 session and/or in patients who wanted to try another treatment . Patients were given hydration, analgesic and antispasmolytic treatments during the sessions and the first post - treatment week . Patients were evaluated with kub and usg at the end of the first week after the procedure and at 3-month follow - ups . Ct was not performed on any patients who did not display significant symptoms or if hydronephrosis was not detected in their usg . Size of the stone(s), auxiliary procedures (with or without stent use), radiologic evaluations at 3-month follow - up, and complications were evaluated retrospectively . Statistical analyses were performed using chi - square, fisher's exact and mann - whitney u tests . Using the efficacy coefficient equation (eq): stone free% / (100% + re - treatment + auxiliary procedures%) x 100 . Characteristics of patients, stones and treatments the grouping of 1361 patients according to the renal pelvis stone size was as follows: 514 patients in group 1, 530 in group 2, and 317 in group 3 . There were 178 patients (13%) who had stent placement prior to the procedure according to the aforementioned criteria . The number of patients in groups 1, 2 and 3 who had pre - swl stent placement was 30 (6%), 44 (8%), and 104 (33%), respectively . Patient and stone characteristics by groups and stent placement are presented in table 2, while treatment features by groups are shown in table 3 . The average number of sessions was found to be significantly higher among the patients with stent placement in all groups (group 1: p = 0.022; group 2 and 3: p = 0.000), while the proportion of stone - free patients was similar across patients with and without stent placement in group 1 (86.4% and 73.3%; p = 0.06), a significant difference was observed in group 2 (without stent 80.2% vs. with stent 56.8%; p= 0.000) and group 3 (without stent 75.1% vs. with stent 64.4%; p= 0.047). Treatment eq for patients without and with stent placement, and for all patients within the groups were 64%, 46%, and 63.7%, respectively, for group 1; 52%, 30%, and 49%, respectively, for group 2; and 43%, 33%, and 40%, respectively, for group 3 . The distribution of patients with and without stent placement according to steinstrasse formation was 6.7% and 5.8% (p = 0.692) in group 1, 11.4% and 15.8% (p = 0.431) in group 2, and 26% and 22.5% (p = 0.500) in group 3, respectively . According to these findings, no significant difference was observed between the patients with and without stent placement within the groups in terms of steinstrasse formation . Gender distribution and side of the stone localization yielded similar results across groups, as well . While the rates of solitary kidney patients were significantly different across patients with and without stent placement in groups 1 and 2 (p = 0.000), no such significant difference was observed in group 3 (p = 0.209). None of the major complications such as stent migration, infection, pyelonephritis or stent breakages were observed in any of the patients with stent placement in this study . Of the 178 patients with stent placement, 68 (38%) complained of frequent urination and pain in the bladder and kidney area that was associated with the stent but these issues were resolved via symptomatic therapies . Comparison of characteristics of patients and stones according to the groups treatment characteristics according to groups ureteral stents are mostly used to enable continuation of drainage in the presence of complications such as stone, tumor or obstruction between the kidney and the bladder . The pieces of the stones broken down still have a risk of causing obstruction in the ureter following swl and this condition is associated with the size of the stone . Use of ureteral stent in swl is generally not recommended and there are various studies on the matter . Although ureteral stent is useful to prevent complications such as obstruction and renal colic following swl, it does not protect from steinstrasse formation or infectious complications and does not increase the proportion of stone - free patients [8, 9, 10]. In our study, the stone - free rate following swl was found to be higher for renal pelvis stones> 1 cm in patients without stent placement, although no difference was observed regarding steinstrasse formation . As for the demographic characteristic of our patient series, the female / male ratio, side ratio, stone load, and the number of swl sessions were in line with the literature findings . Libby et al . Demonstrated that ureteral stents reduced morbidity in case of stones of sizes> 2.5 cm . On the other hand, reported that the swl morbidity with or without stent placement is similar to that of pnl in patients with stones of> 2 cm . Groeneveld recommended ureteral stent placement in case of stones that are greater than 3 cm, if swl with prior debulking or swl alone will be performed . However, the benefits of ureteral stent use prior to swl are still disputable [17, 18]. Low et al ., with their 179-patient retrospective series, determined that there was no difference between the stone - free rates of patients with or without stent placement in their 1-month and 3-month evaluations . Sulaiman et al . Reported that ureteral stents did not make a difference in steinstrasse formation in cases of stones that greater than 2 cm . Preminger et al . Also failed to detect a difference between patients with and without stent placement in terms of their stone - free rates, independent of the stone load and shock strength . Reported that stent use did not cause a significant difference in the stone - free rates of large kidney stones or reduce post - swl morbidity and that their use in routine care is not necessary . However, in hollowel's inquiry of urologic specialists in the united states, it was determined that most of the urologists declared that they use stents in case of stones that are greater than 2 cm in size although there was no objective data about their usage . We believe that the urologists desire to stay on the safe side in terms of colic - type pain or obstructions following swl for relatively larger stones play a role in the decision . In sfoungaristas's study investigating stent use in ureter stones that are 4 - 10 mm in size, stent use was reported to reduce stone - free rates and that it negatively affected the post - swl quality of life . In pettenali et al . 's retrospective study, stent use was reported to reduce swl success in cases of proximal ureteral stones that are larger than 8 mm . . 's retrospective analysis showed that, in cases of kidney stones larger than 30 mm, steinstrasse formation was more common among patients with stent placement, and that the sole removal of the stent would be sufficient for these patients; and in cases of stones that are between 2030 mm, they reported no difference between patients with and without stent placement in terms of steinstrasse formation . They do not recommend stent use in the case of stones smaller than 20 mm in size . Mustafa et al . Also reported that the placement of a ureteral stent for the purpose of improving stone free rates or enhancing the passage of fragments during swl is unnecessary in renal stones with diameters less than 2.5 cm . Though medical literature specifies that stents generally do not affect swl results or at least do not have a negative effect on stone - free rates, we state in our series that ureteral stents were observed to have a negative effect on the stone - free rates among stones greater than 1 cm in size . According to this finding, while stone - free rates do not vary across groups with or without stent placement in case of stones up to 1 cm, stent use was observed to have a negative impact on the stone - free rates in groups 2 and 3 . Again, the eq values were higher among patients without stent placement in all groups . This is contradictory with the predisposition of stent use with increase in stone size and in fact shows that ureteral stents may reduce the efficacy of swl as stone size increases . In all 3 groups of our series, we believe that the difficulty the stent creates in focusing on the stone and the additional sessions performed for the stones traveling down the ureter plays a significant role in these findings . Furthermore, in such cases stents can also block the particles that could migrate downwards under normal conditions . In terms of steinstrasse formation, no significant difference was observed between the patients with and without stent placement in any of the 3 groups and ureteral stents were observed to be unable to prevent steinstrasse formation . Literature reports migration, stent breakage, encrustation, infection, pyelonephritis, and stone formation as stent - related complications [26, 27]. 's evaluation of 290 cases with stent placement that had uretero - renoscopy and swl, the 12-week rates for encrustation was 76.3%, migration was 3.7%, and breaking of stent was 0.3% . Joshi et al . Also reported that 60% of the patients had stent- related symptoms of overactive bladder such as increased urination frequency and urge incontinence . While we did not observe complications such as migration, encrustation, breaking or pyelonephritis in our series, about 38% of the patients had symptomatic (frequency, urgency, hematuria, dysuria, and in some patients a colic - like pain) complaints . We believe that urinary reflux related to stent may play a role especially if the patient with a full bladder experiences colicky pain after urination . These complaints were generally controlled via symptomatic treatments and none of the patients required stent removal due to these symptoms . We believe that informing the patients about the possible symptoms that can be experienced following stent placement will allow them to tolerate the ureteral stents better . The retrospective nature, lack of randomization in terms of ureteral stent use prior to swl and that only 13% of patients had stent placement may be considered the limitations of this study . On the other hand, these limitations are avoided by the sole use of specialists with 25 years of swl experience (n.t .) In the performance of all procedures, which contributed significantly to our study with respect to the standardization provided in ureteral stent preference and adjustment of the number and strength of shock waves . We believe that our patient series with cases of various stone loads has a lot to contribute to the medical literature on this matter . The usage of kub and usg for monitoring the treatment results, but not ct could also be argued . Kub and usg are preferred in the aim to avoid the risk of high radiation levels and financial burden on the health system especially in symptom - free patients . 's study on this topic with 2759 patients between the ages of 1876 years reported that usg, compared to ct, does not increase complications, pain score, admission rates to the emergency department, or hospitalization but does reduce exposure to radiation . In our study, the stone - free rate following swl was found to be higher for renal pelvis stones> 1 cm in patients without stent placement, although no difference was observed regarding steinstrasse formation . Our results showed that ureteral stents were observed to have a negative effect on the stone - free rates among stones greater than 1 cm in size . The results of this study support the idea of limiting the application of stents during swl and prevent possible complications as well as reduce the financial cost of treatment . Considering that ureteral stents reduce stone - free rates, we believe that they should be preferred in special cases such as solitary kidney patients or those with long - term obstruction.
Noncommunicable diseases, including type 2 diabetes mellitus (t2 dm), are considered the leading cause of death in the world and, in brazil, constitute the main cause of diseases . T2 dm assumes an important role in this context given that it is considered a worldwide epidemic disease; in 2011 it appeared among the ten leading causes of death in the world and it is prospected that number of cases will continue to increase . The ones most frequently being discussed are the increase in population life expectancy, changes in lifestyle (including unbalanced diet and physical inactivity), and obesity . Diabetes can be defined as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both . The underlining cause of t2 dm can be attributed to a combination of resistance to the action of the hormone, increased production of insulin in a compensatory manner, and inadequate secretory response . On the other hand, according to a study conducted by kaprio et al ., the heredity has an input around 47% in susceptibility to t2 dm; thus, genetic factors play an important role in the development of the pathology . Some facts corroborate the importance of heritability in t2 dm: the greater concordance between monozygotic twins than among dizygotic twins and a wide variation in the prevalence of t2 dm in epidemiological studies with different ethnic groups as well as positive results in numerous other genetic studies . In this regard, it should be noted that even as more than 30 genes associated with t2 dm have been identified, the contribution of each individual gene in the disease susceptibility is very small . Additionally, most of these genes identified are related to dysfunction of pancreatic cells . Several reports suggest that the characteristics of each individual, including genetic polymorphisms, may confer differences in the occurrence of diabetes and other complex diseases such as cancer and heart disease . Withal, in most cases, a positive association between polymorphisms of these genes and t2 dm are not reproducible in the analysis of a different population . Two polymorphisms in the promoter region of the tnfa gene have been described, one present at position -308 . In this regard, swaroop et al . Demonstrated an association between the adipocytokine tnf- and development of insulin resistance . This systematic review was conducted to provide a comprehensive assessment of the association between the polymorphism in the gene tnfa -308g / a and the presence of t2 dm . The literature search was made through the virtual health library (vhl) which uses as bibliographic databases the publications found in sources such as lilacs, medline, and other information bases such as open educational resources, websites, and scientific events . Any publications with case - control studies, on january 21, 2016, that show the relationship between polymorphisms of tnfa gene -308g / a and susceptibility to type 2 diabetes mellitus (t2 dm) were surveyed . The search terms used were as follows: diabetes and tnf or tumor necrosis factor, and polymorphism . The filters are selected in accordance with the main subjects of the following terms: type 2 diabetes mellitus, tumor necrosis factor alpha, single nucleotide polymorphism, and genetic polymorphism . The filter base on the type of study included solely the term case - control study . Inclusion criteria were as follows: (1) the study must have been case - control; (2) the study should have assessed type 2 diabetes mellitus and -308g / a polymorphism of tnfa gene, even if associated with other comorbidities; and (3) the study should have provided sufficient data, including the number or frequency of alleles and genotypes . Studies were excluded if (1) there were commentaries, case reports, or non - case - control or meta - analyses studies; (2) they did not report sufficient data; (3) they presented data from other types of diabetes or did not specify what kind patients had; and (4) they showed polymorphism in another coding region of the tnfa gene or (5) if the sample was composed of only dm patients . Data from eligible studies were extracted independently in accordance with the inclusion and exclusion criteria . For each study the following characteristics were collected: the authors, the study title, the year of publication, where it was published, the sample size, the tnfa -308g / a genotypes with their corresponding simple frequencies and percentages for the control and the case group, and the study's p value and odds ratio, when calculated . The literature search found articles published starting from 2000, given that the year the complete mapping of the human genome was made available . Advances in molecular biology technology since this project have been quick and, thus, increasing the number of genetic studies published for the association of certain genetic polymorphisms with various pathologies . In the case of literature search for articles with case studies that related polymorphism position -308 of the tnfa gene with diabetes, the result presented forty - three publications between the years 2003 and 2015, ten of which met the criteria inclusion . Thirteen studies presented results for other types of diabetes; eleven related to type 1 diabetes and two to gestational diabetes . Two others did not specify what type of diabetes had patients in the case group . Two of these studies did not show enough genetic data for analysis . In relation to tnfa gene, seven studies presented polymorphisms in region other than the -308 position and three publications had single nucleotide polymorphism in a gene that was not tnfa . Other studies were excluded due to different methodological designs in their inclusion criteria: two meta - analysis, one being observational and the other prospective and, relative to the sample . Furthermore, there was a publication that presented data of diabetic patient's polymorphisms in both the control and the case group . The number and homogeneity of the included studies presented were too limited to allow a meta - analysis . Relate the polymorphism in gene studied with t2 dm and coronary heart disease, while another two (buraczynska et al ., dabhi and mistry) relate association frequencies in diabetic patients with nephropathy or renal failure . Finally, five studies reported data that directly relate polymorphism in question with t2 dm (shiau et al ., bouhaha et al ., guzmn - flores et al . One of which also relates it with obese t2 dm patients (bouhaha et al . ). The general characteristics of the studies included in this review are summarized in table 1 . In three studies conducted in spain, hungary, and morocco significant associations between the tnfa -308g / a polymorphism and the risk of t2 dm were identified [11, 17, 20]: two of them in patients with other comorbidities such as coronary heart disease and atherosclerosis . In the present study, therefore, we infer that although there was no evidence of heterogeneity in the association between polymorphisms in the tnfa -308g / a gene and increased risk for development of t2 dm, the presence of positive results may mean that few studies have not been enough to clarify this relationship, and besides there is no documented evidence in various ethnic groups . Due to contradictory results, this systematic review is aimed at providing a comprehensive assessment of the association between tnfa -308g / a and phenotype involving type 2 diabetes . The concern regarding the quantification and qualification of the genetic impact on phenotypic characteristics started after the first complete human sequence after genomic era . As a consequence, health research delved into the genetic information as a way to find answers to complex disease questions . This type of research has shown high prevalence and increasing prospects in a number of cases, such as diabetes . For this purpose, various portions of the human genome are now analyzed in search for genetic variations among individuals that could explain these diseases . Studies seeking interactions between these variations, in particular, have been very promising . In the case of diabetes, a syndrome whose etiology is complicated and involves several risk factors for its development and progression, some studies on single nucleotide polymorphisms were suggested as causing the phenotypic characteristics and responsible for the increased risk of developing pathology [2527]. To study the risk factors for a disease is to study the possibility of a certain event happening . Within the epidemiological concepts, the term is used to predict the likelihood of healthy individuals, exposed to certain factors, developing a given disease . This does not necessarily mean that it would occur; nevertheless the presence of these factors makes the individual more vulnerable to manifest the disease . A risk factor x may be the trigger of various diseases, and several risk factors can cooperate for the genesis of a common disease . Environmental factors such as eating habits [2931] and physical inactivity [3234], in addition to the concomitant presence of diverse mutations in many genes, need to be considered so that the contribution of each of them is understood . Linkage research studies genetic markers and specific polymorphisms in a family group that has the disease . This, thus, allows the researchers a way to better study the regions of a chromosome that is affected . After the connection is made to the chromosome, one can test for association of polymorphisms for identifying a genotype specific to a given disease in groups of people who have the targeted disease [34, 35]. Several association studies have discussed the importance of clarifying the relationship between genetic polymorphisms and the development of type 2 diabetes . The search for candidate genes was initially focused on possible genes encoding proteins directly involved in the pathophysiology of t2 dm: related encoders, for example, production, secretion or activity of insulin, or development of the pancreas [3638]. Gradually, however, emerging studies suggested that several other factors could contribute to the development of t2 dm, as the expression of proinflammatory cytokines and other molecules that act in the inflammatory process and that appear to play a critical role in the development of chronic complications of dm . Several studies have shown the influence of various polymorphisms in proinflammatory cytokines genes as a risk of development of obesity, diabetes, and metabolic syndrome . One of these cytokines, tumor necrosis factor (tnf-), has been investigated since diabetics have elevated levels of it circulating . Tnf- has also been implicated as an insulin resistance - causing factor associated with the pathogenesis of t2 dm . Tnf- is a proinflammatory cytokine that acts in the regulation of cell proliferation, differentiation, and apoptosis . Among the various polymorphisms describing the tnfa gene, the -308g> a variant located in the promoter region excels by affecting the expression of its gene . The presence of the a allele at nucleotide -308 increases transcription of tnfa gene approximately twofold, therefore increasing the production of this cytokine . Under this circumstance, it is expected that the polymorphic genotype may be associated with an increased frequency of diabetes and, for the proper association, of case - control studies can be used . In this review, several studies with the frequency of genotypes in control and case group were observed for a better understanding of the population genetic profile and a possible association between polymorphism of tnfa gene and t2 dm in groups with different ethnicities . The influence of genetic polymorphisms on certain diseases may be found in a population and yet not in another; this may impact the frequencies and distribution of a given polymorphism in distinct population . The difference in influence may be due to racial variantion as well as other factors . . In addition, some studies have included a proportionally small sample size in relation to the country's population and most of them define the case of groups in t2 dm patients who have concomitant comorbidities . Case - control studies were selected in this review and were made with the people of spain, taiwan, poland, india, tunisia, mexico, hungary, and morocco; nonetheless a positive association was only found in the spanish, hungarian, and moroccan population . Sefri et al . In their meta - analysis, which considers 21 case - control studies published until august 2013, argue that no significant associations were found between the polymorphism in the studied region of the tnfa gene and risk of developing t2 dm . Their finding was consistent with a previous meta - analysis, which included data from 18 association studies . In these studies, publications with different ethnic groups were included; among them were africans, asians, caucasians, and other populations . So, when comparing the frequencies of polymorphic genotypes (ga and aa) in the groups of case - control study (described in table 1), it is possible to observe that these genotypes are presented more frequently in the group that has the disease . The exceptions to this were the studies in tunisia and india; the most frequent genotype in type 2 diabetic patients was the gg . In all other countries this evidence supports the assumption that polymorphisms in the tnfa gene and its association with other aspects, both genetic and environmental, may represent an important risk factor for type 2 diabetes mellitus . Risk factors not modifiable or irreversible in nature, such as genetic profiles, refer to the individual characteristics . Even though these factors may not be changed, the identification of their presence in individuals and families may enable health professionals to advise change in the lifestyle of these patients, avoiding early manifestation of t2 dm.
Of the 130 million babies born worldwide every year, approximately 4 million are stillborn, more than 98% of these occur in developing countries . Stillbirth accounts for more than half of perinatal mortality in developing countries . In sub - saharan africa, while countries in south - east asia report the highest overall numbers of stillbirth, countries in africa report the highest incidence rates per 1000 live births . The average stillbirth rate in developing countries has been reported to be 26 per 1000 live births, about five times higher than in developed countries (5 per 1000). One fourth to one third of all stillbirths is estimated to take place during delivery [5, 6]. Stillbirths occurring in the intrapartum period generally have a normal appearance and are often called fresh the skin not being intact implies death more than 24 hours before delivery (antepartum), often called macerated stillbirths . Stillbirths have not been widely studied, have been under - reported, and rarely have been considered in attempts to improve birth outcomes in developing countries [5, 6]. There are many factors associated with stillbirth including inadequate access to obstetric care, inadequate care, malaria, hypertensive disease, poor nutritional status, history of stillbirth, congenital anomalies, sickle cell disease, and high burden of infectious comorbidities [5, 810]. First, maternal infection may cause severe illness, leading to fetal death [12, 13]. Also, an infection in the uterus or anywhere else in the mother's body may precipitate preterm labor . Last, the placenta may be directly infected, leading to reduced blood flow to the fetus, a likely cause of stillbirth associated with malaria infection . When malaria parasites infect the placenta, placental insufficiency results because of lymphocyte and macrophage accumulation, and increased expression of pro - inflammatory cytokines; these impede maternal blood flow through the placenta [16, 17]. Intestinal helminths, including hookworms and trichuris trichura, have been associated with anemia [18, 19]. It has been suggested that low hemoglobin concentrations can cause a state of chronic hypoxia, which is presumably exacerbated in pregnancy when oxygen demands are particularly high because of the metabolism of the mother and the fetus, and that oxygen transfer to the fetus is probably reduced in anemic women . A strong association has been observed between maternal plasma, cord plasma, and placental folate concentrations, suggesting that transplacental folate delivery depends on maternal plasma folate concentrations . According to the world health organization's opportunities for africa's newborns 2006 report, the stillbirth rate for ghana is 24 per 1000 deliveries . Even though stillbirths represent a large proportion of perinatal deaths, causes of stillbirths are poorly understood in ghana . To our knowledge, the association between malaria and intestinal helminth coinfection in pregnancy and stillbirth has not been studied . Few studies have studied the association between malaria and helminths in pregnancy, with conflicting results . Given that 98% of stillbirths occur in developing countries, especially sub - saharan africa, which also has a high burden of malaria and intestinal helminth infections, it is important to investigate the role of these infections in contributing to stillbirth . The study was conducted in kumasi, the capital of the ashanti region of ghana . The climate in kumasi is humid and tropical, with two rainy seasons, april to june and september to october . Helminth infection is endemic in the ashanti region, which also has an intense perennial malaria transmission, the predominant parasite being plasmodium falciparum . The institutional review board of the university of alabama at birmingham and the committee on human research, publications and ethics, school of medical sciences, kwame nkrumah university of science and technology, kumasi reviewed and approved the study protocol before its implementation . A cross - sectional study of women presenting for delivery at two hospitals in kumasi, the komfo anokye teaching hospital (kath), and the manhyia polyclinic was conducted in november and december 2006 after informed consent was obtained, a questionnaire was administered to collect sociodemographic information, and medical and obstetric histories . Body weight and mid upper arm circumference (muac) were measured for each woman . Obstetric information was also obtained from the mothers' antenatal care (anc) charts . Anc charts provided information on number of antenatal care visits, gestational age as assessed by palpation at first anc visit or ultrasound at first anc, tetanus shots, malaria prophylaxis, antihelminth medications, hemoglobin level, and illnesses and treatments during pregnancy . Blood was drawn by venipuncutre for determination of hemoglobin levels, serum folate level, and malaria antigen tests . State of the newborn (alive or stillbirth), sex, weight, and length were obtained as recorded by the midwives . Determination of malaria antigen in plasma was done using the malaria antigen celisa (cellabs, brookvale, australia). The malaria antigen celisa kit is a monoclonal antibody - based assay specific for p. falciparum malaria . The assay detects a merozoite antigen that circulates in the blood for up to 14 days postinfection . Determination of hookworms, ascaris lumbricoides, and trichuris trichura was done using the kato - katz thick smear technique (who, 1991). Stool samples were processed within 12 hours of collection and examined microscopically within one hour of preparation to avoid missing hookworm ova . For strongyloides stercoralis, samples were processed using the baermann method . Hemoglobin level was measured in an automatic cell counter (sysmex m-2000; digitana ag, hamburg, germany) about 30 minutes after blood sampling . Uncomplicated pregnancy: absence of hypertension, pre - eclampsia, history of a previous caesarean section and hemorrhage, and a normal presentation of the fetus . Malaria infection: presence of malaria antigen in the mother's peripheral blood at the time of delivery . Intestinal helminth infection: presence of helminth eggs or larvae in stool collected at the time of delivery . Anemia: hemoglobin levels <11 g / dl of blood, and severe anemia: hemoglobin level <8 g / dl . Stillbirth: an intrauterine death of a fetus weighing at least 500 grams after 20 completed weeks of gestation occurring before the complete expulsion or extraction from its mother . An intrauterine death of a fetus during labor or delivery was considered a fresh stillbirth, and an intrauterine death of a fetus sometime before the onset of labor, where the fetus showed degenerative changes was considered a macerated stillbirth . Induced abortion: the purposeful interruption of an intrauterine pregnancy with the intention other than to produce a live - born infant, and which does not result in a live birth . Sample size was calculated using unpublished reports on stillbirth from the two study hospitals, which estimated that at least 1%1.5% of 1000 births would be stillbirths . We made the assumption that if we obtained 10 stillbirths, and that 1025% of women with normal births had both malaria and intestinal helminth infections, at a 5% significance level, we would have 80% power to detect an odds ratio of 7.59.0; assuming 15 stillbirths, we would be able to detect an odds ratio of 6.07.5 . Data analysis was performed using sas software version 9.1 (sas institute, cary, nc). Differences in socio - demographic and obstetric characteristics by stillbirth were assessed by chi - square or t - test . Correlation analyses were performed to identify potential multicollinearity between independent variables . To determine factors associated with stillbirth, we used multiple logistic regression . Variables that were significant (p <.05) on bivariate analysis and those that are known to be associated with stillbirth based on previous studies were entered into a regression model . Through this procedure, we calculated odds ratios (or) and 95% confidence intervals (ci). Seven hundred and eighty five (785) women were recruited into the study before delivery in the two hospitals in kumasi . We obtained both malaria and intestinal helminth results from 746 women, and data analysis was limited to these women . Overall, the mean age of the women was 26.8 years (range: 15 to 48 years); 21.1% were single, 30.2% were primigravidae, 30.6% were anemic, 29.5% had a prior induced abortion, and 5.2% had a history of stillbirth (table 1). . A higher proportion of women who were single did not receive sp during pregnancy, had fewer than 5 anc visits, had low folate levels, were anemic, had had a prior induced abortion or a prior stillbirth and delivered a stillborn infant compared to their counterparts (table 1). Of the 746 women, 407 (54.6%) had neither infection, 147 (19.7%) were infected with p. falciparum only, 68 (9.1%) were infected with helminths only, while 124 (16.6%) were coinfected . A higher proportion of women with either organism presented with stillbirth than women with neither infection . Women who were coinfected had a modestly higher rate of stillbirth than women with a single infection (table 2). Low serum folate, severe anemia, prior induced abortion and prior stillbirth were each strongly, independently associated with stillbirth, with increased odds ranging from over 3-fold to a 6-fold increase (table 3). Women with malaria irrespective of whether or not they had intestinal helminths had a 90% increased odds of stillbirth . Although intestinal helminth infection had a stronger association, it was not statistically significant (table 3). This study demonstrated that the study population had a relatively high rate of stillbirth (5% of all deliveries). Factors associated with stillbirth were malaria, severe anemia, low serum folate concentration, past induced abortion, and history of stillbirth . Many stillbirths were fresh (75.7%), an indication that a proportion of these cases could likely have been prevented . It has been suggested that stillbirths occurring in the peripartum period could be prevented through appropriate cesarean section, improved obstetric care, and improved emergency response to obstetric complications . In this study, women who had fewer antenatal care visits had an increased risk of stillbirth, suggesting that stillbirths are closely linked to use and quality of maternal services . Malaria is endemic in many african countries, and is thought to play a role in contributing to stillbirth . Intestinal helminths, especially hookworms and trichuris can cause anemia [18, 19], which in turn leads to adverse birth outcomes including stillbirth . We did not observe an association between intestinal helminths and stillbirth, a finding that has been previously reported . However, our observation could be the result of small numbers, that is, malaria was more common than intestinal helminthes . Coinfection with malaria and intestinal helminths did not increase the risk for stillbirth but as in the case of intestinal helminths, this could be a matter of numbers . Malaria contributes to anemia by hemolysis or destruction of parasitized cells and causes shortened red cell survival [36, 37], while hookworms and trichuris cause anemia through direct blood loss [19, 38]. Since the mechanisms by which malaria and intestinal helminth infections cause anemia differ, it is possible that their impact on anemia are additive and could exacerbate adverse birth outcomes . A strong association has been observed between maternal plasma, cord plasma, and placental folate concentrations, suggesting that transplacental folate delivery depends on maternal plasma folate concentrations . Some studies [40, 41] have reported higher rates of stillbirth in women with megaloblastic anemia than those without . Abortion is legal in ghana only for medical reasons, and is not available upon request (ministry of health, ghana). Most women seeking abortion therefore sometimes attempt illegal abortions, and then go to the hospital for treatment of complications . Removal of retained products of conception in the hospital setting is usually performed by cervical dilation and curettage . There has been concern that this may result in cervical insufficiency, hence future adverse birth outcomes . The tendency to repeat pregnancy outcomes in successive births is well known and includes risk of stillbirth . Previous studies have demonstrated that women with a history of stillbirth may have a 6 to 10-fold increased risk of stillbirth [46, 47]. The causal mechanism may involve impaired placental development and function due to compromised vascular support system . A methodological weakness of our study lies in limited power due to the number of stillbirths, therefore our findings should be interpreted with caution . The fact that it was a cross - sectional study also limits our ability to draw causal or temporal associations . The study however has several strengths including new findings, high participation and consistency with other studies in some risk factors of stillbirth, which strengthens confidence in the new findings . Another strength of the study lies in the fact that the hospitals in which the study was conducted are secondary and tertiary hospitals which cater to large numbers of women of all socioeconomic status from kumasi and surrounding areas . The 2008 demographic health survey for ghana (dhs, 2008) reported that 82.4% of women in urban areas in ghana deliver in a health facility . The fact that most of the stillbirths were fresh suggests that higher quality intrapartum care could reduce stillbirth rates . More studies need to be conducted to further assess the association between stillbirth and malaria and intestinal helminth coinfection . It is important to conduct further studies to investigate risk factors of stillbirth to determine which stillbirths are preventable so that targeted interventions can be developed and tailored for resource - poor settings.
Type 1 diabetes is characterized by insulin deficiency resulting from destruction of pancreatic cells and subclassified into type 1a (autoimmune) and type 1b (idiopathic) diabetes . We previously reported a novel subtype of type 1b diabetes that we referred to fulminant type 1 diabetes . In fulminant type 1 diabetes, remarkably acute and almost complete cell destruction occurs and nearly no insulin secretion remains, even just after the disease onset . Measurement of serum cpeptide level is effective for assessing the ability of endogenous insulin secretions . In particular, it is valuable to presume residual cell capability for type 1 diabetic patients . With a conventional method, enzyme immunoassays (eia), low levels (usually <0.07 nmol / l [0.2 ng / ml]) of serum cpeptide are difficult to detect . However, we can now detect up to 0.003 nmol / l (0.01 ng / ml) serum cpeptide levels using a high sensitivity method, chemiluminescent enzyme immunoassay (cleia). It enables us to know more precise levels of serum cpeptide in patients who have almost no insulin secretion, such as patients with fulminant type 1 diabetes . In the present study, we evaluated serum cpeptide levels measured by cleia and clarified the clinical features of fulminant type 1 diabetic patients based on residual endogenous insulin secretions . We studied 12 patients with fulminant type 1 diabetes (5 males and 7 females). These patients were diagnosed as having fulminant type 1 diabetes between 19882006, and had been followed in our hospital for more than 0.5 years since the disease onset . The diagnosis of fulminant type 1 diabetes was established according to the inclusion criteria proposed by the committee of the japan diabetes society . Namely: (i) the presence of ketosis or ketoacidosis within a week after the onset of hyperglycemic symptoms; (ii) urinary cpeptide excretion <10 g / day or fasting serum cpeptide level <0.10 nmol / l (0.3 ng / ml) and peak serum cpeptide level <0.17 nmol / l (0.5 ng / ml) after glucagon (1 mg) or a meal load soon after disease onset; and (iii) plasma glucose level 16.0 mmol / l (288 mg / dl) and hba1c level <8.5% at first visit . Patients characteristics were as follows: they were aged 2278 years (44.2 18.4), duration of the disease was 0.819 years (4.8 5.2), body mass index (bmi) was 18.524.8 (20.8 1.9), hba1c was 5.710.4% (7.2 1.5), and glycoalbumin was 19.032.5% (25.1 4.1). Eight of the 12 patients were receiving multiple daily injections and three patients were receiving continuous subcutaneous insulin infusion . Every patient was usually followed every month in our outpatient clinic and insulin doses were adjusted for targeting nearly normal glucose levels (hba1c <6.5%, fasting glucose level <5.6 mmol / l [100 mg / dl], 120min postprandial glucose level <7.8 mmol / l [140 mg / dl]). The data of hba1c, glycoalbumin, bodyweight and daily dosages of insulin were measured every 9 months (from january to september in 2006) and expressed as mean of 9 months measurements . Sevenpoint (fasting and 120 min postprandial for each meal and bedtime) capillary blood glucose concentrations were measured in seven of the 12 patients for at least two days, and mean mvalue was calculated in each patient . Mvalue is a logarithmic transformation of the deviation of blood glucose from a selected standard . We used 5.6 mmol / l (100 mg / dl) as the selected standard . The deviation index () of each individual glucose value () is first calculated as = (10 log[/5.0]). Serum cpeptide levels were measured by cleia (lumipulse f, lumipulse cpeptide; fujirevio, tokyo, japan) and two different eia, aia21 (e test cpeptide; tosoh, tokyo, japan) and eclusys 2010 (eclusis cpeptide; roche diagnostics, basel, switzerland) simultaneously by using the same serum samples . Serum cpeptide level was also followed for 9 months by cleia, and the maximal value was determined in each patient . The present study was carried out with patients approvals and informed consent was obtained from every patient . The significance of differences between the two groups was evaluated by pearson s correlation coefficient and mvalue was evaluated by mann we studied 12 patients with fulminant type 1 diabetes (5 males and 7 females). These patients were diagnosed as having fulminant type 1 diabetes between 19882006, and had been followed in our hospital for more than 0.5 years since the disease onset . The diagnosis of fulminant type 1 diabetes was established according to the inclusion criteria proposed by the committee of the japan diabetes society . Namely: (i) the presence of ketosis or ketoacidosis within a week after the onset of hyperglycemic symptoms; (ii) urinary cpeptide excretion <10 g / day or fasting serum cpeptide level <0.10 nmol / l (0.3 ng / ml) and peak serum cpeptide level <0.17 nmol / l (0.5 ng / ml) after glucagon (1 mg) or a meal load soon after disease onset; and (iii) plasma glucose level 16.0 mmol / l (288 mg / dl) and hba1c level <8.5% at first visit . Patients characteristics were as follows: they were aged 2278 years (44.2 18.4), duration of the disease was 0.819 years (4.8 5.2), body mass index (bmi) was 18.524.8 (20.8 1.9), hba1c was 5.710.4% (7.2 1.5), and glycoalbumin was 19.032.5% (25.1 4.1). Eight of the 12 patients were receiving multiple daily injections and three patients were receiving continuous subcutaneous insulin infusion . Every patient was usually followed every month in our outpatient clinic and insulin doses were adjusted for targeting nearly normal glucose levels (hba1c <6.5%, fasting glucose level <5.6 mmol / l [100 mg / dl], 120min postprandial glucose level <7.8 mmol / l [140 mg / dl]). The data of hba1c, glycoalbumin, bodyweight and daily dosages of insulin were measured every 9 months (from january to september in 2006) and expressed as mean of 9 months measurements . Sevenpoint (fasting and 120 min postprandial for each meal and bedtime) capillary blood glucose concentrations were measured in seven of the 12 patients for at least two days, and mean mvalue was calculated in each patient . Mvalue is a logarithmic transformation of the deviation of blood glucose from a selected standard . We used 5.6 mmol / l (100 mg / dl) as the selected standard . The deviation index () of each individual glucose value () is first calculated as = (10 log[/5.0]). Serum cpeptide levels were measured by cleia (lumipulse f, lumipulse cpeptide; fujirevio, tokyo, japan) and two different eia, aia21 (e test cpeptide; tosoh, tokyo, japan) and eclusys 2010 (eclusis cpeptide; roche diagnostics, basel, switzerland) simultaneously by using the same serum samples . Serum cpeptide level was also followed for 9 months by cleia, and the maximal value was determined in each patient . The present study was carried out with patients approvals and informed consent was obtained from every patient . The significance of differences between the two groups was evaluated by pearson s correlation coefficient and mvalue was evaluated by mann maximal concentrations of serum cpeptide evaluated by cleia were 0.020.10 nmol / l (0.060.29 ng / ml) in four patients and <0.003 nmol / l (0.01 ng / ml) in eight patients . Serum cpeptide concentrations of the former four patients ranged from 0.007 to 0.10 nmol / l (0.020.29 ng / ml) and those of the latter eight patients were always less than 0.003 in contrast, serum cpeptide was detectable in just two patients (0.090.10 nmol / l, respectively) by conventional eia . Detectable serum cpeptide levels in 4 patients (,,,) ranged from 0.007 to 0.10 nmol / l, however serum cpeptide levels in eight patients were always <0.003 nmol / l . Patients who kept more serum cpeptide levels needed significantly fewer daily dosages of insulin (p <0.01; figure 2). The patients with detectable serum cpeptide levels showed a significantly lower mvalue (median 2.94, range 0.966.15, n = 4) than those without detectable serum cpeptide levels (median 16.62, range 5.3627.10, n = 3; p = 0.01). Relationship between maximal serum cpeptide level(nmol / l) and daily dosages of insulin (unit / kg). Residual insulin secreting capacity shown as serum cpeptide level and daily dosages of insulin indicated a significant negative correlation (* p <0.01). There were no significant correlations between hba1c, glycoalbumin, bmi, duration of disease and serum cpeptide levels . In the present study, we clarified a significant negative correlation between serum cpeptide levels and daily dosages of insulin . The patients who had more residual endogenous insulin secreting capacity needed fewer daily dosages of insulin injection . In addition, the patients whose serum cpeptide levels were <0.003 nmol / l had a significantly higher mvalue than the patients whose serum cpeptide levels were 0.0070.10 nmol / l . These results suggest that even very low endogenous insulin secreting capacity would improve the doses of required insulin and stability of blood glucose controls in patients with fulminant type 1 diabetes . Fukuda et al . Have reported a negative correlation between endogenous insulin secreting capacity and degree of blood glucose instability in 20 patients with type 1 diabetes whose fasting serum cpeptide levels were 0.010.13 nmol / l . In the present study, compared with the report from fukuda et al ., not fasting but maximal postprandial cpeptide levels were 0.020.10 nmol / l or less, showing that a negative correlation was observed even in the patients with lower levels of serum cpeptide . Second, serum cpeptides were detectable and ranged from 0.007 to 0.10 nmol / l in four patients with fulminant type 1 diabetes throughout the followup period of 9 months . Pathophysiology of fulminant type 1 diabetes is known as almost complete cell destruction around the disease onset, resulting in nearly no cpeptide secretion . However, our results also suggest that endogenous insulin secreting capacity would be preserved by intensive insulin therapy, as shown in data from the diabetes control and complications trial, even in patients with fulminant type 1 diabetes . It is important because preserving cell function, even at a low level, could help to stabilize blood glucose levels in patients with fulminant type 1 diabetes . In conclusion, our present results suggest that even very low levels of serum cpeptide could reduce daily dosages of insulin and stabilize blood glucose controls in fulminant type 1 diabetes.
Two types of cnt were investigated, nc7000 from nanocyl and mwnt sa from sigma aldrich (catalog reference 659258), both produced by chemical vapor deposition (cvd). Nc7000 has a diameter of 710 nm, and mwnt sa has a diameter of 110170 nm . The cnt were functionalized by a 1,3-dipolar cycloaddition reaction of azomethine ylides using a one - pot functionalization procedure described elsewhere13 leading to the formation of pyrrolidine - type groups bonded to the cnt surface . The functionalization was carried out at 250 c over 5 h. the unzipping of the cnt was performed using an ultrasonic processor up100h from hielscher, equipped with a sonotrode ms2 . Cnt suspensions were prepared by mixing functionalized cnt (5 mg) in ethanol (8 ml). A control experiment was conducted using pristine cnt (5 mg) in ethanol (8 ml). The suspensions were centrifuged (8000 rpm, 1 h) to separate the unzipped cnt, and the gnr supernatant solutions were collected . These solutions were analyzed by uv - visible spectroscopy on a shimadzu uv-240 1 pc, using 10 mm light path quartz cells . The micro - raman analysis was conducted in the backscattering configuration on a jobin yvon hr800 instrument (horiba, japan), using a 1800 l mm grating and the 532 nm laser line from a nd: yag dpss laser (ventus, laser quantum, uk). A 50x objective (spot size: 3 m, na=0.75, olympus, japan) was used to focus the laser light onto the sample (<0.15 mw m) and to collect the backscattered raman radiation to be detected by a peltier cooled (223 k) ccd sensor . The spectrometer was operated in the confocal mode, setting the iris to 300 m, while the acquisition time was set to 30 s with 2 accumulations . Transmission electron microscopy (tem) of the gnr mwnt sa was performed on an energy filtered 200 kv transmission electron microscope hr-(ef)tem tem analysis of the gnr nc7000 was carried out on a titan chemistem 80200 kv probe cs corrected microscope . Low - magnification tem and high - resolution tem (hrtem) images were acquired with a gatan ultrascan 1000 p camera controlled with digital micrograph software integrated in the microscopes user interface . The samples were prepared by adding a drop of the gnr solution onto a lacey carbon cu grid (300 mesh, ted pella) and allowing it to dry under vacuum . X - rays diffraction experiments were performed on a panalytical xpert pro xrd system using the cu k1 wavelength of 0.15406 nm from a copper x - ray tube operated at 45 kv and 40 ma . A pixcel-3d detector was used, and the scan range was from 4 to 40 in 2. the gnr samples were deposited on glass lamellae by solvent evaporation . The unzipping process produces functionalized gnrs with widths equal or larger than 35 nm for gnr nc7000 and 350 nm for gnr mwnt sa . For this reason, a model of functionalized graphene (see figure 6), as opposite to finite gnr, was chosen . Graphene layers and their intermolecular interactions were modelled with the mm3 force field that has been found to give accurate intermolecular structures.17 all the calculations were performed with the tinker molecular mechanics suite18 using three - dimensional periodic boundary conditions.19 the shape and dimensions of unit cells containing five functionalized graphene layers with different concentrations of functional groups were systematically obtained by energy minimization . Additional data for the characterization for the gnr: thermogravimetric analysis (tga) and fourier - transform infrared spectroscopy (ftir), is provided as supporting information . As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Periodontal diseases are polymicrobial immune - inflammatory infectious diseases that can lead to the destruction of periodontal ligaments and adjacent supportive alveolar bone . The subgingival plaque contains more than 700 bacterial species, and some of these microorganisms have been shown to be responsible for initiation / progression of periodontal diseases [1, 2]. The red complex, which includes porphyromonas gingivalis, treponema denticola, and tannerella forsythia (formerly bacteroides forsythus), encompasses the most important pathogens in adult periodontal disease . Additionally, fusobacterium nucleatum, prevotella species, eikenella corrodens, peptostreptococcus micros, and campylobacter rectus are increased in deep periodontal pockets and are implicated as possible periodontopathogens [14]. These bacteria are not usually found alone, but in combination in the periodontal pockets, suggesting that some bacteria may cause destruction of the periodontal tissue in a cooperative manner . Studies using animal models have reported the synergistic pathogenicity of mixed infections with p. gingivalis - t . Furthermore, coaggregation, nutrient effects, and modulation of virulence factors by periodontopathogens or by interspecies interactions between periodontopathogenic and nonpathogenic organisms have been reported to contribute to oral microbial pathogenesis . This paper focuses on interspecies pathogenic interactions within the red complex, in particular the combinations of p. gingivalis - t . Potential therapies using normal inhabitants of the oral microbiota that have an antagonistic relationship with the red complex are discussed . P. gingivalis possesses many virulence factors, such as fimbriae, lipopolysaccharides, and proteases [1315]. The arg - gingipain (rgp) and lys - gingipain (kgp) cysteine proteinases are important for the virulence of p. gingivalis as they elicit dysfunction of inflammatory and immune responses and can degrade various connective tissue proteins [16, 17]. Rgp is encoded by two separate genes (rgpa and rgpb), whereas kgp is encoded by a single gene (kgp). In a murine abscess model, rgpa and rgpb double and kgp single - mutants induced smaller abscesses than did the wild type, and the rgpa - rgpb - kgp triple (gingipain - null) mutant showed negligible lesion formation . These findings indicate that gingipains play an important role in abscess formation in mice . Compared to the abscess formation induced by monoinfection with each bacterium in the abovedescribed murine model, mixed infection with wild - type p. gingivalis atcc 33277 and t. forsythia atcc 43037 showed a synergistic effect on abscess formation . On the other hand, mixed infection with p. gingivalis mutants devoid of gingipain (rgpa rgpb, kgp, and rgpa rgpb kgp) with t. forsythia showed only just an additive effect on abscess formation . These findings suggest that the gingipains of p. gingivalis play an important role in the pathological synergism between p. gingivalis and t. forsythia . A combination of t. forsythia strains isolated from periodontitis patients and p. gingivalis also showed synergistic pathogenesis in a rabbit abscess model . The researchers found 100% abscess formation in rabbits with mixed infection using p. gingivalis and t. forsythia but 0% in rabbits with monoinfection with each bacterium . Recently, verma et al . Injected p. gingivalis fdc 381 and t. forsythia atcc 43037 into the oral cavity of rats and evaluated the synergistic effect of mixed infection on periodontal disease . Mixed colonization by p. gingivalis and t. forsythia in the rat oral cavity was confirmed by polymerase chain reaction (pcr). The induction of moderate periodontal inflammation and pronounced apical migration of junctional epithelium, generation of a specific immunoglobulin g (igg) antibody response, and stimulation of both th1- and th2-like immune responses as reflected by the serum igg subclass profiles were evident . Mixed infection with p. gingivalis and t. forsythia resulted in a significant increase in interproximal alveolar bone resorption compared to that in rats infected with a single bacterial strain and controls, but there was no synergy between the bacteria . The researchers indicated that the virulence of p. gingivalis and t. forsythia mixed infection resulted from the immune - inflammatory responses and the lack of humoral immune protection during periodontitis in rats . Mice that were first infected with a wild - type strain and subsequently reinfected with the same wild - type strain showed significantly smaller lesions than control mice that were mock - infected with medium only and then re - infected with the wild - type strain . Assessed the serum igg antibody response in a murine abscess model after mixed infection with various levels of p. gingivalis atcc 33277 or t. forsythia atcc 43037 whole bacteria cell antigens . After mixed infection, igg antibody responses to p. gingivalis increased in proportion to the level of t. forsythia injected . In contrast, igg antibody responses to t. forsythia did not correlate with the level of p. gingivalis injected . Reasons for this difference may be that p. gingivalis and t. forsythia induced different antibody responses after mixed infection in mice; alternatively, these bacteria may exhibit different interactions in terms of growth at the injected sites . T. forsythia, which is a fastidious anaerobic gram - negative rod, is frequently isolated together with p. gingivalis, especially from the active state of periodontitis [2427]. It is well known that the growth of t. forsythia is accelerated on blood agar when cocultivated with p. gingivalis or f. nucleatum, suggesting that a form of symbiosis occurs with respect to nutrition . Sonicated cell extracts of t. forsythia atcc 43037 stimulate growth of p. gingivalis atcc 33277 in nutrition - decreased medium in a dose - dependent manner, whereas the cell extract of t. forsythia had no stimulatory effect on the growth of the rgpa rgpb kgp triple (gingipain - null) mutant of p. gingivalis . These results suggest that gingipains of p. gingivalis play an important role in the digestion or uptake of the growth - promoting factor derived from t. forsythia . The growth - promoting interaction between p. gingivalis and t. forsythia may be partly related to synergistic virulence in a murine abscess model . T. forsythia is a member of the polymicrobial flora that invades buccal epithelial cells taken directly from the mouth . Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses and for forming reservoirs important in recurrent infections . T. forsythia possesses some putative virulence factors, such as a trypsin - like protease, a sialidase, hemagglutinin, components of the bacterial s - layer, and a cell surface - associated and secreted protein (bspa). Bspa has been recognized as a virulence factor important for alveolar bone loss in mice . Inagaki et al . Investigated the epithelial cell adherence and invasion abilities of t. forsythia and reported that these are dependent on bspa . Additionally, they found that p. gingivalis fdc 381 or its outer membrane vesicles enhance the attachment and invasion of t. forsythia atcc 43037 to epithelial cells . T. denticola, a small oral spirochete, is frequently found with p. gingivalis in progressing periodontitis lesions [1, 3335]. T. denticola is located within the surface layers of the subgingival plaque, whereas p. gingivalis is observed predominantly beneath the spirochete layer; a symbiotic nutrient utilization relationship between these two periodontopathogens has been shown in vitro . The growth - stimulating factors produced by p. gingivalis atcc 33277 and t. denticola atcc 35405 have been identified as isobutyric acid and succinic acid, respectively . Coculture of p. gingivalis fdc 381 and t. denticola atcc 35405 induced synergistic biofilm formation and coaggregation . Confocal microscopy demonstrated that p. gingivalis attaches to the substratum first as the primary colonizer followed by coaggregation with t. denticola to form a mixed biofilm . The t. denticola flagellar mutant and cytoplasmic filament mutant exhibit significantly reduced biofilm formation with p. gingivalis . Similarly, the p. gingivalis gingipain mutant and major fimbriae mutant exhibited significantly reduced biofilm formation with t. denticola . Using two - dimensional electrophoresis followed by a ligand overlay assay with p. gingivalis fimbriae, hashimoto et al . Determined that dentilisin, a chymotrypsin - like proteinase of t. denticola, was the p. gingivalis fimbriae - binding protein . These results support the hypothesis that these two organisms assist each other's survival in subgingival sulcus and explain why they are frequently isolated together from subgingival plaque . Synergistic virulence of mixed p. gingivalis and t. denticola infection has been assessed in several animal lesion models . Using a murine abscess model, one group reported that high doses of p. gingivalis w50 (1 - 2 10 cells per dose) together with t. denticola atcc 35404 (1 10 or 1 10 cells per dose) had no effect on the formation and size of the spreading lesion caused by p. gingivalis . However, at low p. gingivalis doses (1 - 2 10 cells), addition of t. denticola (1 10 cells) significantly enhanced the virulence of p. gingivalis compared with monoinfection . Investigation of the synergistic virulence of p. gingivalis and t. denticola using a murine experimental model of periodontitis found that a 1: 1 ratio of p. gingivalis w50 and t. denticola atcc 35405 coinoculum at 5 10 or 1 10 total bacterial cells induced the same level of bone loss as four doses of 1 10 p. gingivalis . Coinoculation induced strong p. gingivalis - specific t cell proliferative and interferon- (ifn-) cytokine responses and induced a strong t. denticola - specific ifn- cytokine response . Another study using a rat model of periodontal disease reported that a mixed p. gingivalis fdc 381 (5 10 cells) and t. denticola atcc 35404 (5 10 cells) infection produced significantly more interproximal and horizontal alveolar bone loss compared to monoinfections (1 10 cells); however, there was no synergy between p. gingivalis and t. denticola . Furthermore, colonization of these bacteria was observed in the rat oral cavity during 7 weeks of periodontal disease, resulting in the generation of a specific serum igg antibody response that reflected oral infection and the induction of an inflammatory response consistent with the established characteristics of periodontitis . These results suggest that p. gingivalis and t. denticola act synergistically (with no synergy between the bacteria) to stimulate the host immune response and to induce alveolar bone loss in a rat model of periodontitis . Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and secretion of inflammatory mediators and matrix metalloproteinases (mmps) by these host cells contribute to periodontal tissue destruction . Investigated the inflammatory response of an in vitro macrophage / epithelial cell coculture model following mono- or mixed infections with whole bacterial cells of the red - complex and their lipopolysaccharide (lps). Mono- or mixed infections of the coculture model induced the secretion of interleukin-1 beta (il-1), il-6, il-8, prostaglandin e2 (pge2), and mmp-9 . All lps mono- or mixed infections induced an increase in chemokine, mmp-9, and pge2 production . Compared to mono - infections with individual bacterial species, no synergistic effects on cytokine, pge2, or mmp-9 production by the bacterial mixtures tested were observed . P. gingivalis and t. forsythia induced the secretion of rantes (regulated and normal t cell expressed and secreted), whereas t. denticola alone or in combination with p. gingivalis resulted in a significant decrease in rantes levels . Rantes degradation by mono- or mixed infections with red - complex bacteria resulted in massive proteolytic degradation of rantes by t. denticola . The ability of periodontopathogens to degrade cytokines and chemokines in vivo may play an important role in their pathogenicity by disrupting the host inflammatory response . Recently, polymicrobial infection with p. gingivalis, t. denticola, and t. forsythia in a rat model of periodontal disease was investigated . A 1 10 cell mixture (1 ml) containing an equal number of cells of each bacterium was injected into the rat oral cavity . Pcr of the bacterial dna in the oral sample revealed that polymicrobial infection enhanced colonization by p. gingivalis, t. denticola, and t. forsythia compared to their levels in monomicrobial infections . Oral infection of rats with a polymicrobial consortium comprising p. gingivalis, t. denticola, and t. forsythia induced significant increases in maxillary and mandibular alveolar bone resorption compared to those resulting from any of the monomicrobial infections (p <0.001). The levels of serum igg against all of the bacteria in the polymicrobial infection were lower than the respective levels induced by monomicrobial infections . This suggested that the host response to the polymicrobial infection was altered, resulting in enhanced evasion of protective immune responses by the bacterial consortium . Most therapeutic modalities for treatment of periodontitis aim to remove pathogens and kill all bacteria in the periodontal pocket . Understanding the effect of interbacterial interactions on the pathogenesis of periodontitis may facilitate development of novel treatment modalities, such as the inhibition of adherence using antagonists, passive immunization, replacement therapy, regulation of levels of nonpathogenic bacteria to modulate virulence, probiotics, and interference with signaling mechanisms . The disruption of the harmonic relationship between the host and commensal microorganisms is considered to be an important factor for the development of oral pathologies ., we discuss the significance of interbacterial antagonism for maintenance and recovery of a healthy oral microbiota . Moreover, antagonistic bacteria have the potential for probiotic action, which may protect against periodontitis . Staphylococcus aureus and streptococcus mutans isolates inhibited the growth of t. denticola and p. gingivalis . S. aureus strains produced a bacteriocin - like inhibitory substance, whereas the inhibitory effect of s. mutans was related to the production of lactic acid . S. sanguinis, s. cristatus, s. salivarius, s. mitis, actinomyces naeslundii, and haemophilus parainfluenzae inhibited the adhesion of standard p. gingivalis strains in vitro . S. cristatus arginine deiminase repressed fima, a major subunit protein of the long fimbriae, and inhibited biofilm formation by p. gingivalis . An analysis of the ability of clinical isolates from healthy and periodontitis patients to inhibit the growth of periodontopathogens showed that the number of isolates from healthy volunteers that inhibited either p. gingivalis or p. intermedia was significantly higher than that from diseased patients . These isolated growth - inhibiting strains included some viridans group streptococcus, actinomyces, and bifidobacterium strains . Compared to these isolates, commercial dietary probiotics showed stronger inhibition of the periodontopathogens . In a study that compared oral lactobacilli from patients with chronic periodontitis and periodontally healthy subjects, the most prevalent species in healthy subjects were lactobacillus gasseri and l. fermentum, whereas the most prevalent species in subjects with periodontitis was l. plantarum . Furthermore, the greatest antimicrobial activities were associated with l. paracasei, l. plantarum, l. rhamnosus, and l. salivarius . The international guidelines for the evaluation of probiotics confirm that these four organisms exhibit both high antimicrobial activity and high tolerance to environmental stress . Some oral bacteria possess the potential for antagonism towards periodontopathogens, which highlights the therapeutic potential of stimulation of oral health using beneficial effector strains . Most evidence indicates that probiotics in the gut do not populate the gastrointestinal microbiota permanently, and they disappear from feces soon after cessation of probiotic ingestion . Probiotic bacteria used in the human oral cavity include bifidobacterium and lactobacillus species, and most of them were not derived from the oral cavity . L. reuteri and l. salivarius colonized the oral cavity of patients; however, the study was of only 2 weeks duration [5759]. Another study reported that oral administration of l. salivarius decreased the proportion of l. salivarius in saliva during the 4- and 8-week intervention periods, although the sampling and analysis methods differed . Organisms antagonistic to periodontopathogens that are derived from typical representatives of the oral microbiota and have probiotic potential may overcome the weaknesses associated with exogenous probiotic bacteria.
Glaucoma is a leading cause of blindness worldwide.1 despite the growing understanding of its pathophysiology and advances in its treatment, challenges remain regarding how to predict and stop its progression . The treatment plan for glaucoma depends on its progression rate . In practice, most ophthalmologists determine the glaucoma progression rate based on the results of examinations of the patient s visual field (vf). It is difficult to predict the rate of glaucoma progression.2 many studies on the progression of glaucoma have been reported . Candidate factors associated with glaucoma progression include increased baseline intraocular pressure (iop), increased mean iop, increased peak iop, and greater iop fluctuation.38 de moraes et al reported that the peak iop is a better predictor of vf deterioration compared to the mean iop.9 the study is supported by an investigation of an animal model of glaucoma which demonstrated a more predictive role of the maximum iop in structural change, compared to the mean iop.10 it is difficult to identify the peak iop because it might occur while the patient is asleep . If the peak iop is to be determined using goldmann applanation tonometry (gat), the iop must be measured many times . The iop is known to fluctuate, and iop fluctuation might be a better predictor of the progression of glaucoma than the mean iop.6,8,11 the existing studies on iop fluctuation can be divided into short - term (throughout the day) and long - term (several years) studies . The relationship between short - term and long - term iop fluctuations has not been clear . Since an assessment of short - term iop fluctuation requires that the iop be measured at night, it burdens both the patient and his or her examiner(s). Short - term (24-hour) iop fluctuation can be measured using a contact lens sensor (cls). The cls was developed to continuously monitor habitual iop fluctuations over a 24-hour period by measuring changes in the eye s circumference in the area of the corneoscleral junction.12,13 once a cls is placed on an eye, the iop fluctuation can be measured automatically without interference with the subject s sleep . The measurement obtained with a cls has been shown to be accurate and reproducible.14 in the present study, we investigated the relationship between short - term and long - term iop fluctuations . This was a retrospective study . Fifty adult glaucoma patients (25 men, 25 women) all 50 patients underwent a comprehensive ophthalmic examination, including test of refraction and visual acuity, goldmann gonioscopy, gat, a fundus examination, and automated perimetry (humphrey field analyzer; carl zeiss meditec ag, jena, germany). The study and the research protocol were approved by the institutional review board of the university of toyama (approval no 23 - 100), and the procedures used conformed to the tenets of the declaration of helsinki . After the nature and possible consequences of the study were explained to the subjects, written informed consent was obtained from each subject . The diagnosis of glaucoma was made if both of the following criteria were satisfied: 1) the presence of glaucomatous optic disk neuropathy (a cup / disk ratio of 0.7 or the presence of notching) accompanied by corresponding vf defects, and 2) a threshold sita 30 - 2 examination showing a glaucoma hemifield test result outside normal limits, and a cluster of three contiguous points on the pattern deviation plot depressed at the p<0.05 level (occurring in age - matched normal subjects) not crossing the horizontal meridian which are compatible with glaucoma . The inclusion criteria for the subjects were as follows: 1) the subject had best - corrected visual acuity 0.2 . 2) the subject had spherical equivalent <+ 6 or> 6 d. in this study, we included patients who had undergone a previous cataract surgery at least 2 years prior to the enrollment in the study . Eyes with myopia over 6 d or hyperopia over + 6 d before cataract surgery were excluded . We defined the start of the follow - up period when the patients were diagnosed with glaucoma . For the patients who had underwent a cataract surgery during the follow - up period, we excluded the iop data obtained before cataract surgery, and used the iop data obtained after the cataract surgery . We included patients who met the requirement of> 2-year follow - up period after the cataract surgery . For the patients who had underwent selective laser trabeculoplasty, we used the iop data in the same way . We also included the cases in whom the glaucoma medications were changed or added during the follow - up period . The exclusion criteria were 1) history of glaucoma operation, 2) history of ocular trauma, 3) retinal diseases, and 4) ocular inflammatory diseases . Physicians who had been trained in cls measurement conducted the placement and removal of cls in each subject in a hospital setting . One eye of each subject was monitored . If both eyes had glaucoma, we measured the eye with the worse vf which also met the inclusion criteria . The 24-hour iop was monitored with a triggerfish cls (sensimed, lausanne, switzerland). The cls consists of a highly oxygen - permeable soft contact lens, the key elements of which are two sensing - resistive strain gauges that are capable of recording circumferential changes in the area of the corneoscleral junction . This device is based on a novel approach to iop monitoring in which changes in corneal curvature and circumference are assumed to correspond to changes in the iop . The unit of measurement used for the monitoring of iop fluctuation with the cls is not mmhg but mveq, which is unique to the cls . The median iop values were obtained every 5 minutes, providing 288 points over the 24-hour period . The range of iop fluctuations, which was defined as the difference between the maximum value (mveq) and the minimum value measured during the 24-hour course, was calculated from the iop fluctuation data . Patients who were taking glaucoma medication continued to take the medication during the measurement period . We defined the number of glaucoma drops as the number of drops prescribed when the subject s iop was measured by cls . The long - term iop fluctuation was defined by four parameters: 1) the mean of the eligible iop (mmhg) determined during the follow - up; 2) the iop difference (mmhg), which we defined as the difference between the maximum iop and the minimum iop; 3) the standard deviation of iop (sd - iop, mmhg) which was calculated from all eligible iop values; and 4) the peak iop (mmhg), which was defined as the maximum iop . All statistical analyses were performed using the jmp 10 software program (sas institute inc ., we used the spearman rank correlation coefficient for determining the correlation of the long - term iop fluctuation index and short - term iop fluctuation . This was a retrospective study . Fifty adult glaucoma patients (25 men, 25 women) all 50 patients underwent a comprehensive ophthalmic examination, including test of refraction and visual acuity, goldmann gonioscopy, gat, a fundus examination, and automated perimetry (humphrey field analyzer; carl zeiss meditec ag, jena, germany). The study and the research protocol were approved by the institutional review board of the university of toyama (approval no 23 - 100), and the procedures used conformed to the tenets of the declaration of helsinki . After the nature and possible consequences of the study were explained to the subjects, written informed consent was obtained from each subject . The diagnosis of glaucoma was made if both of the following criteria were satisfied: 1) the presence of glaucomatous optic disk neuropathy (a cup / disk ratio of 0.7 or the presence of notching) accompanied by corresponding vf defects, and 2) a threshold sita 30 - 2 examination showing a glaucoma hemifield test result outside normal limits, and a cluster of three contiguous points on the pattern deviation plot depressed at the p<0.05 level (occurring in age - matched normal subjects) not crossing the horizontal meridian which are compatible with glaucoma . The inclusion criteria for the subjects were as follows: 1) the subject had best - corrected visual acuity 0.2 . 2) the subject had spherical equivalent <+ 6 or> 6 d. in this study, we included patients who had undergone a previous cataract surgery at least 2 years prior to the enrollment in the study . Eyes with myopia over 6 d or hyperopia over + 6 d before cataract surgery were excluded . We defined the start of the follow - up period when the patients were diagnosed with glaucoma . For the patients who had underwent a cataract surgery during the follow - up period, we excluded the iop data obtained before cataract surgery, and used the iop data obtained after the cataract surgery . We included patients who met the requirement of> 2-year follow - up period after the cataract surgery . For the patients who had underwent selective laser trabeculoplasty, we used the iop data in the same way . We also included the cases in whom the glaucoma medications were changed or added during the follow - up period . The exclusion criteria were 1) history of glaucoma operation, 2) history of ocular trauma, 3) retinal diseases, and 4) ocular inflammatory diseases . Physicians who had been trained in cls measurement conducted the placement and removal of cls in each subject in a hospital setting . One eye of each subject was monitored . If both eyes had glaucoma, we measured the eye with the worse vf which also met the inclusion criteria . The 24-hour iop was monitored with a triggerfish cls (sensimed, lausanne, switzerland). The cls consists of a highly oxygen - permeable soft contact lens, the key elements of which are two sensing - resistive strain gauges that are capable of recording circumferential changes in the area of the corneoscleral junction . This device is based on a novel approach to iop monitoring in which changes in corneal curvature and circumference are assumed to correspond to changes in the iop . The unit of measurement used for the monitoring of iop fluctuation with the cls is not mmhg but mveq, which is unique to the cls . The median iop values were obtained every 5 minutes, providing 288 points over the 24-hour period . The range of iop fluctuations, which was defined as the difference between the maximum value (mveq) and the minimum value measured during the 24-hour course, was calculated from the iop fluctuation data . Patients who were taking glaucoma medication continued to take the medication during the measurement period . We defined the number of glaucoma drops as the number of drops prescribed when the subject s iop was measured by cls . The long - term iop fluctuation was defined by four parameters: 1) the mean of the eligible iop (mmhg) determined during the follow - up; 2) the iop difference (mmhg), which we defined as the difference between the maximum iop and the minimum iop; 3) the standard deviation of iop (sd - iop, mmhg) which was calculated from all eligible iop values; and 4) the peak iop (mmhg), which was defined as the maximum iop . All statistical analyses were performed using the jmp 10 software program (sas institute inc ., cary, nc, usa). We used the spearman rank correlation coefficient for determining the correlation of the long - term iop fluctuation index and short - term iop fluctuation . We classified the subtypes of glaucoma as primary open - angle glaucoma (30 eyes including 15 normal - tension glaucoma eyes), primary angle - closure glaucoma (one eye), and pseudo - exfoliation glaucoma (19 eyes). We defined normal - tension glaucoma as untreated iop that had never been> 21 mmhg among the primary open - angle glaucoma eyes . We were able to successfully measure the 24-hour iop with the cls in all of the subjects . Some subjects had minor complications: conjunctivitis, slight hyperemia, or peripheral corneal edema . These healed within a few days without any additional eye drops . As shown in figure 1 and table 2, the short - term iop fluctuation was significantly correlated with the mean iop (p=0.001), the iop difference (p=0.0207), the sd - iop (p=0.0061), and the peak iop (p=0.0017). The short - term and the long - term iop fluctuations were moderately but significantly correlated . We classified the subtypes of glaucoma as primary open - angle glaucoma (30 eyes including 15 normal - tension glaucoma eyes), primary angle - closure glaucoma (one eye), and pseudo - exfoliation glaucoma (19 eyes). We defined normal - tension glaucoma as untreated iop that had never been> 21 mmhg among the primary open - angle glaucoma eyes . We were able to successfully measure the 24-hour iop with the cls in all of the subjects . Some subjects had minor complications: conjunctivitis, slight hyperemia, or peripheral corneal edema . As shown in figure 1 and table 2, the short - term iop fluctuation was significantly correlated with the mean iop (p=0.001), the iop difference (p=0.0207), the sd - iop (p=0.0061), and the peak iop (p=0.0017). The short - term and the long - term iop fluctuations were moderately but significantly correlated . The prior studies on iop fluctuation were a mix of short - term and long - term studies . It has been difficult to determine the significance of these studies due to lack of standardization regarding the time between assessments, the methods of measurement, and the definition of iop fluctuation itself . To the best of our knowledge, this study is the first to analyze the relationship between long - term and short - term iop fluctuations with a cls . The mean iop as well as peak iop, the iop difference, and sd - iop of long - term iop fluctuation was correlated with the short - term iop fluctuation measured with a cls . Thus, the short - term and the long - term iop fluctuations were significantly correlated . The long - term iop fluctuation was shown as a significant risk factor by multiple logistic regression analysis.8,15 another study showed that peak iop was a better predictor of progression than mean iop or iop fluctuation.9 some studies reported that the short - term and the long - term iop fluctuations were correlated in stable glaucoma patients.1519 these results are in agreement with our present findings . Tan et al reported that short - term iop fluctuation correlated with thickness of retinal nerve fiber layer.17 agnifili et al compared iop fluctuations between healthy subjects and glaucoma patients . They reported that glaucoma patients showed larger iop fluctuation than normal subjects.18 the larger iop fluctuation in both long and short term could predict the rate of deterioration of vf . The only available treatment for glaucoma at present is lowering the iop, but it does not adequately suppress the progression of glaucoma.8,9 since a new treatment modality to reduce the iop fluctuation is required, it may be necessary to use a cls to measure iop fluctuations . Measuring the iop fluctuation with a cls could thus become a standard method of measuring short - term iop fluctuation . This method has several merits: iop fluctuation can be measured every 5 minutes rather than every few hours . No restriction is imposed on the subject s posture during the measurement . Even though the obtained values are objective (mveq), short - term iop fluctuations measured with a cls might help to predict long - term iop fluctuations . It was reported that glaucoma medication could make the circadian iop fluctuation smaller.20 holl et al reported that a cls could not measure the effect of topical medication or body position.21 adherence of a cls to the cornea might alter the effects of topical medications . Second, short - term iop fluctuation was assessed with a cls on different days, although some studies reported a good reproducibility of the results of short - term iop fluctuation with a cls.2225 third, the value in mveq measured with a cls cannot be converted to mmhg . Mottet et al reported that the iop measurement using a cls is an accurate and reproducible method to characterize the iop rhythm but does not allow estimating the iop value in mmhg corresponding to the relative variation of the electrical signal measured.14 to address these problems, further studies are necessary . In conclusion, long - term and short - term iop fluctuations were significantly correlated . Measuring iop fluctuation with a cls could be useful to predict long - term iop fluctuation.
To reproduce highly esthetic tooth - colored fixed dental prostheses, computer aided design / computer aided manufacturing (cad / cam) techniques are becoming frequently used . Since the commercial introduction of polymeric blanks into the dental market, cad / paul, mn, usa) are innovative new cad / cam materials that make it possible to achieve superior esthetic results in easy steps . The milled rnc restorations can be individualized intra- or extra - orally, either before or after insertion . In recent years, researchers have tried to achieve a more effective and longer - lasting adhesion between restorative materials and the dental substrate . The adhesive techniques are based on research on the hybrid layer and on chemical and mechanical adhesion . Some researchers have attempted to shorten the application time and reduce the number of steps, creating new generations of materials and improving their quality . Increasing demand for esthetic restorations has led to greater use of all ceramic materials because of their improved biocompatibility and optical properties, compared with metal - ceramic restorations . Advances in cad and cam systems are providing new options for dentistry, creating an alternative to the conventional impression and casting technique for producing dental restorations . A requirement for the successful function of a cad / cam restoration is adequate adhesion between ceramic and tooth substance; however, the literature is unclear on which cement, ceramic, conditioning treatment, and dentine bonding agent produce the highest bond strength . Resin composite cements are used to lute conventional metal crowns, fixed partial dentures, ceramic crowns, and veneers and to repair fractured metal ceramic restorations . Resin cements have been selected for their advantageous mechanical and adhesive properties compared with conventional luting agents . The use of resin luting agents also appears to be essential in determining an effective stress distribution, which will prevent crack initiation . Bond strength to ceramic material is influenced by the composition of the ceramic substrate as well as by mechanical and chemical interaction between substrate and bonding agent . The aim of this study was to evaluate the influence of three different luting protocols (total - etch, self - etch, and self - adhesive) on shear bond strength of cad / cam rnc material, based on cad / cam technology, to dentin . In the present in vitro study, 30 disks (5 mm in diameter, 3 mm thick) were designed with cerec software 4.2 platform (sirona dental gmbh, salzburg, austria) and obtained by milling from rnc blocks for cad / cam (lava ultimate restorative, 3 m espe, st . Paul, mn, usa) with cerec mc xl (sirona dental gmbh, salzburg, austria) [figure 1]. The disks were obtained by milling from resin nano ceramic blocks for computer aided design / computer aided manufacturing with cerec mc xl . Characteristics of materials tested the disks were subsequently cemented to the exposed dentin of 30 bovine permanent mandibular incisors freshly extracted and stored in a 0.1% (wt / vol) thymol solution, which were used as a substitute for human teeth . The criteria for tooth selection included intact buccal enamel with no cracks caused by extraction, the absence of caries, and adequate dimension of the crown . The teeth were cleansed of soft tissue remnants and debris with periodontal curettes, stored in the thymol solution for 1 week, and later in saline solution at room temperature until testing . Then the roots of the teeth were embedded in self - curing, fast - setting acrylic resin (rapid repair, degu dent gmbh, hanau, germany). Specially fabricated cylindrical teflon mold with an internal diameter of 14 mm were filled with the acrylic resin and allowed to cure, thus encasing each specimen while allowing the buccal surface of dentin to be exposed . Each tooth was oriented so that its labial surface was parallel to the shearing force . The buccal enamel was removed using a high - speed carbide rotary instrument (no . H21 l.314.014; komet, lemgo, germany) under copious water irrigation, to expose midcoronal dentin . The exposed dentin surfaces were finished off with an automated polishing machine (apl-4; arotec s.a . Ind com, cotia, sp, brazil) with a 600-grit silicon carbide abrasive paper disks for 5 s, to obtain a flat and uniform dentin surface and reduce any micromechanical interlocking that could affect the real bonding influence of the tested adhesive cements . Before cementation, to remove the smear layer and to treat the dental surface with cleaning solution, the dentin surface was rinsed for 1 min with a cotton pellet impregnated with tubulicid blue (dental therapeutics ab, saltsj - boo, sweden) without fluorine . The surface was then rinsed and dried before cementation; the labial surface of each incisor was cleaned for 10 s with a mixture of water and fluoride - free pumice and rubber polishing cup in a low - speed handpiece . The dentinal surface was rinsed with water to remove pumice or debris and then dried with an oil - free air stream . The bonding surface of each disk was cleaned with alcohol and rinsed with water to remove oil debris contained in the milling liquid . The specimens were randomly assigned to 3 groups of 10 teeth each [figure 1] according to different luting procedures (total - etch, self - etch, and self - adhesive). Group 1: ult - te: disks were cemented on the dentin surface, which had been acid - etched with scotchbond universal etchant phosphoric acid (3 m espe, st . Paul, mn, usa) for 15 s, the etched substrates were rinsed with water for 15 s, and gently air dried for 15 s to remove excess water and treated with scotchbond universal adhesive (3 m espe, st . Paul, mn, usa) with adhesive resin cement (relyx ultimate/3 m espe, st . Paul, mn, usa)group 2: ult - se: disks were cemented on the dentin surface, which had been treated with scotchbond universal adhesive, with the adhesive resin cement (relyx ultimate)group 3: u2-sa: disks were cemented on the dentin surface with the dual polymerization resin luting agent relyx unicem 2 automix . Group 1: ult - te: disks were cemented on the dentin surface, which had been acid - etched with scotchbond universal etchant phosphoric acid (3 m espe, st . Paul, mn, usa) for 15 s, the etched substrates were rinsed with water for 15 s, and gently air dried for 15 s to remove excess water and treated with scotchbond universal adhesive (3 m espe, st . Paul, mn, usa) with adhesive resin cement (relyx ultimate/3 m espe, st . Paul, mn, usa) group 2: ult - se: disks were cemented on the dentin surface, which had been treated with scotchbond universal adhesive, with the adhesive resin cement (relyx ultimate) group 3: u2-sa: disks were cemented on the dentin surface with the dual polymerization resin luting agent relyx unicem 2 automix . A vinyl ring with an internal diameter of 4.5 mm was applied under the dentin surface to standardize the adhesion area . For all groups, no further procedures were applied in order to avoid bias and confounding factors . During cementation, a thin layer of cement was applied and distributed to the bonding surface of the cylinders by means of a heidemann spatula . On each specimen, five surfaces were identified: mesial, lingual, distal, buccal, and occlusal . As suggested by the manufacturer, every surface was light - polymerized for 20 s at a light intensity of 1000 mw / cm using a light - emitting diode (led) curing light in soft start - polymerization mode (celalux 2 high - power led curing light, voco gmbh, cuxhaven, germany). The power output (light intensity) of the led was measured with a cure rite radiometer (caulk - dentsply mod . All samples were stored in distilled water at room temperature for 24 h. after storing, the specimens were all submitted to a shear bond strength test to check the strength of adhesion between the two substrates, dentin, and rnc . This test is defined as a test in which two materials are connected by an adhesive agent and loaded in shear until separation occurs . Specimens were placed in a universal testing machine (model 3343, instron corporation, norwood, ma, usa). Teeth were secured in the lower jaw of the machine so that the bonded cylinder base was parallel to the shear force direction . Specimens were stressed in an occlusogingival direction at a crosshead speed of 1 mm / min [figure 2]. The maximum load necessary to debond was recorded in newton (n) and calculated in mpa as a ratio of newton to the surface area of the cylinder (the calculated shear bond strength was determined by dividing the strength at which bond failure occurred by the bonding area). Specimens were placed in a universal testing machine, secured in the lower jaw of the machine so that the bonded cylinder base was parallel to the shear force direction . After the testing procedure, the fractured surfaces were examined with an optical microscope (stereomicroscope sr, zeiss, oberkochen, germany). Statistical analysis was performed with stata 12.0 software (stata, college station, texas, usa). Descriptive statistics, including the mean, standard deviation, median, and minimum and maximum values were calculated for all groups . Analysis of variance (anova) was applied to determine whether significant differences in debond strength values existed among the groups . In the present in vitro study, 30 disks (5 mm in diameter, 3 mm thick) were designed with cerec software 4.2 platform (sirona dental gmbh, salzburg, austria) and obtained by milling from rnc blocks for cad / cam (lava ultimate restorative, 3 m espe, st . Paul, mn, usa) with cerec mc xl (sirona dental gmbh, salzburg, austria) [figure 1]. The disks were obtained by milling from resin nano ceramic blocks for computer aided design / computer aided manufacturing with cerec mc xl . Characteristics of materials tested the disks were subsequently cemented to the exposed dentin of 30 bovine permanent mandibular incisors freshly extracted and stored in a 0.1% (wt / vol) thymol solution, which were used as a substitute for human teeth . The criteria for tooth selection included intact buccal enamel with no cracks caused by extraction, the absence of caries, and adequate dimension of the crown . The teeth were cleansed of soft tissue remnants and debris with periodontal curettes, stored in the thymol solution for 1 week, and later in saline solution at room temperature until testing . Then the roots of the teeth were embedded in self - curing, fast - setting acrylic resin (rapid repair, degu dent gmbh, hanau, germany). Specially fabricated cylindrical teflon mold with an internal diameter of 14 mm were filled with the acrylic resin and allowed to cure, thus encasing each specimen while allowing the buccal surface of dentin to be exposed . Each tooth was oriented so that its labial surface was parallel to the shearing force . The buccal enamel was removed using a high - speed carbide rotary instrument (no . H21 l.314.014; komet, lemgo, germany) under copious water irrigation, to expose midcoronal dentin . The exposed dentin surfaces were finished off with an automated polishing machine (apl-4; arotec s.a . Ind com, cotia, sp, brazil) with a 600-grit silicon carbide abrasive paper disks for 5 s, to obtain a flat and uniform dentin surface and reduce any micromechanical interlocking that could affect the real bonding influence of the tested adhesive cements . Before cementation, to remove the smear layer and to treat the dental surface with cleaning solution, the dentin surface was rinsed for 1 min with a cotton pellet impregnated with tubulicid blue (dental therapeutics ab, saltsj - boo, sweden) without fluorine . The surface was then rinsed and dried before cementation; the labial surface of each incisor was cleaned for 10 s with a mixture of water and fluoride - free pumice and rubber polishing cup in a low - speed handpiece . The dentinal surface was rinsed with water to remove pumice or debris and then dried with an oil - free air stream . The bonding surface of each disk was cleaned with alcohol and rinsed with water to remove oil debris contained in the milling liquid . The specimens were randomly assigned to 3 groups of 10 teeth each [figure 1] according to different luting procedures (total - etch, self - etch, and self - adhesive). Group 1: ult - te: disks were cemented on the dentin surface, which had been acid - etched with scotchbond universal etchant phosphoric acid (3 m espe, st . Paul, mn, usa) for 15 s, the etched substrates were rinsed with water for 15 s, and gently air dried for 15 s to remove excess water and treated with scotchbond universal adhesive (3 m espe, st . Paul, mn, usa) with adhesive resin cement (relyx ultimate/3 m espe, st . Paul, mn, usa)group 2: ult - se: disks were cemented on the dentin surface, which had been treated with scotchbond universal adhesive, with the adhesive resin cement (relyx ultimate)group 3: u2-sa: disks were cemented on the dentin surface with the dual polymerization resin luting agent relyx unicem 2 automix . Group 1: ult - te: disks were cemented on the dentin surface, which had been acid - etched with scotchbond universal etchant phosphoric acid (3 m espe, st . Paul, mn, usa) for 15 s, the etched substrates were rinsed with water for 15 s, and gently air dried for 15 s to remove excess water and treated with scotchbond universal adhesive (3 m espe, st . Paul, mn, usa) with adhesive resin cement (relyx ultimate/3 m espe, st . Paul, mn, usa) group 2: ult - se: disks were cemented on the dentin surface, which had been treated with scotchbond universal adhesive, with the adhesive resin cement (relyx ultimate) group 3: u2-sa: disks were cemented on the dentin surface with the dual polymerization resin luting agent relyx unicem 2 automix . A vinyl ring with an internal diameter of 4.5 mm was applied under the dentin surface to standardize the adhesion area . For all groups, no further procedures were applied in order to avoid bias and confounding factors . During cementation, a thin layer of cement was applied and distributed to the bonding surface of the cylinders by means of a heidemann spatula . On each specimen, five surfaces were identified: mesial, lingual, distal, buccal, and occlusal . As suggested by the manufacturer, every surface was light - polymerized for 20 s at a light intensity of 1000 mw / cm using a light - emitting diode (led) curing light in soft start - polymerization mode (celalux 2 high - power led curing light, voco gmbh, cuxhaven, germany). The power output (light intensity) of the led was measured with a cure rite radiometer (caulk - dentsply mod . After storing, the specimens were all submitted to a shear bond strength test to check the strength of adhesion between the two substrates, dentin, and rnc . This test is defined as a test in which two materials are connected by an adhesive agent and loaded in shear until separation occurs . Specimens were placed in a universal testing machine (model 3343, instron corporation, norwood, ma, usa). Teeth were secured in the lower jaw of the machine so that the bonded cylinder base was parallel to the shear force direction . Specimens were stressed in an occlusogingival direction at a crosshead speed of 1 mm / min [figure 2]. The maximum load necessary to debond was recorded in newton (n) and calculated in mpa as a ratio of newton to the surface area of the cylinder (the calculated shear bond strength was determined by dividing the strength at which bond failure occurred by the bonding area). Specimens were placed in a universal testing machine, secured in the lower jaw of the machine so that the bonded cylinder base was parallel to the shear force direction . After the testing procedure, the fractured surfaces were examined with an optical microscope (stereomicroscope sr, zeiss, oberkochen, germany). Statistical analysis was performed with stata 12.0 software (stata, college station, texas, usa). Descriptive statistics, including the mean, standard deviation, median, and minimum and maximum values were calculated for all groups . Analysis of variance (anova) was applied to determine whether significant differences in debond strength values existed among the groups . Anova showed the presence of significant differences among the various groups (p <0.0001). As showed in figure 3, post - hoc tukey testing showed that the highest shear strength values (p <0.001) were reported in group 2 (ult - se). The lowest data (p <0.001) were recorded in group 3 (u2-sa). Descriptive statistics (mpa) of the different groups shear bond strength values (mpa) obtained in the three groups . Patients needs and desires and developments in adhesive dentistry have made the use of all - ceramic restorations increasingly frequent, particularly silica - based ones such as crowns, inlay - onlays, and laminate veneers . In order to improve the impression and casting procedure steps and to produce indirect restorations faster and easier, without the need for provisional restorations and dental laboratories, cad / paul, mn, usa) is a new composite / nanoceramic material for cad / cam manufacturing . This material allows the possibility to use composite materials to characterize and adjust the restoration after milling . Unlike conventional ceramic restorations, customization and glaze firing industrially, prefabricated cad / cam restorations are polymerized by standardized methods and improving material properties, in particular, predictability and consistency . Comparing these machinable prostheses to laboratory - handmade restorations, it has been advocated that, due to a highly homogeneous quality crystalline content, the bond strength to hard tooth tissues and the clinical longevity of these cad / cam restorations have been increased . In contrast, conventional manual polymerization and processing is greatly influenced by the operator and can cause a high level of variations . However, to achieve a long duration of restoration and, therefore, its long - term success, durable bond strength between the tooth and the restorative material is fundamental . To date, no study has evaluated the shear bond strength between the new nanoceramic lava ultimate material and tooth structures . The aim of this study was to investigate the influence of different luting protocols (total - etch, self - etch, and self - adhesive) on the shear bond strength values between dentin and this innovative rnc material, based on cad / cam technology . Although it is preferable to use extracted human teeth for bonding research, it has become increasingly difficult to obtain such samples for laboratory studies in italy . To compare data from the current study with that reported in previous bovine enamel bond strength tests, bovine teeth have some advantages, as they are easy to obtain in large quantities, are in good condition and have less composition variables than human enamel . Bovine teeth also have large, flat surfaces and are unlikely to have undergone prior caries challenges that could affect the test result . The mineral distribution within the carious lesions in bovine teeth is reportedly similar to that found in human teeth, and the structural changes that occur in human and bovine teeth are also similar . For the cementation procedure, three different luting protocols have been evaluated in this study: total etch / etch and rinse protocol (group 1 - ult - te: sco35% phosphoric acid + scotchbond universal adhesive + relyx ultimate conventional resin cement); self - etch protocol (group 2 - ult - se: scotchbond universal adhesive + relyx ultimate conventional resin cement), and self - adhesive protocol (group 3 - u2-sa: relyx unicem 2 automix self - adhesive resin cement). These three different cementation techniques have been selected according to contemporary adhesive systems classification into etch - and - rinse and self - etch adhesives . While the etch - and - rinse approach requires a separate acid - etch step to promote dentin and enamel demineralization before monomer infiltration, demineralization and infiltration occur simultaneously in the self - etch approach, although with no perfect synchronism . In addition, the separate etch - and - rinse step completely removes the smear layer, while the combined etch and bonding step in self - etch adhesive systems only partially dissolve the smear layer . Complete removal of the smear layer may allow for more intimate contact of the hydrophilic primer and hydrophobic bonding agent to the tooth . Many articles related to adhesive procedures used for the cementation of ceramic to tooth structure have shown that the presence of a hybrid layer between adhesive resin and dentin seems to adequately seal the dentinal tubules and allows a cellular reorganization of the pulpal tissues . In the present study, all the tested cements are based on adhesive procedures, which determine the formation of the hybrid layer and lead to the creation of a stronger link between dental structure and composite cement . In general two different types of resin composite cements exist: the conventional and the self - adhesive resin composite cements . To bond cad / cam restoration to dentin, relyx ultimate conventional resin cement was used together with a etch and rinse adhesive or with a self - etch adhesive; while relyx unicem 2 automix self - adhesive resin cement was used alone . A pretreatment of tooth abutment is necessary in case of use of conventional resin composite cements . For this reason, since they do not necessitate any pretreatment of tooth substrate, self - adhesive resin composite cements have been developed . Self - adhesive resin composite cements contain acid monomers, resulting in an initial lower ph value for the infiltration into the demineralized collagen network, in our study significant differences were found between conventional and self - adhesive resin cements . The lowest shear bond strength values were recorded in group 3; i.e., lava ultimate rnc disks bonded to dentin with self - adhesive cements . This is in accordance with a study by stawarczyk et al ., which reported lower tensile bond strength with self - adhesive resin composite cements to polymeric crowns, compared to the bonding with conventional resin cements . While some products have equal bond strength of self - adhesive resin cement to dentin, other products show an inferior bond to enamel . The success of the restoration depends not only on the bond between tooth and resin cement but also on the bond between restoration and resin composite cement . According to some authors to achieve a resistant bond, further conditioning of the restoration material is needed . When comparing the shear bond strength values of conventional resin cements used together with an etch and rinse adhesive or with a self - etch adhesive, significantly higher bond strength values were recorded in group 2 (self - etch protocol). Accurate and meticulous procedures during the cementation phase may play an essential clinical role in achieving a valuable connection between the dentin and the ceramic restoration . Retention form of the preparation, marginal integrity, and clinical micro - leakage are the key parameters used to judge the effectiveness of a resin composite cement system . Further studies should be conducted to test the correlation of bond strength for both tested materials to 48 h, 1 week, 1 month, and 1 year to evaluate the time factor related to the effective quality of bonding between ceramic material and dentin . Additional research should also be conducted to test the correlation between porosity, poor wetting, high viscosity, and failures . Within the limitations of this in vitro study, conventional resin cements (coupled with etch and rinse or self - etch adhesives) showed better shear strength values compared to self - adhesive resin cements . Then, for the tested polymeric cad / cam materials, the use of additional adhesives for conditioning is necessary . Furthermore, conventional resin cements used together with a self - etch adhesive reported the highest values of adhesion . However, future researches are needed to evaluate the bond strength to new ceramic / polymer prosthetic materials . The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article
Uterine cancer is the most common diagnosed gynecologic cancer among women in the united states, with 43,470 estimated new cases and approximately 7,950 estimated deaths in 2010 . Although uterine cancer is found mainly in the older patient population, up to 14% of uterine cancers are found in women younger than 45 years of age . Most studies have demonstrated that younger women have a lower risk of death from uterine cancer than older women independent of stage at diagnosis [4, 5]. Surgical management of uterine cancer entails a staging procedure which includes a total hysterectomy, salpingooophorectomy, and possible lymph node dissection depending on the spread of the disease and histological type . A high index of suspicion must be maintained if the diagnosis of uterine cancer is to be made in the young patient . Young women with or without risk factors may present with abnormalities in their menstrual periods, which can be initially treated with hormonal therapy, thereby delaying the diagnosis of cancer . Some risk factors for uterine malignancy include prolonged exposure to unopposed estrogen either endogenous, (e.g., early menarche, late menopause, nulliparity, polycystic ovarian syndrome, obesity), or exogenous (e.g., estrogen therapy or hormone replacement without progesterone). Uterine cancer in young women is often associated with early - stage disease, well - differentiated tumors, and a good prognosis [68]. The actual care delivered to women diagnosed with uterine cancer appears to depend upon a number of variables including surgeon training, hospital volume, extent of disease, patient characteristics, and physician access . These women are mostly managed by gynecologic oncologists as well as by nongynecologic oncologists (general gynecologists and general surgeons). Gynecologic oncologists are specifically trained to perform the required staging and cytoreductive surgical procedures for uterine cancer . However, patterns of care studies have shown that gynecologic oncologists provided care in 48.8% of the patients and gynecologists in 50.2% of the cases . Roland et al . Have shown that the gynecologic oncologist completed the surgical staging two times more frequently than the general gynecologist (94.0% versus 45.2%, p they concluded that women with uterine cancer managed by gynecologic oncologists are more likely to receive comprehensive surgical staging resulting in an efficient use of health care resources and minimizing the potential morbidity associated with adjuvant radiation therapy . Numerous studies have evaluated the association of surgeon case volume with clinical outcomes for various procedures and have shown that higher surgeon volume is associated with improved outcomes [1013]. While specialty training is critically important to quality cancer care, recent attention has also focused on the positive relationship between surgeon and hospital case volume and clinical outcomes for malignancies treated with technically complex surgical procedures . Recently, there has been an interest in elucidating patterns of care for patients treated with uterine cancer . Most studies evaluating age - based outcomes for uterine cancer have been single institution analysis containing only a small portion of patients . More specifically, prior studies have not evaluated whether care provided by high - volume surgeons at high - volume centers leads to differences in resource allocation and short - term survival among younger patients . Therefore, the goals of this study were to evaluate volume - based care for women with uterine cancer aged 50 years and younger using a statewide population database . Approval to conduct this study was obtained from the johns hopkins medical institutions clinical research committee and joint committee on clinical investigation, and the requirements for informed patient consent were waived . The study design was a cross - sectional analysis of hospital discharge data from the nonfederal acute care hospitals in maryland collected by the maryland health services cost review committee (hscrc). The hscrc database provides information regarding the index hospital admissions and is limited to 30 days of followup . The main study objective was to characterize volume - based care among women 50 years when compared to women> 50 years . A subgroup analysis among women 50 years with regards to surgeon and hospital uterine cancer volume was performed to evaluate the risk of in - hospital related death and associations with the length of hospital stay, hospital - related costs, and icu length of stay . All adult female patients, 18 years of age and older, who underwent a surgical procedure including a hysterectomy for a malignant uterine neoplasm in maryland between january 1, 1994 until december 31, 2005 were included in the study . The international classification of disease, 9th revision (icd-9) code 182.0 (malignant uterine neoplasm) was used for sorting . The surgical procedures included in the analysis were limited to those incorporating hysterectomy as this was felt to be the most likely to capture those patients undergoing initial surgery for uterine cancer . Patient age was modeled as a categorical variable (patients 50 years of age and younger compared to patients over 50 years of age). This age cutoff was designated based on the average age of menopause as 51.4 years [15, 16]. Frequency distribution for variables like ethnicity, insurance payer status, hospital volume, surgeon volume, hospital type, inpatient death, and concordance between attending and operating physician was tabulated among the two groups . Information regarding international federation of gynecology and obstetrics (figo) or american joint committee on cancer (ajcc) stage of disease, tumor grade, histological subtype, extent of disease, or residual disease was not available from the hscrc database . In addition, the hscrc database only provides clinical information for the index hospital admission, so that data on clinical outcomes beyond this time period could not be assessed . Based on previous research in volume - based care in uterine cancer, surgeons performing 100 cases of uterine cancer per study period were categorized as high volume, and those performing <100 cases of uterine cancer per study period as low volume . Surgeons were included in the analysis if they performed at least one uterine cancer surgery during the entire study period . Based on previous research in volume - based care in uterine cancer, hospitals with 200 cases of uterine cancer per study period were categorized as high volume, while those with <200 cases of uterine cancer per study period were categorized as low volume . Similarly, hospitals were included in the analysis if at least one uterine cancer surgery was performed during the entire study period . A community teaching hospital was defined as a nonuniversity hospital with a residency program in obstetrics and gynecology . Hospital - related charges for each index admission were converted to the organizational cost of providing care using cost to charge ratios for individual hospitals . Cost to charge ratios were calculated from data from the health services cost review commission by dividing the average inpatient expense by the average inpatient revenue of each hospital during each year of the study interval . This ratio was then multiplied by each patients' charge to obtain the cost per admission . T - test for simple linear regression was employed to evaluate adjusted cost of hospital - related care, intensive care unit length of stay, and admission length of stay among the two age groups . T - test for simple linear regression was done for the subset group of women aged 50 years and younger to evaluate the variables of adjusted cost of hospital - related care, admission length of stay, and intensive care unit length of stay among hospital's and surgeon's volume . All statistical computations were performed using the sas system, and all reported p values are two sided . A total of 6,181 women who met the criteria for a primary surgical procedure for a malignant uterine neoplasm were identified in the state of maryland during the period of 19942005 . Women aged 50 years and younger comprised 13.6% (n = 844) of the cases . Most of the women aged 50 years and younger were white (78.0%), had insurance coverage through an hmo (40.3%), and were treated in a community - based hospital (67.4%) (table 1). Young women were more likely than older women to have concordance between the operating physician and the attending physician (p = 0.03) (table 1). In other words, young women were more likely to have an operating surgeon that was the same clinician of record . Younger women were more likely to have shorter length of hospitalization stay (p <0.0001) and lower adjusted cost of hospital - related care (p = 0.0002). There were no significant differences between the lengths of intensive care stay between groups (table 2). Four of the reported deaths occur among the group of women 50 years of age and younger . In total, 894 different surgeons provided primary uterine cancer surgical care, although not all surgeons provided continuous care for the entire duration of the study period . Only 9 (1.0%) of the surgeons were categorized as high - volume surgeons . Women aged 50 years and younger were less likely to be managed by high - volume surgeons when compared to women older than 50 years (31.6% versus 35.1%, resp ., p = 0.02). In the subgroup of women aged 50 years and younger, those that were managed by high - volume surgeons were more likely to have longer hospitalization stays (p = 0.003) and higher adjusted cost of hospital - related care (p <0.0001) (table 3). No difference was observed in terms of icu length of stay between high- and low - volume surgeons . No deaths were reported among high - volume surgeons during the immediate 30-day period . Due to the lack of events on one of the groups, a statistical comparison of death rates between high- and low - volume surgeons could not be performed . A total of 49 hospitals provided care for uterine cancer patients during the study period . Only 8 (16.3%) hospitals meet the criteria of high - volume hospitals . No difference was observed among groups in terms of access to high - volume centers (52.0% versus 54.0%, p = 0.22). In the subgroup of women 50 years of age and younger, those admitted to a high - volume hospital were more likely to have higher adjusted cost of hospital - related care (p = 0.01) (table 4). No deaths were reported among high - volume hospitals during the immediate 30-day period . Due to the lack of events on one of the groups, a statistical comparison of death rates between high- and low - volume surgeons could not be performed . Several studies have demonstrated that age is an independent predictor of overall survival [4, 5]. Our analysis of the hscrc database from 19942005 confirms that younger women have lower short - term mortality than older women diagnosed with uterine cancer . The fact that younger women have better outcomes can be explained by a number of hypotheses . It may be that younger individuals have a better health profile, with less morbidity and concurrent medical problems . Younger body / tissues may respond to a surgical insult with less postoperative complications and accelerated healing . Also, uterine cancer in young women is often associated with early - stage disease, well - differentiated tumors, and a good prognosis [68]. Even though the lack of events did not allow performing a statistical analysis, no in - hospital related deaths were reported among the young group of women treated by high - volume surgeons or at high - volume centers . In addition to reduced mortality, lengths of hospital stay and hospital costs for younger women with uterine cancer were lower . The reduced rate of medical comorbidities at baseline in younger women likely contributed to this difference as preoperative comorbidities are an independent predictor of length of postoperative hospitalization and therefore cost . Younger women were more likely to be managed at university - based hospitals and more likely to have an operating surgeon that was the same attending physician of record decreasing the opportunity for miscommunication and repetition of testing that could occur when multiple providers are involved in patient care . Rogers and curtis had shown that continuity of care leads to decreased hospital admissions, decreased length of stay, reduced duplication of diagnostic testing, increased patient satisfaction, and improved compliance . The high level of concordance in the younger women may account for the improved outcomes such as the decreased cost and shorter length of stay . The analysis suggests that younger women were less likely to be managed by high - volume surgeons . For women a perceived lower risk of cancer risk or mortality, limited financial resources, unwillingness to travel long distances for care, and the absence of medical comorbidities may lead fewer younger women to seek out the expertise of a high - volume specialist on their own . Several studies have shown the benefit of women that have been cared for by a gynecologic oncologist [9, 24, 25]. Although the overall peri - operative outcomes were similar, the subspecialists were more likely to perform comprehensive surgical staging, if necessary, and the patient was less likely to receive adjuvant radiotherapy . The overall survival favored those patients managed by the general gynecologist, with no difference in disease - free interval . This may reflect the patient population that is usually managed by the general gynecologist, as compared to the gynecologic oncologist . Similar to the differences in costs of care between younger and older women, younger women treated at low - volume settings had lower costs of care and shorter hospital stays . The reduced rate of medical comorbidities of women treated at these settings is likely to contribute to these cost differences for the same reasons as mentioned previously . The hscrc database cannot be queried to clarify whether high - volume surgeons are located exclusively at high - volume hospitals; therefore it is unclear whether higher costs are impacted by more frequent care by gynecologic oncologists . Low - volume hospital costs may also be reduced due to the different resources of these facilities . Surgeons may be less inclined to perform aggressive procedures if these low - volume facilities do not have adequate support (i.e., access to consultants or high - acuity icu care) and thus refer complex patients to high - volume centers . Improved efficiency at low - volume hospitals cannot be excluded as an alternative cause for lower costs . The strength of this study lays in the relatively large number of patients included form institutions across the state of maryland . Unfortunately, the maryland hscrc database is limited in ways typical of large population - based studies as it does not provide followup beyond 30 days from the index admission and contains no information on either ajcc or figo stage of disease, tumor grade, or histological subtype . Given these restrictions, a meaningful analysis of long - term surgical outcome was not practical from the available data . Also, the hscrc does not provide information in terms of performance status, asa scores, and comorbidities . These factors may have had an impact on the overall morbidity and mortality between the two populations studied . Furthermore, additional information regarding the baseline medical condition of the populations was not examined, and this could be a confounding factor on the results . A second limitation is that there is a possibility that the grouping by case volume could be too selective and there could be subgroups in the lower - volume level that still are able to achieve excellent outcomes . Due to the lack of previous reports of case volume among uterine cancer, a third limitation of the current study is that our selected volume criteria have not been widely validated as outcomes measures . Despite these limitations, the current study provides the most extensive evaluation on volume - based care among young women that underwent surgery for uterine cancer . The information obtained in this study increases our current knowledge of volume - based care of young women diagnosed with uterine cancer and may be useful in developing strategic planning in order to improve the care offered to this population.
In iden 2012, many diverse experiences associated with basic procedures to advanced techniques of endoscopic retrograde cholangiopancreatography (ercp) were highlighted in great enthusiastic lectures by world renowned experts . Interesting cases entitled " interesting cases in pancreatobiliary endoscopy " were introduced in video forum . In this highlight summary of interesting presentations, i will present a summarized review about basic procedures of ercp like balloon dilation for common bile duct (cbd) stones, advanced techniques like endoscopic necrosectomy for necrotizing pancreatitis, recently proposed management for the prevention of post - ercp pancreatitis, and spyglass direct visualization system through which we can directly observe the cbd and pancreatic duct . Eplbd is easy to use and effective for the removal of common bile duct (cbd) stones, but still debates exist on safety issue . Since eplbd requires only a small endoscopic sphincterotomy (est) or none at other occasions, eplbd is generally believed to avoid the complications of a full est . Lee and han1 conducted a large retrospective multicenter study, in which a total of 946 consecutive patients with large cbd stones were enrolled in this study . Serious adverse events occurred in 95 patients (10%), after which the following guidelines of eplbd in order to pursuit zero mortality were suggested; 1) eplbd should be avoided in patients with distal cbd strictures; 2) full est should be avoided immediately before eplbd; 3) the balloon should be inflated gradually; 4) inflation should be discontinued in cases of persistent balloon waist (75% rule); 5) not to be inflated beyond the maximum diameter of the dilated cbd; and 6) convert to an alternative stone removal or drainage method any time there is difficulty in removing the stone . Because it was multicenter study analyzing the safety of eplbd based on large numbers of cases, experienced experts will agree with the above referred recommendations, though it was studied retrospectively . Kahaleh and freeman2 recently made recommendations to minimize risks of ercp in international digestive endoscopy network (iden) 2012 as follows; adequate selection of patients undergoing ercp, skilled operators using novel techniques for prompt identification, which is key to successful prevention and management . Pancreatitis is the most common complication associated with ercp procedure with average rate of about 5% . Risk factors for the occurrence of post - ercp pancreatitis include younger age, indication of suspected sphincter of oddi dysfunction, history of previous post - ercp pancreatitis, absence of elevated serum bilirubin levels, and female sex are usually at increased risk . Techniquerelated issues have long been recognized to be important in causing post - ercp pancreatitis . He also mentioned about specific techniques to reduce risk of post - ercp pancreatitis, such as pancreatic stents, more promising pharmacological agents including non - steroidal anti - inflammatory drug, and enema for prevention of post - ercp pancreatitis . New possibility that eus - guided endoscopic pancreaticobiliary drainage can replace the percutaneous techniques and obviate surgery was shown in iden 2012 . Kahaleh3 referred that endoscopic debridement and stent insertion can reduce high morbidity and mortality of surgery in severe necrotizing pancreatitis . Endoscopic necrosectomy using repeats session of debridement and plastic stents insertion has been more frequently used within the last decade and half . Fully covered self - expandable metal stents might provide a safer and more efficient drainage through a larger diameter stent . Additionally he described techniques of transmural drainage and endoscopic debridement, how to make the transenteric access into the pancreatic necrosis, how to make active endoscopic irrigation with a gastroscope and debridement of cystic contents using biopsy forceps, and roth nets and polypectomy snares in detail . This technique is evolving continuously as we attempt to optimize the post - procedural outcomes . There were attractive video lectures during iden 2012 dealing with advanced techniques for pancreaticobiliary visualization including direct peroral cholangioscopy, spyglass direct visualization system, forward - viewing echoendoscopy, and contrast - enhanced eus and elastography accompanied with actual interesting cases for each . Kahaleh4 mentioned that the single operator cholangioscopy (soc) system (spyglass; direct visualization system, natick, ma, usa) may offer an interesting compromise in terms of size (10 fr diameter) and complexity of use . Soc is challenged by the size of the biopsy obtained and the stiffness of the forceps (spybite; boston scientific, natick, ma, usa) fitting within the working channel of the system . Sensitivity of forceps biopsy through the cholangioscope was far higher for intrinsic (66%) than extrinsic (8%) malignant lesions . Conclusively, spyglass (direct visualization system) has not only been used as a platform for advanced intraductal imaging with probe based endomicroscopy and spyglass - guided stone fragmentation; but also for photodynamic therapy to treat bile duct cancer . Soc can be a great step to realize intraductal visualization as well as therapy, but the best is yet to come . Pancreatobiliary endoscopy is continuously evolving area with new techniques and we attempt to optimize the postprocedural outcomes.
Parvovirus b19 is a small, uncoated dna virus belonging to the family parvoviridae (1). Parvovirus b19 associates with a wide range of clinical symptoms and their pattern is under effect of age and immune status of the host (4). Most of b19 infections are asymptomatic or only associate with mild non - specific illness . This virus can also be transmitted through blood transfusion and blood products (5). B19 infects humans widely and is almost endemic in all parts of the world (2). Parvovirus b19 can also be transmitted through vertical transformation (from mother to fetus) (6). Its prevalence in pregnancy is about 15%, but in epidemic situation it receives to 10% (78). Its incidence in pregnancy is about 15% and it can cause complications in 3% of infected pregnant women (9, 10). The virus is transmitted through vertical transmission to the fetus and creates some complications to fetus including anemia, spontaneous abortion, hydrops fetalis, intrauterine fetal death and congenital anomalies (11). Parvovirus infection of mothers is diagnosed using serologic or an immune assay enzyme b19 igm and b19 igg (12). B19 igg positive serologic evidence of previous infection with the virus that leads to lifelong immunity . In this study, we tried to determine prevalence of serum parvovirus infection in pregnant women and its association with some of the parameters such as number of lived children, number of family members, number of commensalts and amount of hemoglobin . The risk of adverse fetal outcome increases if maternal infection occurs during the first two trimesters of pregnancy but may also happen during the third trimester . It is significant cause of fetal loss throughout pregnancy, but has a higher impact in the second half of pregnancy when spontaneous fetal loss from other causes is relatively rare (13). For example, the risk of infection in pregnant women with one child are 3 times more than nulliparous women, but this risk for women with three or more children are 7.5 times more . The other risk factors are working in the school, care centers and other full stress jobs (1416). Igg antibody levels elevated with the increase of age (17, 18) at the age of 15, about 50% of persons have detectable igg levels and its amount in older people, has increased to more than 90% (2). The importance of b19 investigation in pregnant women is due to vertical transmission of this virus . The complications related to this virus are anemia, hydrops fetalis, fetal death, and unintended abortion (11). Intrauterine growth retardation, myocarditis, pleural effusion, pericardial effusion and brain involvement of the fetus may occur following infection with the virus . Although, parvovirus b19 is not related to congenital malformations (19, 20), in case of pregnant women infection, the prevalence of transmision to the fetus is 30% (21). Acute parvovirus b19 (b19v) infection is a proven risk for pregnant women and their fetus (22). Regarding to complications related to b19 virus infection during pregnancy and lack of study about its infection prevalence in ardabil county, this study was conducted with the aim of determination of the prevalence of parvovirus and igg in pregnant women in ardabil . The numbers of samples were calculated 350, based on statistical formula for sample size . These numbers were divided into 39 prenatal care centers, according to population covered by each center . 5 ml of blood was taken from participant in care centers and was referred to the reference laboratory, immediately . The serum of blood samples were isolated and were stored at 20 c, until analysis . The questionnaire consisted of two parts: part i including mother s age, location, number of family members (father, mother, sister, brother), number of children, number of commensalts, and in part ii information about blood test of pregnant women, were recorded . For measurement of igg antibodies in serum samples against b19 virus, the elisa kit euroimmune, (germany) was used as follows: 100 l of serum 1/201 diluted patients and 100 l of each of the standards were added to micro plate wells in which the viral antigen was coated . The samples were incubated for 1 h at room temperature, then the wells were washed and 100 l goat, anti - human, anti - igg antibody binded to the peroxide was added to each sample and they kept at room temperature for one more hour . Then, after washing the wells, 100 l of substrate (3, 3, 5, 5 tetra - methyl benzidine) was added to each well and allowed to create color within 30 minutes at room temperature . Subsequently, 100 l of fixing solution (sulfuric acid) was added to each well and a maximum light absorption of the double wavelengths of 450/630 nm were read using a plate reader . The chi - square and t - test and descriptive methods were used for data analysis . The significant levels of 0.05 were used for all statistical analysis . In order to consider the principles of medical ethics, in this study, of 350 pregnant women, 64.6% (226/350) were ardabil citizen and the rest were from rural area (124/350). The youngest participant was 15 years old and the oldest was 34 years (average of 23 3.92 years). Overall, 242 (69.1%) had positive serology b19 igg and 124 (30.9%) were seronegative . Distributions of seroprevalence of parovirus b19 among pregnant women in residence location are shown in table 1 . Of 108 people that living in rural areas, 85 (68.5%) were seropositive and 39 (31.5%) were negative . There was no significant relationship between residence location and prevalence of serum parvovirus b19 (p=0.475). Distribution of serology parvovirus b19 prevalence among pregnant women based on residence location the average age of participants with positive serology was 24.774.26 years, and the mean age of those with negative serology was 22.983.58 years . Result showed there was a significant correlation between age and the prevalence of parvovirus (r=0.268) and the immunity against the virus was higher in older pregnant women (table 2). Distribution of serology parvovirus b19 prevalence among pregnant women according to age group the average number commensalts seropositive pregnant women was 3 1.74 and those with negative serology 2.83 1.16 that showed no significant relationship between the number of commensalts and prevalence of parvovirus (p=0.377). The average family members of participants with positive serology was 6.86 2.4 and those with negative serology was 6.61 2.23, which showed no significant relation between family size and prevalence of parvovirus (p=0.369) (table 3). Relationship between the number of people breaking bread together, family size and the number of children living with an outbreak of parvovirus b19 the average number of lived children in participants those with positive serology was 0.57 0.655 and average number of lived children in those with negative serology was 0.51 0.634 which presented the relationship between family size and sero prevalence of parvovirus was insignificant (p= 0.417). The average amount of hemoglobin in participants with positive serology was 12.51.12 (p=0.177) and the average amount of hemoglobin in those with negative serology was 12.261.06 (p=0.169) which showed no significant relationship between hemoglobin and serum parvovirus . Results of this study showed that the prevalence of b19 in pregnant women in ardabil was 69.1% . (2005) in shiraz showed that parvovirus infection providence was 69.01% (17). In a cross - sectional study, sohrabi et al . (2007) reported that 55.7% of pregnant women referred to ahvaz imam khomeini hospital were b19 positive (had specific igg of b19). According to their findings, more than 40% of pregnant women had not igg b19 and were at the risk of the virus infection and its fetus complications (23). In a study conducted in nigeria by emiasegen et al . (2011) parvovirus infection prevalence among pregnant women attending to prenatal care clinics and its relation to occupation, number of children and transfusion history were studied . They reported that 273 pregnant women were studied and 27.5% of them had igg antibody for b19 and the relationship between the numbers of living children, occupation and achieved transfusion history were significant (24). In another study, elnifro (2009) found that its prevalence in libya was 69% (25). The other hand, khameneh (2014) reported that in orumiyeh prevalence of parvovirus was 75.6% (26), that was further from the results of our study . For example, sohrabi in a study in ahvaz showed that its incidence was 55.7% (19); abiodun (2013) reported 20% in nigeria (27), and cohen (1995) declared 53% of pregnant women are immune to the virus (28). The difference between rural and urban areas in the prevalence of b19 virus was not significant . Of 226 people lived in urban area, 157 patients (69.5%) were positive while 68.5% of people (85 of 108) lived in rural areas were positive in serology . The significance of difference among mean age between positive serology and negative serology indicates that relationship between age and the prevalence of parvovirus . Therefore, its incidence was higher in older women . In the ziyaeyan (2005) and sohrabi (2007) studies, there was no significant correlation between age and the prevalence of parvovirus, that is contrast to our results (17,23). In this study, the relations between seropositive incidences with number of children, number of family members, number of commensalts, and hemoglobin content were not significant . (2001) (29). In a study from nigeria, emiasegen et al . (2011) showed that the number of children was related to the outbreak of parvovirus (24) that is not consistent with the results of our study . Comparison between the average number of sero - positive children and seronegatives revealed that the number of children did nt related to the prevalence of parvovirus that is in accordance with the shahraki et al . The relation between the number of children and outbreak of parvovirus was significant (24) that is not consistent with our results . The results of this study showed a high susceptibility of pregnant women to parvovirus b19 in ardabil region and immunity to the virus increases with age . In our study, a significant correlation was not found between the prevalence of parvovirus and the number of living children, number of family member, number of commensalts and hemoglobin content . Considering that a high percentage of infected pregnant women with parvovirus b19 in the ardebil region, health education and screening for the virus, especially in pregnant women with anemia is recommended to prevent fetal complications.
There is both growing recognition and scientific evidence that interventions of physical activity and exercise programs (paeps) have a favorable impact on patients with psychiatric disorders.17 these findings have been reported among groups of patients with major depressive disorders (mdds),6,8,9 schizophrenia,10,11 post - traumatic stress disorder (ptsd),12,13 alcohol abuse,14,15 sleep disorders,16 or autism spectrum disorders.17 the neurophysiological mechanisms underlying the beneficial effects of pa on psychological functioning and wellbeing are complex, although there is increasing evidence that regular, acute, and chronic pa does have an impact at the molecular level18,19 and on brain plasticity.1,20,21 in parallel, psychological mechanisms are involved; specifically, regular pa has a favorable influence of self - esteem among patients with psychiatric disorders,22 on body image and physical self - concept,23 and, most importantly, on dimensions of anxiety and depression.6,9,24 however, it is widely reported that patients with psychiatric disorders are less physically active and do exercise less than healthy peers,2527 potentially increasing and maintaining the risk for further physical and mental health problems . Among those seeking treatment, psychiatric hospitals provide an excellent environment to initiate physical activity, as they offer supervised free training during a phase in which patients are under few time constraints . In view of the potential of paeps as adjunct treatment reported in the literature to date, our aim in this study was to explore the extent to which these programs are implemented in the inpatient treatment of psychiatric disorders in the german - speaking part of switzerland . An understanding of the implementation of paeps in practice, and the experiences of program staff, is an important factor for the future translation of research findings to clinical practice . During spring 2012, all psychiatric hospitals of the german - speaking part of switzerland (n=55) were identified via publicly accessible lists on the internet . To be enrolled in the study, the minimum requirement was that psychiatric hospitals treated not only outpatients but also inpatients and that the official language was german; that is to say, staff and inpatients communicate in german or swiss german . Next, nb contacted all identified hospitals by phone calls and asked for a brief talk with the staff member responsible for the paeps . The aim of the phone call and the study were explained, and, if agreed, the url of an online survey was transmitted via email . All participants were informed that participation was voluntary and that all data would be gathered anonymously; written informed consent was obtained on the first page of the questionnaire . Furthermore, they were informed that they could withdraw from the study at any time . To improve participation, participants could take part in a prize draw (ten vouchers of chf 30.00 [approximately usd 30.00] from a known national supermarket), although to do so they needed to provide an email address . Ethical approval was not required by the institutional review board of the university of basel because the survey was not addressed to patients or underage people . Data collection was done online, as this was judged to be both time saving for participants and a reliable method to gather data.28 we used commercially available software (unipark; http://www.unipark.de), as this is an easily applied tool for the creation of internet - based studies . Furthermore, the software provider guarantees that all data are stored on a server that is not accessible to any third party . Importantly, the user of the software receives the raw data of the answers but not participants ip address, ensuring both data security and participants anonymity . Moreover, the online survey was designed to avoid repeated participation (ip addresses already used are automatically blocked). Participants needed ~1520 minutes to complete the survey . A questionnaire with multiple choice response options was developed expressly for this study, in order to address the specific areas of inquiry . The findings are based on descriptive statistics such as mean, standard deviation, median, or percentage . Furthermore, chi - square tests were performed to calculate associations between dimensions as specified in more detail in the text . All statistical analyses were performed with spss 22.0 (ibm corporation, armonk, ny, usa) for apple mac . During spring 2012, all psychiatric hospitals of the german - speaking part of switzerland (n=55) were identified via publicly accessible lists on the internet . To be enrolled in the study, the minimum requirement was that psychiatric hospitals treated not only outpatients but also inpatients and that the official language was german; that is to say, staff and inpatients communicate in german or swiss german . Next, nb contacted all identified hospitals by phone calls and asked for a brief talk with the staff member responsible for the paeps . The aim of the phone call and the study were explained, and, if agreed, the url of an online survey was transmitted via email . All participants were informed that participation was voluntary and that all data would be gathered anonymously; written informed consent was obtained on the first page of the questionnaire . Furthermore, they were informed that they could withdraw from the study at any time . To improve participation, participants could take part in a prize draw (ten vouchers of chf 30.00 [approximately usd 30.00] from a known national supermarket), although to do so they needed to provide an email address . Ethical approval was not required by the institutional review board of the university of basel because the survey was not addressed to patients or underage people . Data collection was done online, as this was judged to be both time saving for participants and a reliable method to gather data.28 we used commercially available software (unipark; http://www.unipark.de), as this is an easily applied tool for the creation of internet - based studies . Furthermore, the software provider guarantees that all data are stored on a server that is not accessible to any third party . Importantly, the user of the software receives the raw data of the answers but not participants ip address, ensuring both data security and participants anonymity . Moreover, the online survey was designed to avoid repeated participation (ip addresses already used are automatically blocked). A questionnaire with multiple choice response options was developed expressly for this study, in order to address the specific areas of inquiry . The findings are based on descriptive statistics such as mean, standard deviation, median, or percentage . Furthermore, chi - square tests were performed to calculate associations between dimensions as specified in more detail in the text . All statistical analyses were performed with spss 22.0 (ibm corporation, armonk, ny, usa) for apple mac . Of the 55 psychiatric hospitals in german - speaking switzerland approached, staff members of 48 (87.3%) took part in the survey . In all, 42 respondents (87.5%) were physiotherapists, three (6.25%) had a master s degree in sports science, and three (6.25%) did not answer to this item . Of the 48 respondents, 32 (66%) were working in a general psychiatric hospital, eight (17%) were working in a university psychiatric hospital, and eight (17%) were working in psychiatric wards of a somatic hospital . Twenty (43%) hospitals had> 200 beds for inpatients, nine (19%) hospitals had 99200 beds, 15 (32%) had between 50 and 99 beds, and three hospitals reported <50 beds for psychiatric inpatients . Seventeen (35.5%) psychiatric hospitals were located in the canton berne, eight (17%) were located in the canton zrich, five (10%) in the canton thurgau, four (8%) each in the cantons st gallen and aargau, three (6%) each in basel city and in aarau (city and close surroundings), two (4%) each in the cantons basel country and grisons, and one (2%) each in the cantons of appenzell - ausserrhoden and zug . Regarding the disorders most frequently treated at the hospital, 80% of respondents reported personality disorders and mood disorders, ~78% reported anxiety, stress - related, and somatoform disorders, ~65% reported substance use disorders, 55% reported schizophrenia and nonmood psychotic disorders, 50% reported mental disorders due to existing physiological conditions, and 5%15% reported intellectual disabilities, pervasive and specific developmental disorders, behavioral and emotional disorders, and unspecific mental disorders . A chi - square test revealed that there was no association between most frequently treated disorders and the number of beds of the hospital ((n=48, df=27)=0.45; p=0.67). As depicted in figure 1, all psychiatric hospitals (100%) offered relaxation techniques, and 47 (97%) offered sports therapy (fitness, swimming, ball sports, and endurance training), 46 (96%) offered general physical activity, 40 (85%) offered physiotherapy / remedial gymnastics, 28 (59%) offered so called body therapies such as feldenkrais or bioenergetics, 27 (57%) offered far - east techniques (tai chi and qigong), and ten (22%) offered hippotherapy . Therefore, the majority of hospitals offered more than one type of paep content . As regards the frequency with which paeps took place, responses ranged from once per week to five times per week . Factors that were reported as limiting the frequency of sessions were staff availability, sports hall and/or equipment availability, and the size of the hospital: hospitals with a lower number of beds offered fewer and less frequent paeps ((n=48, df=18) = 16.45; p<0.05). As regards the group size of participants, a range of one to seven participants was the most frequently reported group size . One - to - one sessions were chiefly offered as personal training or for therapeutic interventions such as physiotherapy or other body therapies . Of the 48 participants, 40 (83.3%) answered that <25% of all inpatients participated in the paeps, and eight (16.7%) estimated that between 25% and 50% of all inpatients participated in the paeps (figure 2). All paeps were attended on a voluntary basis (100%), although 40 (83.3%) respondents emphasized that patients were made aware of the programs and encouraged to attend the paeps . Furthermore, participants reported that patients seemed generally moderately to rather highly motivated to participate in the paeps . Thirty - six (75%) participants reported that the availability and equipment of the current paep infrastructure were sufficient, five (10.4%) reported that the infrastructure was well to very well equipped, whereas seven (14%) reported that the availability and equipment of the infrastructure were not or not at all sufficient . All participants responded that in their view, paeps improved patients physical and psychological status . Regarding the hypothesized importance of the programs, answers ranged from a little importance (n=8; 16.6%), to moderate importance (n=38; 79%); two (4.16%) answered that the importance of paeps was large (figure 3). All participants reported that after discharge, their patients no longer had access to the paeps offered as part of inpatient treatment . Thirty - five (72.9%) answered that the main goal of a paep was to improve patients psychological state, 45 (93.75%) answered that a further main goal was to improve patients social skills, and 17 (35.42%) answered that a further main goal was to improve patients physical status . None of the participants answered that a paep was aimed at improving patients coping strategies (note that multiple answers were possible; figure 4). No associations between the main goals of paeps and the most frequently treated disorders were observed ((n=48; df=27) = 0.45; p=0.78). All participants answered that they rely on their own experience and vocational training to plan and organize the paep sessions . Furthermore, the majority (n=40, 83.3%) also reported benefiting from the experiences and advice of their peers . No respondent stated that they sought or obtained information from specific internet sites or scientific publications . All participants answered that paeps were an integrated treatment modality of the hospital . In all, 38 (79.16%) reported that they were particularly motivated to lead paeps and 30 (62.5%) reported that the hospital s medical staff recognized the beneficial effects of paeps . Nobody answered that paeps were offered because supplementary vocational training was available, because the hospital had the existing infrastructure, or because of scientific evidence supporting the benefits of such programs . All participants reported that the main treatment regimen of the hospital comprised medication (100%), psychotherapy (100%), and occupational therapy (100%). Thirty (62.5%) answered that within the main treatment regimen, the potential benefits of paeps was fairly well accepted; ten (20.83%) answered that it was well accepted, four (8.3%) answered that it was not well accepted, and four (8.3%) did not know . All participants reported that in order to improve the paeps offered by the hospital, staff would require advanced vocational training in sport and exercise sciences and advanced vocational training in adapted physical activity for psychiatric patients . Of the 55 psychiatric hospitals in german - speaking switzerland approached, staff members of 48 (87.3%) took part in the survey . In all, 42 respondents (87.5%) were physiotherapists, three (6.25%) had a master s degree in sports science, and three (6.25%) did not answer to this item . Of the 48 respondents, 32 (66%) were working in a general psychiatric hospital, eight (17%) were working in a university psychiatric hospital, and eight (17%) were working in psychiatric wards of a somatic hospital . Twenty (43%) hospitals had> 200 beds for inpatients, nine (19%) hospitals had 99200 beds, 15 (32%) had between 50 and 99 beds, and three hospitals reported <50 beds for psychiatric inpatients . Seventeen (35.5%) psychiatric hospitals were located in the canton berne, eight (17%) were located in the canton zrich, five (10%) in the canton thurgau, four (8%) each in the cantons st gallen and aargau, three (6%) each in basel city and in aarau (city and close surroundings), two (4%) each in the cantons basel country and grisons, and one (2%) each in the cantons of appenzell - ausserrhoden and zug . Regarding the disorders most frequently treated at the hospital, 80% of respondents reported personality disorders and mood disorders, ~78% reported anxiety, stress - related, and somatoform disorders, ~65% reported substance use disorders, 55% reported schizophrenia and nonmood psychotic disorders, 50% reported mental disorders due to existing physiological conditions, and 5%15% reported intellectual disabilities, pervasive and specific developmental disorders, behavioral and emotional disorders, and unspecific mental disorders . A chi - square test revealed that there was no association between most frequently treated disorders and the number of beds of the hospital ((n=48, df=27)=0.45; p=0.67). As depicted in figure 1, all psychiatric hospitals (100%) offered relaxation techniques, and 47 (97%) offered sports therapy (fitness, swimming, ball sports, and endurance training), 46 (96%) offered general physical activity, 40 (85%) offered physiotherapy / remedial gymnastics, 28 (59%) offered so called body therapies such as feldenkrais or bioenergetics, 27 (57%) offered far - east techniques (tai chi and qigong), and ten (22%) offered hippotherapy . Therefore, the majority of hospitals offered more than one type of paep content . As regards the frequency with which paeps took place, responses ranged from once per week to five times per week . Factors that were reported as limiting the frequency of sessions were staff availability, sports hall and/or equipment availability, and the size of the hospital: hospitals with a lower number of beds offered fewer and less frequent paeps ((n=48, df=18) = 16.45; p<0.05). As regards the group size of participants, a range of one to seven participants was the most frequently reported group size . One - to - one sessions were chiefly offered as personal training or for therapeutic interventions such as physiotherapy or other body therapies . Of the 48 participants, 40 (83.3%) answered that <25% of all inpatients participated in the paeps, and eight (16.7%) estimated that between 25% and 50% of all inpatients participated in the paeps (figure 2). All paeps were attended on a voluntary basis (100%), although 40 (83.3%) respondents emphasized that patients were made aware of the programs and encouraged to attend the paeps . Furthermore, participants reported that patients seemed generally moderately to rather highly motivated to participate in the paeps . Thirty - six (75%) participants reported that the availability and equipment of the current paep infrastructure were sufficient, five (10.4%) reported that the infrastructure was well to very well equipped, whereas seven (14%) reported that the availability and equipment of the infrastructure were not or not at all sufficient . All participants responded that in their view, paeps improved patients physical and psychological status . Regarding the hypothesized importance of the programs, answers ranged from a little importance (n=8; 16.6%), to moderate importance (n=38; 79%); two (4.16%) answered that the importance of paeps was large (figure 3). All participants reported that after discharge, their patients no longer had access to the paeps offered as part of inpatient treatment . Thirty - five (72.9%) answered that the main goal of a paep was to improve patients psychological state, 45 (93.75%) answered that a further main goal was to improve patients social skills, and 17 (35.42%) answered that a further main goal was to improve patients physical status . None of the participants answered that a paep was aimed at improving patients coping strategies (note that multiple answers were possible; figure 4). No associations between the main goals of paeps and the most frequently treated disorders were observed ((n=48; df=27) = 0.45; p=0.78). All participants answered that they rely on their own experience and vocational training to plan and organize the paep sessions . Furthermore, the majority (n=40, 83.3%) also reported benefiting from the experiences and advice of their peers . No respondent stated that they sought or obtained information from specific internet sites or scientific publications . All participants answered that paeps were an integrated treatment modality of the hospital . In all, 38 (79.16%) reported that they were particularly motivated to lead paeps and 30 (62.5%) reported that the hospital s medical staff recognized the beneficial effects of paeps . Nobody answered that paeps were offered because supplementary vocational training was available, because the hospital had the existing infrastructure, or because of scientific evidence supporting the benefits of such programs . All participants reported that the main treatment regimen of the hospital comprised medication (100%), psychotherapy (100%), and occupational therapy (100%). Thirty (62.5%) answered that within the main treatment regimen, the potential benefits of paeps was fairly well accepted; ten (20.83%) answered that it was well accepted, four (8.3%) answered that it was not well accepted, and four (8.3%) did not know . All participants reported that in order to improve the paeps offered by the hospital, staff would require advanced vocational training in sport and exercise sciences and advanced vocational training in adapted physical activity for psychiatric patients . The key findings of the present survey are that all psychiatric hospitals of the german - speaking part of switzerland whose staff participated in this survey offer a broad variety of paeps . Furthermore, ~25% of all patients staff involved in these programs also felt that specific extended vocational training would allow them to improve their skills and optimize the existing programs . The present survey is the first to give an overview of the existence and role of paeps in psychiatric hospitals of the german - speaking part of switzerland . Specifically, the pattern of results shows that paeps are well established and integrated in the treatment regimen . All respondents reported that they felt that participation in peaps impacted favorably on patients psychological status . While anecdotal, this observation is in line with the increasing wealth of studies showing a favorable influence of paeps on a broad variety of psychiatric disorders,17 such as mdds,6,8,9 schizophrenia,10,11 ptsd,12,13 alcohol abuse,14,15 sleep disorders,16 or autism spectrum disorders.17 next, in our opinion, a major issue is patients participation rate: 83% reported that only up to 25% of patients participated in the offered peaps, and further 16.7% reported that 25%50% of patients did so . Or simply put: we estimate that 50%75% of inpatients do not attend the peaps, and we claim that this rate of participation is low . The quality of the data does not allow a deeper understanding of patients underlying decisions, although if we combine the result that participating patients seemed moderately to rather highly motivated to participate in the peaps, the key factor of patients (non-) participation may be their motivation . Nevertheless, the low participation rates were not surprising . For instance, studies consistently show that people with psychiatric disorders have lower physical activity29,30 and cardiorespiratory fitness levels.31,32 this is critical, because low physical activity and cardiorespiratory fitness are considered important factors which may link psychiatric and comorbid somatic disorders.33 a recent meta - analysis showed that exercise interventions have the capacity to improve cardiorespiratory fitness in people with mdd in clinically meaningful ways.34 taken together, the use of paep in the treatment of psychiatric disorders is supported by strong empirical evidence.3537 for instance, studies show that among patients with clinical depression, exercise seems to have similar effects to antidepressants and psychotherapy.38 thus, integrating exercise as an add - on to standard care during inpatient treatment, as well as efforts to promote lifestyle physical activity to prevent relapses after the end of hospitalization, seem promising . A major challenge, however, is that it is particularly difficult to initiate and maintain regular exercise among patients with psychiatric disorders, because their psychiatric symptoms interfere with their motivation and capacity to self - regulate health - related behaviors.33,39 given this background, we claim that intervention programs specifically tailored to motivate psychiatric patients to achieve a more physically active lifestyle should be key among psychiatric hospitals . In this regard, from intervention studies on motivational processes among patients with somatic complaints, we learned that specific psychoeducational inputs were able to increase patients motivation to exercise also after discharge.40 similarly, future studies might focus on applying such motivational programs in patients with psychiatric disorders . In this vein, all participants reported that after patients discharge, no further options of aftercare were available . In light of the fact that all respondents felt that paeps were beneficial for patients and comprise activities, which can be protective against psychological complaints, the potential to extend paep offers for patients after the inpatient phase could represent a long - term element of health promotion for this population . Since many psychological disorders are relapsing conditions, long - term interventions must be explored as potentially protective against recurrence . The knowledge that peaps can be extended following the inpatient phase may also be an important motivator for patients to begin with such programs, as they will not have to face a loss of facilities as soon as their treatment ends . Furthermore, several participants reported that an aim of the peaps was to bolster patients social skills . This observation is interesting, as, to the best of our knowledge, research on this particular aspect of functioning is sparse . Data from a finnish study suggest that individuals who reported frequent exercise also felt more socially integrated than their less active peers, while participation in exercise has been reported to both foster social integration and be a means of maintaining existing social contacts . Specific research into the possible mechanisms for this effect, and with clinical populations, is currently lacking . This issue is of particular importance, as compared to adults without psychiatric disorders, patients with psychiatric disorders have a fourfold risk of suffering from social anxiety and adverse social interactions.4145 future research should focus therefore on the extent to which paeps in psychiatric hospitals might favorably impact on patients social skills . Finally, a further result demands particular attention: participants reported wanting and needing extended vocational training, specifically as regards the state - of - the - art of peaps for patients with psychiatric disorders . Accordingly, one might claim that institutions such as departments of sports science, in collaboration with psychiatric institutions, might organize specific extended vocational trainings . The strengths of the present results should be balanced against the limitations of the study . First, the present data do not reflect the current application of paeps in psychiatric hospitals in non - german - speaking swiss areas, as we sought to avoid the risk of methodological issues related to the translation of the items into french, italian, and rhaeto - romanic . Second, only hospitals and participants willing and able to complete the questionnaire took part on the study, and accordingly, results might also be biased . Third, the quality of the data did not allow a deeper understanding of which patients with which psychiatric disorder did participate and not participate in the paeps . Such a distinction would have allowed more insight into the possible relations between patients psychiatric characteristics (their disorders), motivation and (non-)participation in specific peaps . Specifically, one might assume, that for instance, the framework of paeps of patients with addiction disorders is different from that of paeps of patients with anxiety and affective disorders . Fourth, the online survey did not assess patients perception of how useful or detrimental peaps were . Finally, it is questionable to what extent domains such as relaxation techniques, physiotherapy, or far - east (meditation) techniques should be considered as peaps, or if they should be considered and discussed apart . The pattern of results of the present online survey suggests that psychiatric hospitals of the german - speaking part of switzerland offer a broad variety of physical activity and exercising programs to their inpatients, although only 25%50% of inpatients take part in programs . Staff are unanimous in the view that these programs are physically and psychologically beneficial . After discharge staff responsible for paeps state that specific extended vocational training is required to offer activities suited to patients with psychiatric disorders . Finally, given that many psychiatric patients may have motivational and volitional deficits, more systematic efforts seem warranted to increase exercise motivation and promote behavioral skills to maintain a more physically active lifestyle among inpatients in psychiatric care settings.
As the silver tsunami continues through the us and globally, the number of drivers over the age of 75 will increase significantly . As of 2012, there were 36 million older americans licensed to drive, which was a 34% increase from 1999 . Accidents or unintentional injuries are the 7th leading cause of death in persons 65 and older and are often the result of impairment . In the 64 to 75 age group, injuries from motor vehicle accidents (mvas) are the leading cause of injury related death and the second leading cause in the 75 to 84 age group . Drivers who are 80 years old and older have 9 times greater fatality rate than drivers aged 25 to 69 . Assessment of driving capability in the older adult is becoming an essential aspect of the comprehensive geriatric exam, even though many primary care providers (pcps) do not view themselves as experts in this area and may defer driving assessment and capability to the local department of motor vehicles (dmv) office . However, a survey conducted by the american association of retired persons (aarp) showed that 40% of people surveyed aged 50 and older believed that their pcp could best determine their ability to drive safely [5, p. 92]. Earlier assessment and intervention lead to prolonged health maintenance and independence, which is an essential aspect that the pcp must consider for the older adult driver . According to the american association of nurse practitioners (aanp) (2012), 80% of nurse practitioners (nps) practice in a primary care setting . Currently, there are three np graduates to every one primary care physician graduate in the united states (us), and nps are improving access to care, especially in rural areas where alternative transportation methods may not be available . Driver safety is considered a public and personal safety issue and the np has a duty to routinely assess and intervene when appropriate . Variations across state laws and regulations regarding drivers make it challenging to have a nationally accepted clinical guideline or standard of care specific to assessment of safe driving . Driving regulations, laws, and reporting policies vary according to state and many providers are not fully aware of these laws and policies within the state where they practice . In one state, a healthcare provider can be sued for alerting the dmv of a person's medical diagnosis, whereas, in another state, the healthcare provider is negligible if they do not report it . These extremes in variability make it confusing and difficult for nps to know what to do and what to follow . Report a lack of specific guidelines for primary care providers to use when caring for an older adult driver in the office setting . Nps caring for older adults do not have an efficient evidenced - based screening instrument for use during a routine 15-minute office visit . The current guidelines published by the american medical association in collaboration with the national highway traffic safety administration (nhtsa) are considered opinion - based according to one author ([5, 9]). Another source describes the guidelines as comprehensive but lengthy and cumbersome to use during a routine office visit and they are not supported by evidenced - based research . The purpose of this critical incident study was to identify strategies utilized by nps in the assessment and intervention of older adult drivers in southeastern north carolina . The aims of this study were to (1) determine current practice of np's assessment of and intervention strategies with older adult drivers in southeastern north carolina; (2) assess the nps knowledge deficits in assessing safe driving capability of the older adults; and (3) assess nps current knowledge of resources available for older adult drivers and their families . Assessment and intervention of safe driving in the older adult healthcare providers have standards of care or published guidelines to assess, treat, and evaluate chronic diseases such as the jnc guidelines for hypertension . A similar guideline for assessment, intervention, and evaluation of driving safety in the older adult would prove beneficial . The aging baby boomer generation started turning 65 in 2011 and, by 2030, 1 in every 5 persons will be 65 years old or older in the united states . Persons 85 years old and older are growing at four times the rate of the us population . American's average life expectancy has increased at the same time death rates for the 65 to 84 age group have decreased . As our population ages, there is an increased need for specialized geriatric care that includes a comprehensive, holistic approach . Personal safety behaviors, including safe driving habits, are part of the shuler nurse practitioner model . This theoretical framework guides the np to focus on prevention of disease and disability through maintenance of maximal function, improvement in healthcare status, rapidly responding to declines, and periodic routine assessments . The schuler model assesses the physiological and the psychological needs and status of the older adult . Through this holistic approach, the np assesses various areas of functional capability that affect the older adult's ability to drive safely . A literature review was performed using these databases: ebsco host, pubmed, nursing @ ovid, proaquest nursing and allied health, science direct, and web of science . Keywords used were as follows: older adult, elderly, geriatric, driving, safety, assessment, and nurse practitioner . Johnson interviewed 25 nps on their perceptions of their role in assessing driving ability in the older adult driver who lives in an urban region . Johnson found nps to be comfortable performing the physical assessment as it relates to driving ability such as vision, hearing, and cognitive status, but they reported being uncomfortable approaching or discussing the topic of driving safety and/or driving termination . Johnson also concluded that early assessment, discussions, and intervention of safe driving were preferred by the older adult, rather than a discussion of driving termination . Even though this study focused on nps, many other healthcare disciplines such as physicians, social workers, and occupational therapists have a vested interest in driving safety of the older adult . A qualitative research method developed by flanagan, the critical incident technique, was selected as the design for this study . The critical incident technique is a tool used to create a functional description of behavioral activity . According to flanagan, obtaining a description of the activity can be achieved by asking the persons who actually perform the work a series of brief, precise questions aimed at identifying behaviors . Flanagan stressed that, in a critical incident study, the sample size is not determined by the number of participants but rather by the number of critical incidents observed or reported and whether the incidents represent adequate coverage of the activity being studied . The primary objective of the critical incident technique is the development of a behavioral classification system or taxonomy to find solutions to practical problems or to determine the prevalence and distribution of critical behaviors . Critical incident studies have been widely used by health service researchers to identify and categorize behavioral responses of healthcare providers in significant and decisive situations [17, 18]. Critical incident interviews aimed at identifying specific behaviors and strategies used by nps when assessing and intervening with older drivers were conducted with a sample of 21 nps recruited through the north carolina nurses association (ncna) council of nurse practitioners - coastal region . The open - ended self - administered critical incident survey consisted of the following questions: (1) think of the last time you cared for an older adult driver in your practice . What assessment strategies or parameters did you use to determine if the driver was safe or not? (2) what strategies did you use to approach that driver about the issue of driving safety? (3) how did you make the determination to intervene if you felt that the older adult was not safe to drive? Permission was obtained through the institutional review board (irb) at an appropriate institution prior to initiating the study . Data analysis was performed using statistical package for the social sciences (spss, version 20.0) to assess the demographic characteristics of respondents . The narrative data obtained through the critical incident surveys was analyzed through an inductive classification process developed by flanagan . The critical incidents were initially reviewed by an advanced practice nurse with a specialty in gerontology and an expert in using critical incident methodology to determine which incidents should be included in the analysis . Incidents that were judged to be nearly identical or very similar were grouped together to form subcategories of behaviors . The incidents were resorted and discussed by the research team to refine and determine the final set of categories . Percentages of agreement between the researchers were calculated for each critical incident analysis . As an estimate of the importance of each category, the number of incidents sorted by the researchers was counted and placed into a hierarchical structure or taxonomy that identified categories and their related frequencies . A total of 21 nurse practitioners in the southeast region of the united states (us) participated in this study . The predominantly female sample (n = 20, 95%) had a mean age of 48.3 years (sd 11.3 years; range 2968 years). The average length of employment as a nurse practitioner was 9.1 years (sd 8.4 years). The majority of the participants earned master's degree (n = 18, 85.7%). One held bachelor's degree with a family nurse practitioner certificate, one had a doctorate of nursing practice (dnp), and one received ph.d . Approximately one - half of the participants (47.6%) achieved specialty certifications reflecting widely varied areas of advanced nursing practice including: adult, geriatrics, psychiatric mental health, diabetes, oncology, hospice, and wound, ostomy, and continence nursing . None of the study participants had reported attendance at a continuing education program on dealing with older drivers . Ninety - five percent (n = 20) reported that they see older adults (> 65 years) who are still driving at least once a week or more often in clinical practice settings . When asked what age they usually start screening for driving ability, 76% (n = 16) indicated that there was no specific age level . Two participants (10%) reported initiating driver's safety screening between the ages of 50 and 65 years, while three (n = 14%) began screening at the age of 66 years and above . Ninety percent (n = 19) involved family members in discussions about driving safety . Six categories of driver behavior prompted nps to consider performing an evaluation for driving safety . Examples of the circumstances that trigger an assessment of driving capabilities are presented in table 1 . The participants' self - rating of competence in assessing older driver's safety reflected a moderate level of success at 6.6 (sd 1.3, 110 scale with 1 very poor and 10 excellent). Self - ratings of comfort in approaching older adults about their driving safety were higher at 8.0 (sd 2.3, 110 scale). A total of 89 incidents described strategies that nurse practitioners used in assessing an older driver's safety . When the incidents were analyzed, 11 major categories emerged with an interrater agreement of 95% . A listing of assessment categories and circumstances that trigger an assessment of driving capabilities is presented in table 1 . The largest category, assessing diminished short - term memory, forgetfulness, or dementia, accounted for 30 incidents (34%). The use of the mental status exam or the montreal cognitive assessment (moca) was mentioned most frequently . Nurse practitioners shared the following:i ask them about how many times they are getting lost [while driving]. I also ask them how many times do you beep' your horn at other people, and how many times do other people beep their horns at you? That's a big red flag to me . If they can't tell me how they drove here from their house, that's another red flag.i had one patient whose daughter mentioned a concern about driving . I asked her [the patient] to draw a clock [i used the clock drawing test to assess cognition] and this was really abnormal even though the geriatrician had just done a mental status exam 8 months prior . There was a significant change.i [conducted a] mental status exam . The trigger for me was having to repeat the same conversation over and over again to a patient who was asking the same questions over and over again, day to day, and week to week . I ask them about how many times they are getting lost [while driving]. I also ask them how many times do you beep' your horn at other people, and how many times do other people beep their horns at you? That's a big red flag to me . If they can't tell me how they drove here from their house, that's another red flag . I asked her [the patient] to draw a clock [i used the clock drawing test to assess cognition] and this was really abnormal even though the geriatrician had just done a mental status exam 8 months prior . There was a significant change . The trigger for me was having to repeat the same conversation over and over again to a patient who was asking the same questions over and over again, day to day, and week to week . The second largest category, identifying physical frailty, included 21 or 55% of the incidents (n = 21). Specific elements included conducting a fall assessment, assessing [the patient's] ability to turn from side to side, including limited range of motion in the neck, how well they can feel their feet, and assessing whether or not the patient is able to use the dominate foot to control the pedals of the car . Participants added the following.i assess their physical ability on walking by watching them walk in' [to the office] and also look at their ability to get up on the exam table and also their balance.i [look for] patients who describe a poor balance and i assess vertigo or syncope . I also look for people who are losing visual field related to macular degeneration or other visual changes . Most people with this are already limiting their own driving.i observe my patients getting out of and then back into their car . I assess their physical ability on walking by watching them walk in' [to the office] and also look at their ability to get up on the exam table and also their balance . I [look for] patients who describe a poor balance and i assess vertigo or syncope . I also look for people who are losing visual field related to macular degeneration or other visual changes . I observe their gait, walking, balance, flexibility, and overall mobility . Behavioral incidents in the third largest category, assessing changes in sensory impairment, i ask every patient when they had a last eye exam and will [evaluate their vision] with the snellen chart . If a person cannot seem to hear me in a normal conversation, i'll check their ears for impaction and the whisper test . A fourth category, obtaining collateral information from family members, accounted for 11% (n = 10) of the total number of incidents . As one participant stated that a trigger is when an adult child makes a comment such as i won't ride with him' or that they will not allow their grandchildren to ride with the patient . A fifth category, assessing for impulsivity / lack of judgment, accounted for 6% (n = 5) of the total number of incidents, while a sixth category contained incidents that described evaluating medications for drowsiness side - effects (n = 3, 3%). To a far lesser extent, nurse practitioners described retrieving a self - report of impaired driving or motor vehicle accident (n = 2, 2%), evaluating symptoms of new onset neurologic disease (n = 2, 2%), assessing changes in functional status (n = 2, 2%), assessing history of drugs and/or alcohol abuse (n = 1, 1%), and evaluating postmyocardial infarction status (n = 1, 1%). The two most common strategies were using therapeutic communication to initiate a nonthreatening conversation about driving safety and expressing concern for the older adult's driving safely . A total of 66 critical incidents were reported which described strategies that nurse practitioners use in practice to increase safety in older adults . When the incidents were analyzed, nine categories of behaviors were determined with an interrater agreement of 100% . Categories included the following: involving family members, using therapeutic communication, referring patient to occupational / physical therapy, conducting a vision examination, using a step approach to driving termination, using objective data, referring patient to the department of motor vehicles, referring patient to a medical specialist, and limiting medications on the beers list . One study has shown that clinicians and older adult drivers are open to the idea of using a tiered approach to driving assessment in the older adult . The largest category, involving family members, accounted for 24% (n = 16) of the incidents . As one nurse practitioner stated the following.bring the family into it . Ultimately, they are the ones to take the keys if the patient is resistant.others added the following . Sometimes i will voice safety concerns that have been brought up by the family . If the family is present, i try to talk to them and coach them to do the right thing like take the keys or put the care in another place.i tell families to stop being the caregiver and go back to being a family member and make me the bad guy' . I don't want to ride with you' and that is a pretty strong message.it's important to work with families and ask them, why are you letting the most unable person in the room drive down the street?' It usually has to do with the way this family has made decisions in the past as well as cultural issues, and family dynamics . When i brought up the situation to the family, they agreed and the patient stopped driving until his medical situation improved . He's doing a lot better now.the second largest category involved the use of therapeutic communication strategies (n = 14, 21%). One participant stated ask the older adult about how they feel about their ability to drive . I show empathy toward him and his loss of independence and having to rely on family for transportation, and i try to be honest about safety concerns.i used therapeutic communication . I told him the concerns such as him not being able to recall the names of the roads that he just drove on to get here.i talked to her conversationally about how she's been doing and how her family has been doing . I started asking about driving when i asked her about her vision and eye glasses . This made her feel comfortable first so then i asked about this [the driving]. Bring the family into it . Ultimately, they are the ones to take the keys if the patient is resistant . Sometimes i will voice safety concerns that have been brought up by the family . If the family is present, i try to talk to them and coach them to do the right thing like take the keys or put the care in another place . I tell families to stop being the caregiver and go back to being a family member and make me the bad guy' . I don't want to ride with you' and that is a pretty strong message . It's important to work with families and ask them, why are you letting the most unable person in the room drive down the street?' It usually has to do with the way this family has made decisions in the past as well as cultural issues, and family dynamics . When i brought up the situation to the family, they agreed and the patient stopped driving until his medical situation improved . I show empathy toward him and his loss of independence and having to rely on family for transportation, and i try to be honest about safety concerns . I told him the concerns such as him not being able to recall the names of the roads that he just drove on to get here . I talked to her conversationally about how she's been doing and how her family has been doing . I started asking about driving when i asked her about her vision and eye glasses . This made her feel comfortable first so then i asked about this [the driving]. A third largest category contained incidents that described incidents where the nurse practitioner referred the patient to occupational or physical therapy (n = 10, 15%), while 12% (n = 8) involved conducting a vision screening examination . Eleven percent of the incidents (n = 7) described using a step approach to driving termination . First thing is to eliminate driving at night and then i try to find out what's the most important thing they want to drive to . I have one patient who only wants to drive 2 miles from his home to the gas station where he goes into buy his coffee and newspaper and then he drives home . For the longest time this is the only driving he would do and then came the day when i didn't feel he was safe to do that anymore and we had him stop driving i don't think you can just take it all away from them unless it's really imminent that they are going to hurt themselves.work with patient to drive only at certain times of the day and to certain areas that are familiar . I had one patient who had very slow response times so we placed a restriction that he could only drive on certain roads and stay off main roads . First thing is to eliminate driving at night and then i try to find out what's the most important thing they want to drive to . I have one patient who only wants to drive 2 miles from his home to the gas station where he goes into buy his coffee and newspaper and then he drives home . For the longest time this is the only driving he would do and then came the day when i didn't feel he was safe to do that anymore and we had him stop driving i don't think you can just take it all away from them unless it's really imminent that they are going to hurt themselves . Work with patient to drive only at certain times of the day and to certain areas that are familiar . I had one patient who had very slow response times so we placed a restriction that he could only drive on certain roads and stay off main roads . Additional categories reflected the importance of using objective data when discussing driving safety with older adults (n = 5, 7%) and referring the patient to the department of motor vehicles (n = 3, 5%). Two incidents (3%) described referring the patient to a medical specialist, while one incident (2%) described the importance of avoiding the use of inappropriate, high - risk medications with older adult drivers . When participants (nurse practitioners) were asked to list perceived barriers for driving cessation, the most frequently mentioned barriers included resistance from the patient with denial, lack of alternative transportation, spousal denial, and increased isolation . A listing of the most frequent barriers that nurse practitioners face in working with older adult drivers is presented in table 4 . The participants in this study closely represent the national np demographics . Compared to the 2012 national aanp sample survey, the demographics of nps nationally were a mean age of 51 years, white (85.4%), black / african american (3%), female (9.4%), having a graduate level degree (97.2%), and having practiced as a np for 11 years . This study identified strategies used by nps in the assessment and intervention of older adults in one geographical region . Obvious limitations of this study are the limitation to one geographical area . To the author's knowledge, this is one of the first studies to examine self - report behaviors and strategies of nps on this topic . Clinical practice for assessing and intervening with older adult drivers varies significantly from provider to provider . Nps are educated to practice holistically and place great importance on building rapport and trust with their patients and their families . This trusting relationship will prove beneficial when a difficult conversation such as driving cessation is needed . Impaired cognition was the most common reason a np determined a need to intervene . Assessing cognitive status, physical frailty, and sensory impairments were the most common physical exam strategies used . Obtaining collateral information from family members ranked just below obtaining physical exam or objective data . Using therapeutic communication techniques and a concern of safety for the older adult were the most common strategies used in approaching the older adult about driving . Nps ranked the most helpful intervention was to involve family members and the least helpful intervention was the lack of family guidance from the provider . Dealing with patient concerns about lack of transportation, especially in rural areas, and resistance to driving termination were the two most common perceived barriers . The wide variation in ways which nps make the determination to address driving in older adults and the lack of consistent guidelines for dealing with this issue should be noted . As the supply of primary care physicians is expected to decrease, the number of nurse practitioners (nps) is expected to increase 94% between 2008 and 2025 . Nps are trained to provide a holistic approach to patient care and approximately half of all nps practice in the primary care setting . Standardized curriculum for nurse practitioner students does not require faculty to address concerns of older adult drivers; therefore, few nps graduate with this training . This could be due to lack of clinical guidelines, and faculty are unclear as to what should be taught . As this is a public safety concern having difficult conversations with older adults such as driving cessation is time consuming and possibly threatens the patient - provider relationship . There is a need for the development of clinical guidelines that nps can use efficiently during a routine office visit . Nps feel comfortable using evidenced - based clinical guidelines to guide their clinical practice with common chronic disease management . Development of an evidenced - based clinical guideline for assessment and intervention with older adult drivers would be used similarly . Our social work and occupational therapist colleagues have been working on this issue for quite some time . Occupational therapists have developed guidelines for them to use in assessment of older adult drivers . Nps and np students should also be aware of community resources available in their areas of clinical practice . Simply making themselves aware of resources available to them and the older adult, such as the online aarp driver safety program that can be found at http://www.aarp.org/home-garden/transportation/driver_safety/ would prove beneficial . This study shows the varied and inconsistent clinical practice of nps assessing and intervening with older adults drivers . Who is to say that one strategy is better than the other? It is clear that nps value the relationship they have with their patients and they value the need to intervene when public safety is at risk . Development of evidenced - based clinical guidelines would prove beneficial for nps to use in clinical practice, as long as the guidelines were able to be used quickly and efficiently.
Left ventricular noncompaction cardiomyopathy (lvnc) is a very rare cardiomyopathy characterized by an increase in the noncompacted, trabeculated myocardium adjacent to compacted myocardium in the left ventricular (lv). Though current research suggests a developmental arrest in embryogenesis as the underlying pathology, the etiology of lvnc are not fully understood . The patients will often present with a spectrum of disease severity ranging from no symptoms to cardiac arrhythmias, cardiac failure, thromboembolism or even sudden cardiac death . Historically, lvnc has been diagnosed by echocardiography when the ratio of noncompacted to compacted myocardium is> 2 . Echocardiography may not visualize the apical region optimally, leading to underestimation of the degree of lv noncompaction . However, cardiac magnetic resonance imaging (cmri) provides a comprehensive depiction of cardiac morphology in any imaging plane . Recent cmri reports suggest a ratio of the noncompacted myocardium to compacted myocardium of> 2.3 yield the highest sensitivity (86%) and specificity (99%) in diagnosis . We present a case of lvnc in an adult patient with its clinical and imaging findings . A 24-year - old female patient was initially referred to our vascular department for investigation of bilateral lymphedema . Her echocardiogram showed mildly impaired lv with evidence of segmental wall motion abnormality and hypertrabeculation in the apex suggestive of noncompaction . The history of the patient started at birth when she had difficulty swallowing and several delayed milestones such as walking and talking at the age of 5 . Due to socioeconomic issues as the patient matured, there was an additional history of palpitations, which were occasionally associated with syncopal attacks . Lower limbs bilateral nonpitting edema up to the knees, proximal bilateral upper and lower limbs weakness with intact sensation, waddling gait, and easy fatigability with few steps were noted . Computed tomography coronary angiogram was performed to exclude coronary artery disease . Results showed segmental wall motion abnormality and revealed normal coronaries, lv dilatation, and lateral wall and apical noncompaction with the noncompacted myocardium to compacted myocardium ratio in the range of 2.53.5 [figure 1]. Her cmri showed mildly dilated lv with mild global hypokinesis and a marked decrease in longitudinal shortening . End - diastolic volume was 120 ml, end - systolic volume was 61 ml and the ejection fraction was 50% . In addition, multiple areas of noncompacted myocardium, particularly in the apex and most of the lateral wall extending from apical lateral wall to the mid - to - basal segment were seen . The ratio of maximum thickness of the noncompacted myocardium to compacted myocardium was more than 2.5 in multiple areas [figure 2]. Gated cardiac computed tomography scan was performed to evaluate the coronary arteries and showed patent coronary arteries of normal anatomy (not shown). Axial image shows left ventricle lateral wall noncompaction with prominent trabeculation (arrows) noncompaction cardiomyopathy in a 24-year - old female . A four chambers view dark blood using horizontal long axis t1 fast spin echo - magnetic resonance imaging . (a) apparent thickening of the left ventricle lateral wall (arrows). A static image of four chamber view from cardiac magnetic resonance imaging cine, using the balanced steady state free precession technique . (b) shows mild dilatation of the left ventricle, prominent left ventricle wall trabecular network (arrowheads) and only a thin compacta (arrows) further investigation showed elevated creatine kinase and electromyography findings consistent with myopathy . Thus, the clinical impression was proximal myopathy mostly mitochondrial or congenital, congenital lymphedema, lv noncompaction with lv ejection fraction of 40%, and normal rv function . As her lv impairment is mild, the decision was made to keep her on beta - blockers and on angiotensin - converting enzyme inhibitor in the form of metoprolol and lisinopril, respectively . On subsequent follow - up visits, she reported a history of interval improvement apart of occasional palpitations with no history of shortness of breath, syncope or dizziness . She has been advised to do regular physiotherapy and to wear compressive stockings for her lymphedema . Bellet and gouley described the first case of noncompaction in an autopsy of a newborn infant with aortic atresia and coronary ventricular fistula . Lvnc without other cardiac abnormalities (isolated noncompaction cardiomyopathy) was first described by an echocardiogram performed by engberding and bender . Historically, the prevalence of lvnc has been underestimated due to the lack of knowledge about this rare condition and its similarity to other diseases of the myocardium and endocardium . A study of pediatric patients with primary cardiomyopathy showed that 9% of the patients had lvnc . The median age at diagnosis of isolated noncompaction cardiomyopathy in the initial case series of isolated noncompaction was 7 years ranging from 11 months to 22 years . The prevalence of lvnc in the adult population ranges between 0.01% and 0.3% of all adult patients referred for echocardiography studies . These numbers are mostly extracted from the referred patient for abnormal echocardiographic findings or congestive heart failure (hf). Therefore, because of this potential selection bias, the true prevalence is not clear . Male patients are predominately affected accounting for 63%, 70%, and 74% of cases in three different reported series . In 2001, jenni et al . Evaluated seven patients with lv noncompaction with pathologic correlation (four postmortem patients and three cardiac transplant patients). Histologic analysis demonstrated ischemic lesions in the thickened endocardium and thickened trabeculae with accompanying fibrosis . Clinical manifestations are highly variable, ranging from no symptoms to disabling congestive hf, arrhythmias, and systemic thromboemboli . According to a french registry, the diagnosis of lvnc diagnosis was confirmed in 105 cases through the use of echocardiogram performed in a laboratory between 2004 and 2006 . In that study, lvnc was first detected in 12 patients with rhythm disorders, 45 patients with hf symptoms, and eight patients through familial screening . During the follow - up study, patients suffered several complications including hf occurring in 33 of the patients, ventricular arrhythmia in seven, embolic events in nine, and nine of the patients received heart transplantation while death occurred in 12 of the patients . A swiss registry recorded a total of 34 cases over 15 years among a patient population who underwent echocardiogram . In this study, major complications included hf (53%), ventricular tachycardia (41%), thromboembolic events (24%), and death (35%). Six of the reported 12 deaths were sudden and four due to end - stage hf in four and the other two cases were due to unrelated causes . The presented case had a family history of sudden death in two brothers, with one of them presumed to have an unknown type cardiomyopathy . Sporadic and familial forms of noncompaction have been described . In the original report of isolated noncompaction ventricular cardiomyopathy that predominantly affected children, half of the patients had a familial recurrence . However, a larger reported adult population with isolated noncompaction ventricular cardiomyopathy showed only 18% familial recurrence . Lower percentage compared with the earlier report might be attributed to incomplete screening of siblings in oechslin et al . This patient has a progressive myopathy, which raised the question of other association with her left ventricular noncompaction cardiomyopathy . About 82% of lv noncompaction cases were found to be associated with neuromuscular disorder including becker muscular dystrophy, metabolic myopathy, myotonic dystrophy, barth syndrome, and other rare genetic disorders . The ratio of noncompacted myocardium to compacted myocardium at the end of systole is> 2:1 is the most often used echocardiographic criteria that were proposed by jenni et al . The criteria also include presence of segmental thickening of the myocardial wall of the lv with two layers: prominent trabeculations and deep recesses with a thin epicardial layer and a thick endocardial layer . The absence of coexisting cardiac abnormalities is required to fulfill the criteria . Lv diastolic dysfunction, reduced global lv systolic function, abnormal structure of papillary muscles, and lv thrombi are nonspecific findings that can be seen on echocardiography . Cardiac magnetic resonance (cmr) can be used in correlation with an echocardiogram to localize and quantify the extent of noncompaction . Cmr offers a detailed view of the cardiac morphology of the noncompacted myocardial layer in the lv in any image plane including the apical and lateral segments - segments, which are not well visualized by echocardiogram . Recent reports suggest that echocardiography diagnostic criteria are strict and mri enhances the detection of more subtle forms of noncompaction . Further, cmr identifies a higher rate of two - layered structures in segments such as the anterior, inferior, anterolateral, and inferolateral segments compared with echocardiogram . However, contraindication after implanted devices, cost and availability of cmr are considered barriers on the way of implementing cmr . According to the american heart association, 1.5 tesla is the minimum acceptable magnet strength to perform cardiac imaging in order to be able to visualize both short and long axis views in approximately 17 segments . Acquiring three diastolic long axis views best identifies the nc / c ratio of the most prominent myocardial trabeculations segment . The nc / c ratio in cmr is measured at the end of diastole and should be more than 2.3, more than echocardiography, which is 2.1 . Stagnant blood flow, within the myocardial trabeculae may be detracted as a high signal in cmr black blood imaging, supporting the diagnosis lvnc . The efficacy of cmr was evaluated in a report of seven patients with lvnc in whom other features supported the diagnosis; the results were compared with 170 healthy volunteers, athletes, or patients with dilated cardiomyopathy or hypertrophic cardiomyopathy, aortic stenosis, and hypertensive heart disease . The most distinguishing feature was a ratio of noncompacted to compacted myocardium during diastole (sensitivity 86% and specificity 99%). Direct imaging of myocardial fibrosis is possible with the use of an inversion recovery prepared t1-weighted gradient - echo sequence and the extracellular fluid tracer gadopentetate dimeglumine . This technique has been termed delayed hyperenhancement and shows nonviable tissue as hyperenhanced or bright . Nevertheless, the early and precise diagnosis is mandatory to rule out other underlying diagnoses and to allow a timely start of standard hf and anticoagulation therapy, thus preventing further complications . Some trabeculae show delayed hyperenhancement despite having a normal compacted to noncompacted myocardial ratio, suggesting that lv noncompaction may be a more diffuse disease process than previously suspected . The use of delayed hyperenhancement sequences improves the correlation between cmri and the parameters of the clinical stage of the disease . Cmri follow - up examinations would be helpful to assess a potential change of noncompacted or compacted mass in a chronological sequence . In conclusion, cmr can distinguish lvnc from other cardiomyopathies and normal hearts with high sensitivity and specificity.
Lung cancer is the most common cause of cancer mortality in the western world, accounting for approximately 5% of all deaths in many countries . Five - year survival for those with pathological stage ia non - small cell lung cancer (nsclc) is 73% whereas metastatic disease has a dismal prognosis (13% 5-year survival). Results from several studies suggest that frequent chest radiographic screening does not result in reduced lung cancer mortality, a conclusion reinforced by the prostate, lung, colorectal, and ovarian (plco) cancer screening trial [3, 4]. In fact, some studies suggest that frequent chest radiographic screening is associated with an 11% relative increase in lung cancer mortality compared with less frequent screening . Randomized trials of screening low - dose computed tomography (ldct) scans demonstrate that computed tomography (ct) is far more sensitive than chest radiography . The national lung screening trial (nlst) showed that, in heavy (30 pack - years or more) current or former (within 15 years) smokers between the ages of 55 and 75, three annual ldct screens reduced lung cancer - specific mortality from 309 to 247 deaths per 100,000 person - years . There are still many unanswered questions about the benefits and harms of those programs that could determine the ultimate success of the mass screening implementation . Additionally, despite expert guidelines for screening high - risk populations, most national health service providers have not implemented (and probably will not implement in the near future) mass lung cancer screening programs . One of the main concerns is that the extrapolation of findings from tightly controlled trials to real - life mass screening programs requires uniform standards and high quality controls not easily achievable in most institutions . Consequently, the current practice is that the patients themselves or their physicians may choose early lung cancer detection on an individual basis . There is little information, however, on the clinical characteristics and outcomes of patients with incidentally detected early stage lung cancer from strictly controlled randomized trials . The objective of this study was to analyze the clinical records of lung cancer patients who underwent surgical resection to evaluate the clinical characteristics and outcomes of patients with incidentally detected lung cancer and patients with symptomatic lung cancer . All patients undergoing pulmonary resection with a curative intention for non - small cell lung cancer (nsclc) in the british hospital in buenos aires between january 1986 and july 2009 were eligible for inclusion in this retrospective study . Our thoracic oncology centre keeps a database of all patients evaluated, with data entered prospectively at the time of their initial evaluation . Patients were excluded if they had exhibited small cell lung cancer or a rare histological result . Preoperative data included methods of diagnosis and a symptoms questionnaire, tobacco exposure history, and medical history . Preoperative staging was performed according to the 7th tnm classification system of the international association for the study of lung cancer using chest computed tomography (ct) and abdominal ct or ultrasonography in all patients . Brain computed tomography or magnetic resonance imaging was done only in case of clinical suspicion of brain metastases . In cases of uncertain clinical or radiologic findings, pet was included only during the last 3 years and not on a routine basis . Mediastinoscopy has not been performed routinely in this series unless the ct scan demonstrated mediastinal lymph node enlargement, pet suggested a malignant involvement of hilar or mediastinal nodes, or high- risk criteria of n2 were present . Patients were classified into two groups: group 1 (asymptomatic): patients who had no symptoms attributable to lung cancer at the time of imaging (patients whose cancer was detected by a medical checkup or under evaluation for other diseases), and group b (symptomatic): patients with lung cancer - related symptoms . The charts of patients classified as having asymptomatic incidentally detected lung cancers were reviewed to check if the indications for imaging really were not based on any potentially cancer - related symptom . Postoperative follow - up included office visits, quarterly chest x - rays, and yearly chest - ct . Operative or in - hospital mortality was defined as death occurring within 30 days after the operation or during hospitalization, respectively . The analysis of differences in categorical outcomes was determined using the chi - squared test or fisher's exact test . Probabilities of survival rates were estimated using the kaplan - meier method and asx and sx patients were compared by using the log - rank test . Of 593 patients included in this study (68.3% male, median age 60.9, and range 2386 years) 320 patients were asymptomatic (asx) (53.9%). Two hundred and thirty (71.8% of the asx patients) were diagnosed incidentally on chest x - ray and the remaining on ct scan . Amongst the patients with symptoms, the leading complaints that resulted in the indication for imaging were the appearance of new cough or the increase of a previously manifested clinical picture suggestive of pneumonia and haemoptysis (table 1). Amongst the 320 asx patients, once the initial chest - x ray (71.8%) or ct scan (28.2%) showed an abnormal image, the usual workup for pulmonary nodules was started . Patients in the asx group were older than patients in sx group (median age 61.9 9.9 versus 59.51 years / old 10.2, p = 0.007), without differences in sex (men 66 versus 73.5%, p = 0.084). They had a higher prevalence of previous malignancy (13.2 versus 4.8%, p = 0.002). The frequency of presentation as spn (49.5 versus 19.4%, p <0.001) or peripheral location (80.3 versus 63.7%, p <0.001) was higher in this group, without differences in clinical suspicion of n2 (8.8 versus 12.9%, p = 0.146). Patients with incidentally detected lung cancer were more likely to have earlier - stage disease, smaller cancers (3.00 2.2 versus 4.3 2.9 cm, p = 0.0001). Clinical characteristics of both groups are shown in table 3 . When the last ten years were analyzed, a higher prevalence of incidental detection compared to previous years was observed (51.7 versus 39.8%, p = 0.008). The overall 5-year survival rates were higher for asx patients: 66.2% and 46.0% for asx and symptomatic patients, respectively (p = 0.001) (figure 1). Amongst the stage i patients, the 5-year survival rates were 81.2% in asx patients and 58.6% in sx patients (p = 0.014) (figure 2). When only stage ia was considered, 5-year survival rates were not different (71.2 versus 84.1%, p = 0.191) (figure 3). When analysis was restricted to t1a tumors there were no differences either in 5-year survival (94.7 versus 93.2, p = 0.489). Median survival times in pathological stages iiib (41.6 m in asx versus 22.0 m in sx patients, p = 0.065) and iv (13.7 versus 12.7 m, p = 0.964) were not significantly different . Our study shows that the incidental finding of non - small cell lung cancer occurred more frequently in smokers and in patients with a history of previous malignancy . The mortality of patients with nsclc as an incidental diagnosis was lower, and this difference persisted into stage i. more than a half of patients who underwent surgical resection of lung cancer at our institution had incidentally detected cancers and the most common indication for the initial imaging was a routine checkup . That proportion of asx patients is higher than that reported by raz et al . In san francisco but far lower than that published by hanagiri et al . In japan . In the absence of a uniform policy appertaining to the role for screening in clinical practice, the indication of imaging asymptomatic patients relies on the preferences and beliefs of both patients and physicians . Different levels of awareness and access to healthcare may justify differences amongst different studied populations . Also, our asx patients were slightly older (contrary to the study by raz et al .) And more frequently smokers which may mean a higher degree of awareness of their risk for lung cancer as the higher prevalence of previous malignancy may have been one of the reasons for routine radiological surveillance . However, it is noticeable that more than 70% of patients in our group (as in other series) were studied by chest - x - ray, a method that has proved to be ineffective and that is not recommended as a screening tool by any major medical organizations . The proportion of early stages of lung cancer was higher amongst our patients with incidental findings . That stage i cases accounted for 65.3% and stage iii cases for 22% in their mass screened group, while, in the symptom group, there was only half that percentage of stage i cases (32.2%). Similarly, in the two more recently published series [7, 8] and in the korean lung cancer registry study asymptomatic patients had higher proportions of stages i - ii . However, there were still 20% of our patients that did not have any symptom and had a stage iii or iv lung cancer . Interestingly, in a retrospective review of coronial autopsies even when the median tumor size of previously undetected cancers was 3 cm, the range was 110 cm and there were several tumours over 5 cm and even some large endobronchial and hilar tumours undetected before death . We found that survival time in symptomatic cases was worse than in incidentally detected lung cancer patients . The 5-year overall survival was lower for the whole group and for pathological stage i. that better outcome has been consistently demonstrated in all the previous reports; however, the causes for those differences are still unclear . The korean registry has shown that absence of symptoms at diagnosis significantly reduced the risk of death from nsclc, regardless of age, gender, stage, smoking history, or whether treatment was performed . Similarly, hanagiri and colleagues showed that their patients with incidentally diagnosed nsclc had significantly better prognoses than the symptomatic group even in stage ii even when they had a larger proportion of stage iv amongst asymptomatic patients than our series (6.7 versus 3.4%) they were still a small number of patients (n = 18) and exact figures of survival rates for those advanced patients were not provided . In our series, whilst pathological stage i patients had a better survival in asx patients, median survival times in stages iiib and iv were similar . It resembles the results of raz et al . Who found that their patients with completely resected incidental lung cancer had similar long - term survival rates as patients with symptomatic lung cancer, after adjusting for stage . In the present study, stage ia disease was diagnosed less frequently in the symptomatic group, similar to what has been reported in other studies [7, 8, 10]. A study by kashiwabara et al . (published in 2002, before the publication of the 7th tnm edition) compared the outcomes in patients with one - year delayed detection of lung cancer on mass screening with chest - x - ray and in patients with no delay (patients with tumours which could versus could not be detected on past chest roentgenograms). They found that one - year delayed detection of lung cancer on mass screening did not affect outcome, but that, according to the maximum dimension of the tumours on the overlooked chest roentgenogram, the 5-year survival rates in patients with missed tumours were different and that survival in early stages (i - ii) for missed tumours> 20 mm was worse than that in patients with missed tumours <10 mm . We had previously shown that tumors over 15 mm are associated with shorter 5-year survival in all tnm stages and several studies have reported tumor size may have an independent predictive value on survival in stage i patients [14, 15]. The impact of the tumor size was finally made evident by the analyses of the database of the iaslc and generated the reclassification of t1 in t1a and t1b and t2 in t2a and t2b . When we analyzed separately the pathological stage ia cases, differences in survival in stage i between the two groups disappeared, suggesting that the size was the most important factor in determining survival . Our study shows that patients with incidentally detected lung cancer had a better survival because they had smaller cancers and earlier - stage disease . The conclusions of clinical studies like this or any of the previously published studies should not be extrapolated to the potential value of mass screening . This sort of study design does not allow demonstrating if there is a survival benefit of treatments in asymptomatic patients or how large the proportion of invasive procedures for benign lesions is performed . One of the main concerns about any screening program is that a proportion of screen - detected cases will be overdiagnosed simply because of competing mortality, a hypothesis that cannot be excluded by a population study like this one . On the other hand, many of the patients in this and the other clinical series were not represented in the clinical trials about mass screening programs: 15% of our asymptomatic patients were never smokers and many of them were under 55 years old and would have not filled criteria for being included in a screening program . This study shows the importance of identifying risk at an individual level as many subjects different from the nlst participants may have a risk similar to or greater than the level of risk observed in nlst . Several studies have previously recognized that there is wide variation in lung cancer risk even amongst those who are smokers [18, 19] and we do not know yet how to identify other risk factors for lung cancer that could potentially justify extending screening to those individuals . Future research to develop clinically useful risk model might include molecular or genetic indicators of risk in order to answer these questions . The national lung screening trial (nlst) showed that, in heavy (30 pack - years or more) current or former (within 15 years) smokers between the ages of 55 and 75, three annual low - dose computed tomographic (ldct) screens reduced lung cancer - specific mortality from 309 to 247 deaths per 100,000 person - years (relative risk of 0.8). But at the moment to make individual decisions (such as screening of certain nonsmokers) it is necessary to take into account the potential effectiveness of such measures . Whilst the number needed to screen (nns) to prevent one death for the entire nlst population was calculated as 320, according to bach and gould for very low risk individuals (defined by the authors as a 40-year - old former smoker) the nns was over 35,000 to prevent one lung cancer death . In order to minimise the potential for harm when screening large populations for a condition that is very rare (derived not only from the costs associated with screening but also from the impact on quality of life of the potential for invasive procedures for incidental findings) but at the same time not to miss other high - risk subjects out of the nslt criteria, better risk models must be developed to have the greatest predictive accuracy for lung cancer risk . Firstly, the classification of a cancer as incidentally detected is a potential bias, once the spontaneous patient consultation may not exclude the presence of some nonspecific symptom that prompted the patient to seek medical consultation . It is remarkable that, in the study by kashiwabara et al . About patients that did not consult a physician after the discovery of a shadow in a radiological screening (almost 25% of the asymptomatic screened patients in their series), when asked about the reason why patients did not consult a doctor, two - thirds answered that it was because they did not have any respiratory symptoms . It shows that a screening program must assure that the health care system can provide all the necessary resources to treat the incidental findings and also the education to guarantee the availability of well - qualified primary care providers trained to encourage patients to follow diagnosis and treatment recommendations once a suspicion of lung cancer is raised from the imaging studies . In summary, our study shows that lung cancer as an incidental finding is not uncommon even amongst nonsmokers and that the better survival of patients with asymptomatic nsclc is related to the greater number of patients with earlier - stage disease . Future research is needed to prospectively identify those patients not represented in the nslt who might benefit from ldct screening.
Hip arthrodesis as a solution for hip - related problems is mainly superseded by total hip arthroplasty (tha) and, in the current treatment of hip disorders, there is hardly room left for arthrodesis of the hip . However, we must realise that in certain scenarios in young adults joint preserving therapy is not an option and total hip arthroplasty is a difficult procedure with an increased risk of failure [13]. In these selected young patients with severe hip pathology due to trauma or infectious causes an arthrodesis may be a suitable solution with an acceptable outcome [14]. There has been considerable literature in the past about the technique and outcome of a hip fusion . Several studies report of pain in adjacent joints in the long term; the most frequent being lower back pain based on degenerative changes reported between 15% and 100% of cases [59]. Following in percentage are complaints of the ipsilateral knee and less frequent complaints of the contralateral hip and knee [911]. The frequency of these complaints seems to be predicted by the alignment of the fusion which should be optimal [3, 6, 12]. In our clinic, we perform hip fusions with our standard technique using a cobra plate; however, in those complex cases where optimal alignment and optimal fixation cannot be achieved by this method we use the technique with a subtrochanteric pendulum osteotomy to achieve the correct alignment . Even in the most difficult cases this method enables the optimal combination between a stable connection of the remaining femoral head neck and pelvis and an optimal alignment of the leg . Although a hip fusion is not the most ideal solution it remains an important option for selected scenarios . Apart from the short - term results, attention should be paid to the long - term results in adolescents and young adults . When performing an arthrodesis the main objective of our study was to show the long - term effects of fusion on the adjacent joints using our technique . In the period between 1974 and 1994, 47 hip arthrodesis were performed by the senior author (rkm). The average age at the time of the arthrodesis was 29 years (range 1255). The hip disorder which led to arthrodesis was septic arthritis in ten patients (including tuberculosis), aseptic necrosis in one, post - traumatic in 21, congenital luxation in three patients, four patients were secondary to childhood hip disorders and there were other indications in eight . We prefer to perform a cobra plate technique, which was initially developed by schneider and later modified by several others, eliminating the need for the pelvic osteotomy [13, 14]. The technique we use is similar to this reported technique; however, we pay extra attention to the abductor muscle since it is mandatory to preserve it if a later total hip arthroplasty is a possibility . The abductors are spared by performing a trochanteric osteotomy which is fixed on / over the cobra plate after the arthrodesis is performed . For complex cases in which the optimal combination of fixation of the arthrodesis and perfect limb alignment is not possible, for instance in septic arthritis with severe destruction of the joint or severe osteonecrosis, we performed an arthrodesis with a subtrochanteric pendulum - osteotomy . The arthrodesis is performed in such a position that optimal contact between femoral head and acetabulum is present, regardless of the position of the leg . The arthrodesis is then relieved by a subtrochanteric osteotomy which also allows optimal positioning of the extremity . After - treatment consisted of traction immobilisation for two weeks followed by six weeks of plaster immobilisation . In this study 33 hips were fused with the cobra plate technique and 14 with the arthrodesis with subtrochanteric pendulum - osteotomy technique . The clinical follow - up consisted of a thorough clinical investigation and the short musculoskeletal function assessment (smfa) questionnaire . The smfa is a well - validated, universal, self - reported health status questionnaire . The smfa questionnaire is developed for clinical assessments of the impact of treatment in groups of patients who have musculoskeletal disease or injury . The smfa questionnaire consists of the dysfunction index, which has 34 items for the assessment of patient function, and a bother index, which has twelve items for the assessment of how much patients are bothered by functional problems . Since a hip arthrodesis can have a negative influence on the contralateral hip joint, the back and knees, we included these adjacent joints in this analysis . The clinical investigation consisted of: range of motion of hips and knees, mobility of the back, laxity of the knee joint, leg length discrepancies and quadriceps strength . The mobility of the back was measured using a standardised mildenberg finger - floor method and the schber test . The schber test was conducted with the patient standing upright; a mark was made at the height of l5 . A second mark was made 10 cm above and a third mark 5 cm below the first mark . The patient was asked to bend forward with the knees straight, reaching as far as possible towards his / her toes . The increase between the upper and lower mark was taken as a measurement of lumbar flexion . The finger - floor measurements were taken upon the first attempt of the patient to bend forward with extended knees . Quadriceps strength was graded from 0 to 5 according to the british medical research council scale . Subjective outcome measurement visual analogue scores (vas) were obtained for pain in hips, knees and back . Vas scores were also obtained for satisfaction with the results of the arthrodesis and for the eagerness to have the arthrodesis converted to tha . Additional questions concerning work, walking distance, problems with sitting and problems with socks and shoes were posed . For both knee joints, the international knee documentation committee (ikdc) knee examination form was used to evaluate secondary knee problems . This form results in four groups: group a is normal knees, group b nearly normal knees, group c abnormal knees, and group d severely abnormal knees . For the radiological analysis, weight bearing x - rays of pelvis, both knees and lumbar spine were used . The contralateral hip and both knees were scrutinised for signs of osteoarthritic changes which were scored according to kellgren and lawrence for the medial compartment, the lateral compartment and the patello - femoral compartment . For the knees, the ahlback score was also used for the medial and lateral compartment separately . On the lumbar spine x - rays the presence of osteophytes as an indicator for osteoarthritic changes were scored; the percentage of joint - space narrowing between the vertebrae and the presence of olisthesis and scoliosis were documented . After an average follow up of 18.9 years (range 5.631.4), six patients had died . Seven hips were eventually converted to tha of which one was performed to facilitate a total knee arthroplasty on the same side . The remaining 30 patients were invited for follow - up and intensively screened . In this group of difficult hip disorders one patient suffered from a deep venous thrombosis which was treated with anticoagulants . In two patients a deep infection occurred, of which both received the arthrodesis for a destructive septic arthritis . In one of them a revision operation was performed with debridement of the infection and surgical drainage; the infection resolved but resulted in a nonunion . The second infection was treated conservatively, and after 24 years of follow - up a fistula and deep infection were still present . Besides the nonunion after infection, in five other patients a nonunion was diagnosed, and in three patients one re - arthrodesis was sufficient to achieve union, while in one patient two re - arthrodesis were performed to achieve union . One patient was left untreated because of co - morbidity for which this arthrodesis was performed for severe post - traumatic destruction; however, the same trauma led to a post - traumatic ankylosis of the ipsilateral knee, a paresis of the contralateral arm and permanent brain damage (mainly cognitive). Clinical and radiological follow - up was obtained for 30 patients after an average of 18.2 years (range 6.230.5) (fig . 1). Clinically, the smfa showed a mean total score of 31.2 (range 9.070.0), the subscore dysfunction an average of 29.1 (range 8.070.5) and the subscore limitations an average of 37.3 (range 9.084.0). The clinical and radiological results for the arthrodesis and adjacent joint will be discussed separately . H x - rays of pelvis, knees and lumbar spine 28 years postoperatively . The arthrodesis was converted to a total hip replacement (thr) 20 years postoperatively (on the contralateral hip a shortening at intertrochanteric level was performed 1 year after the fusion) a a 17-year - old boy with a central hip luxation . H x - rays of pelvis, knees and lumbar spine 28 years postoperatively . The arthrodesis was converted to a total hip replacement (thr) 20 years postoperatively (on the contralateral hip a shortening at intertrochanteric level was performed 1 year after the fusion) the clinical and radiological investigation of the fused hip joint showed an average vas for pain of 1.9 (range 0.08.0). The average alignment of the arthrodesis was 19 flexion (5; 30), 1 of abduction (10; 10) and 2 external rotation (10; 15). An average leg length discrepancy of 3.3 cm (range 11.5 to 2) was present . Walking distance averaged 115 minutes (range 10unlimited) for which one patient needed two crutches and two patients needed one crutch for support . Twenty - three patients experienced difficulties in putting on their shoes, in 17 of them this was caused by the inability to tie their shoelaces . Of these, three had no complaints of the fused hip (vas pain 0,0,3), and one patient with a nonunion after infection had a vas of 6 . The two remaining patients reported vas of 6 and 7 for pain; however, they performed full time heavy labour (north sea fisherman and factory worker). The vas for eagerness to have their fusion converted to a tha was 5.4 average (range 010). The contralateral hip joint on clinical examination showed an average vas for pain of 2.0 (range 08). One contralateral hip underwent an intertrochanteric shortening osteotomy and one was converted to a tha . The average range of motion was: for flexion 104 (range 30140), extension 0.2 (0 5), abduction 25 (1040), adduction 17 (030), external rotation 27 (560) and internal rotation 16 (0 30). On clinical examination the ipsilateral knee joint showed an average vas for pain of 2.0 (range 08). Average range of motion for flexion was 125 (20160) and for extension 2 (010). On radiological examination three knees showed severe osteoarthritic changes with a grade 4 k - l and iv ahlback in both compartments, and in one knee only in the lateral compartment . A grade 3 or 4 k - l oa in the patello - femoral compartment was present in four knees . The ikdc knee examination form showed a grade a in nine knees, grade b in 14 knees, grade c in four knees and grade d in eight knees . On clinical examination the contralateral knee joint showed an average vas for pain of 1.8 (range 08). Quadriceps strength was optimal (grade 5) in all . Average range of motion for flexion was 140 (100160) and for extension 1 (10 to 20). On radiological examination one knee showed severe osteoarthritic changes with a grade 4 k - l and a grade iv ahlback in both compartments, and in one knee only in the lateral compartment . A grade 3 or 4 k - l oa in the patello - femoral compartment was present in three knees . The ikdc knee examination form showed a grade a in 23 knees, grade b in five knees, grade c in five knees and grade d in three knees . There was a significant differences in the outcome of the ikdc examination form (laxity) for the ipsilateral and contralateral knees (chi - square p <0.001). Clinical investigation of the lower back showed an average vas for pain of 3.6 (08) (table 1). The average schber test was 6.1 (310.5), and the standardised mildenberg finger - floor method averaged 32.5 cm (1551). Radiological examination of the sacro - iliacal joint showed three joints with severe arthrosis (one grade 3 and two grade 4 k - l) on the ipsilateral side, and two (both grade 3 k - l) on the contralateral side . The lumbar spine showed signs of oa in the form of osteophytes in 12 backs . In two spines an olisthesis was present, one grade 1 and one grade 2 . Ten ap lumbar spine showed a scoliosis, of which eight had the convexity on the opposite side of the arthrodesis . Results per operation technique sfma short musculoskeletal function assessment, avg average, vas visual analogue score although arthrodesis has been forced into the background by the successful tha, it still remains a valid option for specific difficult problems . This descriptive study of our fusion techniques shows a good outcome measured by the smfa and a good vas for pain for all related joints after an average of 19 years of follow - up . Especially lower back pain seems to occur frequently after a hip fusion in the long term, since 42% of our patients had a vas for pain of 3 and higher for lower back pain . The cobra head plate technique is our first choice since it provides a high fusion rate and allows early mobilisation . For some indications, including severe unilateral destruction of the femoral head in young patients with severe loss of bone stock, which can occur in chronic septic arthritis or osteonecrosis, a cobra head plate arthrodesis does not provide the necessary stability . This method enables the optimal combination between a stable connection of the remaining femoral head - neck and pelvis and an optimal alignment of the leg even in the most difficult cases . The fact that an arthrodesis is only indicated in difficult cases is probably the best explanation for the relatively high complication rate in this group of patients . A second important complication is the occurrence of nonunions in our patient group . At follow - up, six hips showed signs of a nonunion, although in three of them a real nonunion was unlikely due to the absence of symptoms . Two other patients with signs of a nonunion indicated pain in the fused hip (vas 6 & 7), but were able to perform full time heavy labour . Using these two techniques for these selected patients our nonunion rate of 6% it is known that an arthrodesis of any joint has influence on the adjacent joints . These effects can be minimized by performing an optimal alignment of the affected extremity . In an ideal arthrodesis this alignment is adjusted to the patient's preferences . In patients with a mainly sitting occupation more flexion both knee joints did not suffer from the arthrodesis in a way that they caused pain or discomfort . However, we noticed that significantly more knees on the ipsilateral side showed an increased laxity (ikdc knee examination form). No differences were found in the vas scores for the contralateral and ipsilateral knees . In 2000, karol et al . Showed in a detailed gait analysis that excessive motion of the lumbar spine and the ipsilateral knee were present in patients with a fused hip . These excessive movements caused complaints in the ipsilateral knee and lower back in their group of seven patients . Our data supports the fact that a fusion causes more lower back complaints in the long term (42% with a vas higher than 3); however, we could not confirm the occurrence of more complaints in the ipsilateral knee . In our study seven arthrodeses (15%) were eventually converted to a tha (fig . 1), all with a good result . In the literature several studies can be found regarding conversion of an arthrodesis to a tha, showing identical results to primary tha in patients older than 50 [1, 19, 20]. Although primarily tha may not be a solution for these patients, an arthrodesis can bridge the gap to a future tha, while generating a good quality of life in the meantime . We asked our patients how eager they were to convert their arthrodesis to a tha and 13 (43%) saw no reasons for conversion at long - term follow - up, indicating that these patients were content with their current quality of life . We found that the mobility and quality of life were good for patients with an arthrodesis . The average walking time was almost two hours (115 minutes), while ten patients were able to walk for at least three hours, and one patient even participated in marathon running . Although hip arthrodesis has lost it popularity it still is an option for young patient with severe hip disorders, while leaving the possibility to perform a tha at a later stage . If the arthrodesis is performed with an optimal alignment of the leg, complaints from the adjacent joints are minimal, even in the long term, and an acceptable quality of life can be obtained.
Leukotrienes (lts), first described by samuelsson's group [1, 2], are a class of lipid mediators involved in several diseases but classically known for their effects on asthma and allergy . The generation of leukotrienes (lts) is dependent upon the action of 5-lipoxygenase (5-lo) in association with membrane - bound 5-lipoxygenase - activating protein (flap) on arachidonic acid (aa). Aa is derived through the action of cytosolic phospholipase a2 (cpla2) and/or secreted phospholipase a2 (spla2) on membrane phospholipids . Lta4, an unstable precursor of all leukotrienes, is quickly metabolized to one of the two different classes of lts, ltb4 (by lta4 hydrolase) or ltc4 (by ltc4 synthase) and its metabolites (ltd4 and lte4). Collectively, ltc4, ltd4, and lte4 were previously known as the slow - reacting substance of anaphylaxis (sr - a) and are currently termed the cysteinyl lts (cyslts) [3, 4]. The receptors for ltb4 (btl1 and btl2) and cysteinyl lts (cyslt1 and cyslt2) are cell surface g protein - coupled receptors . Additionally, some studies support the existence of other cyslt receptors [5, 6]. Some cells express both btls and cyslts; however, the expression of these receptors differs in different cells types . In addition, these receptors are also expressed on peripheral blood leukocytes [7, 8]. Lt receptors and 5-lo are expressed mainly in immune cells, and lts play important roles in innate and adaptive immune responses and are involved in several inflammatory and infectious diseases [4, 9]. For example, cyslts increase vascular permeability and edema, and ltb4 is involved in leukocyte chemotaxis, lysosomal enzyme secretion, neutrophil degranulation, adhesion molecule expression, defensins and nitric oxide (no) production, phagocytosis, and other functions . Lts are produced during the interaction of phagocytes and microorganisms in vitro and experimental infections in vivo . Pharmacologic or genetic approaches to reduce or block the lt biosynthesis pathways decrease the phagocytic and antimicrobial activities against bacteria, fungi, and parasites [12, 13]. In addition, immunodeficient individuals, such as hiv patients, are characterized by low lt production, which has been associated with impaired immune responses and infection control . Lts play important roles in both th1 and th2 immune responses, which are involved in the defense against protozoan and helminth infections, respectively . In light of the current research on the role of lts in infectious diseases, we have divided the current review into two sections focusing on (1) protozoan infection and (2) helminth infection . Each year, protozoan parasites infect many people worldwide, mainly in developing countries, causing serious health, political, social, and economic problems . The major protozoan parasites with clinical importance for human diseases are plasmodium ssp, leishmania ssp, trypanosoma cruzi, toxoplasma gondii, trichomonas vaginalis, and entamoeba histolytica [1517]. The first three of these organisms are obligate intracellular protozoan parasites that are transmitted to vertebrate hosts by insect vectors . T. gondii is also an obligate intracellular protozoan parasite; however, its transmission to human hosts occurs by ingestion of raw or undercooked meat containing tissue cysts or food or water contaminated with oocysts . T. vaginalis is transmitted sexually (trophozoites) and e. histolytica is transmitted through food and water contaminated with cysts [1517]. Protective immunity against protozoans is mediated mainly by t helper 1 (th1) responses which are characterized by the production of inflammatory cytokines, such as il-12, which is required for the development of the th1 immune response, and interferon gamma (ifn-) and tumor necrosis factor alpha (tnf-), which activate macrophages to produce no, which is involved in the control of parasite replication [16, 1820]. Reiner and malemud [21, 22] conducted the first studies to demonstrate the role of leukotrienes in protozoan infection (leishmania spp). The main effects of lts, in both innate and adaptative immune responses, during the protozoan infections are illustrated in figure 1 . Mouse strains resistant (c57bl/6) to leishmania infection mount th1 immune responses against leishmania . In contrast, infection of susceptible mouse strain (balb / c) is associated with the development of a th2 immune response . In vitro studies have demonstrated increased ltc4 production in splenocytes and macrophages from l. donovani - infected or uninfected balb / c mice upon stimulation with nonspecific (phytohemagglutinin) or specific (l. donovani amastigotes) stimuli [21, 22]. In another study, splenocytes from balb / c mice stimulated with antigens from l. major promastigotes displayed increased ltb4 and il-4 production with concomitant decreases in ifn- and tnf- production . . Demonstrated an increase in the parasite burden of balb / c macrophages infected with l. amazonensis when compared to macrophages from the resistant mouse strain c3h / hepas . This effect was associated with lower levels of ltb4 in macrophages from balb / c mice . In agreement with this finding, macrophages from either susceptible or resistant mice treated with mk0591 (flap inhibitor) and u75302 (blt1 antagonist), but with not mk571 (cyslt1 antagonist), as well as macrophages derived from 5-lo - deficient mice, exhibited decreased leishmanicidal activity . Interestingly, treatment with exogenous ltb4 or ltd4 favored parasite killing by macrophages from balb / c mice . Supporting these in vitro results, susceptible and resistant mice treated with zileuton (inhibitor of 5-lo) or 5-lo - deficient mice infected with l. amazonensis displayed larger footpad lesions than nontreated or wild type animals . The success of lutzomyia longipalpis, an insect vector of the leishmania ssp, at blood feeding on mammals depends on the inhibition of the immediate inflammatory response (e.g., increased vascular permeability, swelling, pain, and itching). It is well known that active substances in the saliva of hematophagous arthropods facilitate the uptake of blood by counteracting host hemostatic, inflammatory and immunological defenses [2528]. Mixed lysates from the salivary glands of l. longipalpis significantly increased the cutaneous lesions and/or parasite loads in the footpads of mice infected with l. major or l. braziliensis when compared to infected animals not exposed to the saliva lysates [29, 30]. In addition, the modulation of infection by saliva was il-4-dependent . In agreement with these results, the salivary gland extract of l. longipalpis exhibited anti - inflammatory activities by decreasing tnf- and ltb4 production, neutrophil numbers, and ltb4-induced chemotactic activity in a murine ovalbumin - induced peritonitis model ., these findings suggest that lts, and particularly ltb4, play a role in immune response to leishmania infection by promoting leishmanicidal activity and consequently, control of infection . Therefore, the modulation of ltb4 during infection in association with the modulation of the immune system during leishmania transmission (by saliva from the insect vector) in synergism with genetic factors (susceptibility; th2) could markedly affect leishmania infection in humans . The components derived from the saliva of the arthropod vector of malaria (e.g., anopheles stephensi) have also pharmacologic effects, such as inhibition of inflammation and coagulation, similar to those observed in the saliva of insect vectors of leishmania . In addition, these proteins also have the ability to neutralize inflammatory small molecules by rapid binding . The anst - d7l1 protein produced by a. stephensi binds cyslts (ltc4, ltd4, and lte4) but does not chemically modify them . Anst - d7l1 effectively inhibited ltc4-induced ileal contraction by binding ltc4, thereby preventing interactions between this molecule and its appropriate cellular receptor . The effects of ltc4 inhibition on the course of malaria infection as well as the influence in the malaria pathogenesis are not known . In the experimental cerebral malaria model, mice infected with plasmodium berghei showed increased ltb4 production in the serum . Interestingly, treatment with aspirin, which may direct arachidonic acid metabolism away from the cyclooxygenase (cox) pathway and toward the lo pathway, induced increased parasitemia and death of infected mice . This effect was associated with the overproduction of ltb4 in the serum . In agreement with these results, children with cerebral malaria treated with salicylate demonstrated complications of severe malaria (metabolic acidosis, hypoglycemia, and death). Although ifn- plays a protective role in malaria infection, it has also been associated with the immunopathology of cerebral malaria [36, 37]. Besides playing a role in initiating the th1 immune response mediated by dendritic cells, therefore, the overproduction of ltb4 after aspirin treatment in experimental and human cerebral malaria could be associated with the overproduction of ifn-. Further studies are needed to evaluate this hypothesis . Eryptosis, or suicidal death of erythrocytes, which occurs in a wide variety of diseases including malaria, is characterized by cell shrinkage, membrane blebbing, and exposure of phosphatidylserine (ps) at the cell surface . Like apoptotic cells, ps - exposing erythrocytes are identified by macrophages and are engulfed, degraded, and removed from the circulation . Demonstrated increased phagocytosis of mutant red blood cells infected with trophozoites of p. falciparum, which may represent a protective mechanism against infection . Remarkably, an in vitro assay demonstrated that erythrocytes were able to produce cyslts upon energy depletion . These effects were inhibited by cyslt1 receptor antagonists and by the 5-lo inhibitor (bw b70c). These results suggest that ltc4 might confer protection during the course of malaria by accelerating the clearance of infected erythrocytes . On the other hand, excessive eryptosis might favor the development of anemia; thus, ltc4 might have a dual effect in malaria pathogenesis . During t. gondii infection, an efficient immune response is important to contain dissemination of the parasite and to prevent mortality of the host . Ltc4, ltd4, and free aa were detected when murine macrophages from swiss mice were cultured with viable t. gondii . In contrast, when macrophages from resistant mice (balb / c; major histocompatibility complex haplotypes h2) or human macrophages were cultured with viable t. gondii, no 5-lo products were observed . Accordingly, prior incubation of human macrophages with viable t. gondii decreased the ltb4 release induced by the calcium ionophore a23187, suggesting that t. gondii inhibits ltb4 production . Treatment with zileuton (an inhibitor of 5-lo) decreased the toxoplasmacidal activity of ifn- in human macrophages, whereas exogenous ltb4 promoted intracellular killing of ingested t. gondii in human monocytes . This effect might be associated with the effect of ltb4 on the induction of cytotoxicity (surface membrane vesiculation, extravasation of cytoplasmic contents into a space between the intermembrane spaces and cytoplasmic vacuolization) in t. gondii tachyzoites [47, 48]. In agreement with these results, 5-lo - deficient mice infected with t. gondii displayed decreased survival as a consequence of an excessive inflammatory response characterized by elevated il-12 and ifn- concentrations in the serum and cd4 and cd8 t - cell infiltration in the brain tissue and not of increased parasitic burden . The increased inflammation in the absent of lts might indicate a compensatory mechanism to control the parasite infection . Taken together, these findings suggest that the downregulation of lts production, and particularly of ltb4, by t. gondii might be considered an evasion mechanism, as this lipid mediator can promote cytotoxicity and toxoplasmacidal activity studies by our group and others have demonstrated reduced lt synthesis (e.g., ltb4) in hiv - infected subjects [14, 50]. Although the clinical manifestation of t. gondii infection is usually asymptomatic in immunocompetent individuals, immunocompromised individuals, such as hiv - seropositive patients, exhibit reactivation of latent tissue cysts (bradyzoites become tachyzoites) and consequent toxoplasmic encephalitis or retinochoroiditis [51, 52]. Interestingly, in agreement with these results, the ltb4 and ltc4 concentrations in the cerebrospinal fluid of hiv-1-seropositive patients with toxoplasmic encephalitis but not those of hiv-1-seropositive patients without inflammatory disease or encephalitis were below the detection limit . These results support those described above and suggest that the reduced basal production of lts in hiv-1-seropositive patients synergizes with the suppression of lts by t. gondii . Moreover, this synergistic decrease in lt production might contribute to the pathogenesis of cerebral toxoplasmosis through the increased reactivation of bradyzoites from tissue cysts and the reduced control of the parasitic infection . Protective immunity against toxoplasmosis and chagas disease is mediated by th1 cells, cd8 t cells, and ifn- . Chagas' heart disease is a severe clinical manifestation of trypanosoma cruzi infection . In chronic chagas disease, cardiomyopathy is observed as an inflammatory process characterized by the infiltration of t cells and macrophages, resulting in myocarditis, fibrosis, and heart fiber damage . Treatment with lt inhibitors has demonstrated beneficial effects in cardiovascular pathologies [55, 56]. T lymphocytes from patients with chronic chagas' heart disease or from chagasic mice show increased contractile activity (positive inotropic and chronotropic effects) of heart (atrial) in an in vitro assay . Interestingly, pretreatment with lipoxygenase inhibitors (ndga) or a cyslt receptor antagonist (fpl 55712) decreased this effect . In a separate study, ltc4 production was observed in the supernatants of murine atria cocultured with t lymphocytes from chagasic mice . In accordance with these results, ltb4 induces chemotaxis of lymphocytes (cd4/cd8 t cells) [8, 59]. Therefore, lts might modulate the cardiac pathology of chagas disease by modulating the immune response profile during this infection . Ltb4 and ltc4 also increased the phagocytic and trypanocidal activity of murine macrophages incubated with t. cruzi trypomastigotes in vitro . In addition, ltb4 restored no and tnf- levels, which were decreased by an ltb4 receptor antagonist (cp-105,696). Cp-105,696 treatment also decreased the trypanocidal activity of ifn- in murine macrophages . With the use of pharmacologic (ltb4 receptor antagonist and lo inhibitors) and genetic approaches (5-lo - deficient mice), in addition, the following anti - inflammatory profiles were observed in t. cruzi infection: (1) decreased leukocyte infiltration in the heart; (2) reduced numbers of cd4, cd8, and ifn--producing cells in the heart; (3) decreased fibrosis in cardiac tissues; (4) decreased inos expression and no production in the heart; (5) decreased tnf- and ifn- in the heart; (6) increased il-10 in the heart; and (7) decreased oxidative stress in erythrocytes [6365]. The survival of 5-lo - deficient mice was greatest when the animals were infected with low number of parasites when compared to animals infected with higher number of parasites . Taken together, these findings suggest that lts, and specifically ltb4, play important roles in the control of chagas disease . The supernatant of viable t. vaginalis induced increased ltb4 production in neutrophils in an igg- and complement-(c5-) dependent manner . This effect was decreased by sc-41930 (ltb4 antagonist) treatment . In the vaginal discharges from patients with vaginal trichomoniasis, shaio and lin demonstrated a positive correlation between neutrophils and ltb4 production in symptomatic patients when compared to asymptomatic patients . Peritoneal and splenic macrophages from nave mice incubated directly with e. histolytica trophozoites or with their excretory / secretory products show increased ltc4 production . On the other hand, peritoneal and splenic macrophages from e. histolytica - infected mice produced low levels of ltc4 . The downregulation of ltc4 by e. histolytica in inflammatory but not nave macrophages might be associated with the pathogen's evasion mechanisms . Over one - third of the human population is infected with one or more species of helminths [69, 70]. Although host immune responses attempt to control or expel the parasites, these organisms can develop evasion strategies to modulate the innate and adaptive immune responses, allowing them to survive . The most prevalent human helminthiases are caused by nematodes (e.g., ascaris lumbricoides, strongyloides spp ., enterobius vermicularis, and trichuris trichiura), including filarial worms (e.g., brugia malayi and wuchereria bancrofti), hookworms (e.g., ancylostoma duodenale and necator americanus), and trematodes (schistosoma spp). Asthma and helminthiasis present similar features and are both controlled by a cd4 t - cell immune response . Initial exposure of the immune system to allergic or parasitic antigens leads to the activation of a subset of t cells known as th2 cells, which orchestrate the immune response to these exogenous antigens by secreting cytokines, including il-4, il-5, and il-13 [7174]. In addition, the accumulation of eosinophils in the blood (eosinophilia), as well as in different organs and tissues, is a hallmark of both diseases . Eosinophils are multifunctional cells that are involved in tissue damage as a consequence of the release of cationic proteins [7679]. In addition, eosinophils are important sources of various inflammatory and regulatory cytokines, chemokines, and lipid mediators, such as lts [78, 80, 81]. During a helminth infection such as a nematode infection, most of the ige produced binds to mast cells and basophils through their high - affinity ige fc receptor (fcri) [82, 83]. Subsequent exposure of immune cells to parasitic antigen induces the degranulation of ige - sensitized mast cells and the release of both preformed and newly generated mediators . These mediators, such as lts, function alone or in conjunction with th2 cytokines to increase the contractility of smooth muscle cells, the permeability of epithelial cells and the production of mucus, thereby contributing to worm expulsion . The experimental gastrointestinal infection of rats with the nematode trichinella spiralis demonstrated that preimmune rats (previously infected with t. spiralis) expelled the nematode t. spiralis more rapidly than nonimmune rats . This effect was associated with the increased production of ltb4 and ltc4 in the gut homogenate as well as the release of rat mast cell protease ii (rmcpii) in the serum [85, 86]. Ltc4 causes smooth muscle contraction, increases vascular permeability, and stimulates mucus hypersecretion, and ltb4 recruits and activates inflammatory cells such as eosinophils to favor the expulsion of helminths . Therefore, leukotrienes released from mast cells may effectively participate in protective immune responses resulting in the rapid expulsion of t. spiralis and possibly other helminths . The main effects of lts, in both innate and adaptative immune responses, during the helminth infections are illustrated in figure 3 . Parasitic worm survival in the host for longer periods depends on the ability of the parasite to evade the host immune system . The aba-1 protein from ascaris lumbricoides (human parasite) and ascaris suum (pig parasite) the interaction between aba-1 and leukotrienes might be associated with an evasion mechanism; however, further studies are needed to evaluate the ability of this interaction to inhibit the biologic effects of lts in vitro or in vivo . Brugia malayi is a nematode (roundworm) that can cause lymphatic filariasis in humans . The infective larvae (l3) of brugia malayi are transmitted to a vertebrate host by an insect vector and undergo two molts to develop into adult worms and complete the life cycle . Interestingly, treatment with inhibitors of lipoxygenases (aa861) or cyslt biosynthesis (ethacrynic acid or acivicin) or with a cyslt1 antagonist (zafirlukast) inhibited the brugia malayi l3 larvae from molting to the l4 stage without altering their survival or motility . In contrast, u-75302, an antagonist of the ltb4 receptor btl1, failed to inhibit molting . The -glutamyl transpeptidase, the enzyme that converts ltc4 to ltd4, has been cloned from brugia malayi (adult worms). In another filaria that causes human infection, dirofilaria immitis, the glutathione s - transferase, which can function as an ltc4 synthase, these results demonstrated that a lipoxygenase pathway involved in the generation of cyslts could be required for molting of the infectious larvae and may possibly have some role in the adult worm . In vivo models of infection with b. malayi could be used to better understand the role of cyslts in the pathogenesis of filariasis . It is widely known that some types of infections in immunocompromised individuals are critical in determining the severity of the disease . The immunosuppression observed in hiv - seropositive subjects has been associated with strongyloides spp infections of abnormally high intensity . Interestingly, reduced lt production was observed in hiv - seropositive patients . In an experimental model that mimics human strongyloidiasis (mice infected with strongyloides venezuelensis), an increase in the concentration of ltb4 but not of ltc4 was observed in the lung and small intestines . In addition, increased larvae recovery in the lung and/or increased worm burdens in the intestines were observed in animals treated with mk886 (a selective inhibitor of 5-lipoxygenase - activating protein (flap)) and in 5-lo - deficient mice than in control animals . Moreover, treatment of animals with mk886 resulted in decreases of igg1 and ige levels in serum, eosinophil numbers in the blood, peritoneal cavity and bronchoalveolar fluid volumes and il-5 concentrations in the lung homogenate as well as increased levels of il-12, which is involved in the th1 response . Il-5 is the major cytokine involved in the accumulation of eosinophils in the blood during allergic inflammation and parasitic infections . This cytokine is essential for eosinophil migration from the bone marrow to the blood [72, 95] and specifically supports the terminal differentiation and proliferation of eosinophil precursors as well as the activation of mature eosinophils [9699]. Ltb4 regulates il-5 production by human t lymphocytes and consequently contributes to parasite elimination . These findings suggest that lts, and specifically ltb4, might be necessary to control s. stercoralis infection . Thus, the reduced levels of ltb4 observed in hiv - seropositive subjects might favor opportunistic hyperinfection with s. stercoralis; however, further human studies are needed to evaluate this association . Toxocara canis is an intestinal parasite of dogs and is the etiologic agent of toxocariasis, also known as visceral larva migrans syndrome (vlms). Infection of both humans and animals with t. canis is characterized by eosinophilia in the blood and tissues, increased total serum ige, and inflammation of the upper respiratory system [72, 95, 101104]. During the inflammatory response, leukocyte recruitment is directly related to the expression of adhesion molecules, which allows the transmigration of these blood cells to the tissues . It has been proposed that the 2 integrin mac-1 (cd11b / cd18) and the 1 integrin vla-4 (cd49d / cd29) adhesion molecules are the major molecules involved in cytokine- and chemokine - induced adhesion and migration of eosinophils in vitro [106, 107]. T. canis infection causes early upregulation of mac-1 with late changes in vla-4 profiles on both peritoneal cavity fluid and bronchoalveolar lavage fluids, whereas mk886 treatment promoted the opposite effect . In addition, lt inhibition had a clear impact on eosinophil recruitment to tissues and on blood eosinophilia throughout the course of infection . In another study, in addition to increased eosinophil numbers, the researchers showed increased numbers of mast cells in the peritoneum, lungs, and small intestines of t. canis - infected rats . Interestingly, these animals increased the concentration of ltb4 in the serum and this was correlated with mast cell and eosinophil accumulation and/or recruitment . Thus, lts might play an important role in eosinophilic inflammation during toxocariasis by inducing leukocytes recruitment and modulating the expression of adhesion molecules . In schistosomiasis, lts can control parasite infection by modulating immune responses and through direct cytotoxic effects on the parasite . Ltb4, but not cyslts (ltc4 and ltd4), enhanced the ability of neutrophils and eosinophils to kill the schistosomula of s. mansoni in a complement - dependent manner . The cytotoxicity of eosinophils against helminths has been associated with the expression of cellular receptors (high affinity ige receptor, fcri) and adhesion molecules and with degranulation and the release of cationic proteins . In an in vitro assay, ige - coated schistosomula induced eosinophil adherence, resulting in the death of the parasites . In addition, the release of ltc4 was observed during this interaction . In agreement with this finding, schistosomula can produce ltb4 and ltc4 . The function of lts in schistosomula is not known; however, their production might accelerate parasite elimination and/or modulate the pathogenesis of schistosomiasis . In addition to the proteolytic enzymes produced by cercariae, host - derived skin essential fatty acids and lts including ltb4 also play important roles in the penetration of the skin by the parasite . In an in vitro assay, increased penetration rates were correlated with increased lts levels . In addition, penetration was reduced upon treatment with a 5-lo inhibitor [116, 117]. Hepatic stellate cells (hscs) are involved in liver remodeling due to collagen production and deposition of extracellular matrix as a consequence of proliferative and fibrogenic phenotypes induced by several mediators (cytokines, lipid peroxide, and others). Mrna for 5-lo, flap and ltc4-synthase and 5-lo expression was observed in hscs from schistosomal granulomas of s. mansoni - infected mice . Consequently, these cells produced cyslts, but not ltb4, and the production of cyslts was increased upon treatment with transforming growth factor beta (tgf-, a fibrogenic cytokine). The proliferation induced by tgf- in hscs from schistosomal granulomas of s. mansoni - infected 5-lo - deficient mice or wild type mice treated with zileuton (5-lo inhibitor) was reduced . In addition, ltc4 induced tgf- production, suggesting a synergic effect in schistosomal granulomas . In another study, dipeptidases were isolated from extracts of hepatic granulomas of mice infected with s. mansoni; these enzymes increased the hydrolysis of ltd4 to lte4, potentially accelerating the metabolism of lts and decreasing their effects on liver remodeling . Moreover, ltb4 and ltc4 are produced by schistosomula and adult females, while males produced only ltb4 . Together, these results suggest that cyslt inhibition might influence liver remodeling in s. mansoni infection . In this way, cyslt1 antagonists (such as montelukast, zafirlukast, and pranlukast) [4, 122, 123], which are currently used in asthma treatment, could be evaluated for their effects on schistosomal granuloma remodeling in experimental or human schistosomiasis . Similar to schistosomiasis, fasciolosis causes liver alterations, which can range from fibrosis to cirrhosis . Fasciolosis is considered both a human health concern and a veterinary problem (zoonoses). During the course of f. hepatica infection in sheep, a reduction in serum ltb4 was observed when compared to control animals . Interestingly, ltb4 was produced in both the culture supernatant and the homogenate of f. hepatica adult parasites recovered from the bile duct 20 weeks after infection . Moreover, recruitment of leukocytes consisting mainly of eosinophils, macrophages, and lymphocytes was observed in the livers of goats infected with f. hepatica . In this way, ltb4 produced by host inflammation in synergy with that produced by the parasite could contribute to liver alterations and consequent pathology . Lts are associated with the control of helminth and protozoan infections through their ability to modulate inflammatory processes and/or to promote direct cytotoxicity of protozoans . In addition, lts may also be associated with exacerbated pathogenesis in protozoan diseases, such as cerebral malaria, and helminthic diseases, such as schistosomal granulomas . Interestingly, some helminths (b. malayi) might use the lts to complete their development to adult worms . In addition, other parasites produce lts (s. mansoni and f. hepatica) or produce enzymes involved in lt biosynthesis (dirofilaria immitis). Taken together,
The term replication stress describes the slowing or stalling of replication forks by endogenously or exogenously derived impediments to dna polymerization (zeman and cimprich, 2014). Replication stressors can be local factors, such as dna damage or secondary structures that affect forks randomly as they are encountered, or global ones, such as nucleotide pool depletion or imbalance that simultaneously slows all forks (poli et al ., 2012, it is now recognized that replication stress induced by nucleotide pool imbalance is an important consequence of the activation of some oncogenes, which drive cells into s phase without upregulation of nucleotide supply (bester et al ., 2011). The resulting loss of polymerase processivity is thought to lead to localized uncoupling of the replicative helicase and polymerase and formation of tracts of single - stranded dna (byun et al ., 2005, pacek and walter, 2004). While this normally induces checkpoint activation and senescence (bartkova et al ., 2006,, in cells that can bypass the checkpoint, such replication stress provides a fertile source of genetic instability, particularly in the vicinity of fragile sites and sites capable of forming secondary structures (de and michor, 2011, tsantoulis et al ., 2008). In addition to the extensive genetic changes that have been well documented in many types of cancer, there are also extensive local and global alterations in histone and dna modifications . The consequent changes in chromatin structure are accompanied by significant dysregulation of gene expression (timp and feinberg, 2013, berdasco and esteller, 2010), which, since it is not accompanied by changes in the dna sequence, may be considered epigenetic (berger et al ., 2009). These epigenetic changes could act alongside genetic instability to produce clonal variation within a tumor, upon which selective pressure can act, and so may contribute to tumor evolution . Mutations in histone and dna - modifying enzymes, and even histone proteins themselves, have been found in several cancers and are likely to explain at least some of the observed epigenetic instability (timp and feinberg, 2013). However, it is not clear that mutations in histone - modifying enzymes account for all the alterations observed in different cancer types . We recently provided evidence that deficiencies in enzymes responsible for replicating g quadruplex (g4) structures, such as the specialized dna polymerase rev1 and helicases fancj, wrn, and blm, can lead to localized changes in histone modifications and gene expression (sarkies et al ., 2010, sarkies et al ., 2012, schiavone et al ., 2014). G4s can form within motifs comprising four short runs of dg bases, separated by linker sequences . The dg bases in the motif form planar hoogsteen - bonded quartet structures that can stack on top of each other, resulting in an often highly thermodynamically stable secondary structure, the g4 (reviewed in maizels and gray, 2013). We proposed that persistent replication fork stalling at g4s in mutants such as rev1 or fancj leads to pathologically long daughter strand gap formation, resulting in local uncoupling of dna synthesis from parental histone recycling . This, in turn, leads to loss of the histone modifications present on the parental chromatin, which, if in the vicinity of a gene promoter, results in changes in transcription (sarkies et al ., 2010, sarkies et al ., 2012, schiavone et al ., a prediction of this model is that global replication stressors that lead to loss of processive dna polymerization with uncoupling of the replicative helicase and polymerase also should promote epigenetic instability by dissociating dna synthesis from histone recycling . Here we test this hypothesis by examining the effect of hydroxyurea (hu)-induced nucleotide pool depletion on the epigenetic stability of a sensitive reporter locus, bu-1, in chicken dt40 cells (sarkies et al . We show that chronic treatment with low - dose hu induces stochastic instability of bu-1 expression, characterized by loss of the chromatin marks h3k4me3 and h3k9/14ac seen in the normally active locus . This instability depends significantly on the presence of a g4 motif 3 of the promoter, oriented to stall the leading strand of a fork heading toward the transcription start site (tss). The presence of this g4 motif not only increases the rate at which bu-1 expression is lost, but is additionally associated with phosphorylation of h2ax and appearance of the heterochromatic mark h3k9me3 . This is consistent with the g4 acting to focus dna damage induced by the global replication stress imposed by hu, with the damage leading to repression of the locus . Further, we show that, across the genome, chronic exposure to hu results in an altered pattern of gene expression similar to that seen in cells lacking the g4-unwinding helicases fancj, wrn, and blm, and that affected genes are enriched in g4 motifs . Together, these observations indicate that nucleotide depletion can combine with naturally occurring dna secondary structures to promote epigenetic instability . We first sought conditions in which we could culture dt40 cells in low - dose hu such that replication is slowed but completed (alvino et al ., 2007). We therefore exposed wild - type dt40 cells to a range of hu concentrations and monitored their doubling time . The cells were able to proliferate for over a week in up to 150 m hu (figure 1a). At this dose, their doubling time increased from 12.3 to 32.7 hr, recovering when the hu was washed out (figure 1a). To determine the effect of low - dose hu on replication dynamics, we performed dna molecular combing after pulse labeling the cells with halogenated nucleotides (figure s1a) 3 days after initiating culture in hu . Average fork velocity decreased from 1.26 to 0.71 kb / min (figure 1b), with a compensatory decrease in average interorigin distance from 72 to 40 kb (figure 1b). Consistent with these perturbed replication dynamics, cell - cycle profiles revealed a significant accumulation of cells in s phase while in hu (figure s1b). We have reported previously that replication - dependent transcriptional instability associated with g4 motifs can be monitored by following expression of a surface marker, bu-1a, in dt40 cells (sarkies et al ., 2012, schiavone et al ., 2014). The bu-1 locus contains prominent g4 motifs 3.5 kb downstream of the tss and 3 kb upstream . Both are orientated to be g - rich on the feature strand with respect to the bu-1 transcript (figure 1c). Epigenetic instability of bu-1 in rev1 cells is entirely dependent on the + 3.5 g4 motif, and it requires the motif to be orientated such that its g - rich strand forms on the leading strand of a replication fork entering the locus from the 3 end (figure 1c; schiavone et al ., 2014). We have reported previously that the bu-1 locus is bidirectionally replicated, meaning that during any given s phase there is a 50% probability of the + 3.5 g4 being replicated on the leading strand template (schiavone et al ., 2014). Growth of wild - type dt40 cells in 150 m hu resulted in the appearance of a bu-1a population as cells divided over the course of 7 days (figure 1d). Surface expression of bu-1a correlates closely with transcript abundance (sarkies et al ., 2012), and this held true for bu-1a clones recovered after hu treatment (figure s1c). To estimate the rate at which bu-1a variants are formed in hu, we performed a fluctuation analysis by expanding multiple parallel populations of 10 bu-1a cells in hu for 7 days, after which we monitored the appearance of bu-1a variants . This revealed a striking degree of expression instability despite the small number of cell cycles through which the cells had passed (figure 1e). Using our previously described monte carlo simulation of bu-1a loss as a replication - dependent phenomenon (schiavone et al ., 2014), we estimated a per - division probability of generating of a bu-1a state during culture in hu of c. 0.15 . To obtain additional evidence that this induced transcriptional instability of bu-1 reflected decreased dna polymerase processivity, we asked whether bu-1 variants could be induced by aphidicolin . Aphidicolin slows replication by directly inhibiting dna polymerases, particularly pol (oguro et al ., 1979), and a low dose induces replication stress (pacek et al ., 2006). Dt40 cells were able to proliferate in up to 150 m aphidicolin for 10 days and, as with low - dose hu, this resulted in substantial instability of bu-1a expression (figure 1f). We have shown previously that removal of the + 3.5 g4 motif from both alleles of bu-1 in rev1-deficient cells results in complete stabilization of expression of the locus (schiavone et al ., 2014). We therefore examined the extent to which this motif also accounted for the observed hu - induced instability of bu-1a expression in wild - type dt40 . We grew wild - type cells lacking the + 3.5 g4 on both alleles, bu-1 (schiavone et al ., 2014), in hu and assessed the frequency of bu-1a variants after 7 days by fluctuation analysis (see figure e3 in schiavone et al this revealed that removal of the + 3.5 g4 motif resulted in a significant reduction in the rate at which bu-1a variants were generated . However, it did not result in complete stabilization of the locus (figures 2a and 2b, i and ii). We considered the possibility that the residual instability could be due to the 3.0 g4 upstream of the tss . However, deleting this motif (figure s2) had no impact on hu - induced instability (figure 2b, iii). To confirm the contribution of the + 3.5 g4 motif, this resulted in the return of the high - level hu - induced instability of bu-1 expression observed in wild - type cells (figure 2c, i). However, this was not seen if the motif was mutated to render it incapable of forming an intramolecular g4 (figure 2c, ii) or when it was inverted so that the g4 structure would form on the lagging strand template (figure 2c, iii). Thus, hu treatment alone can induce instability of bu-1a expression, but its effect is significantly potentiated by the presence of a g4-forming sequence orientated to stall the leading strand replication of a fork heading toward the tss . We next investigated the basis for the hu - induced generation of bu-1a variants . To test whether the bu-1 state is permanent, we isolated five bu-1 clones at the end of 1-week growth in hu and cultured them for a further 3 weeks in hu - free medium . The clones remained stably bu-1 with no evidence of reversion to bu-1, suggesting that this was a permanent change . We considered the possibility that bu-1 cells resulted from genetic changes in the locus, although the observed rate of mutation would be extraordinarily high for this to be the case . We therefore sequenced the region around the + 3.5g4 to look for mutation of the motif and used pcr with restriction digestion to detect larger deletions (figure s3). Neither assay revealed any evidence of genetic instability consistent with the formation of bu-1 variants being an epigenetic event . We therefore examined the pattern of histone modification at the bu-1 promoter by chromatin immunoprecipitation (chip) from bulk populations of cells exposed to hu . Bu-1 is a transcriptionally active locus characterized by high levels of h3k4me3 around its tss . After 48-hr treatment with hu, we observed a small but not significant loss of h3k4me3 at the bu-1 promoter (figure 3a), consistent with the size of the population of bu-1a cells generated by this time point (figure 1d). However, after 7-day treatment, we observed a more significant loss of h3k4me3 correlating with the much larger population of bu-1a cells at this time point (figures 1d and 2a). The loss of h3k4me3 was accompanied by a reduction in h3k9/14 acetylation (figure s4). Seven days of hu treatment also induced a marked increase in h3k9me3 at the bu-1 promoter (figure 3a). It has been proposed previously that hu - induced displacement of parental h3/4 and its buffering by the histone chaperone asf1 may lead to unscheduled heterochromatinization by ectopic deposition of pre - marked histones upon their release from asf1 (jasencakova et al ., 2010, schwab et al ., 2013). Alternatively, the appearance of h3k9me3 may result from dna damage - induced heterochromatinization, which has been observed following double - strand breaks (ayrapetov et al . Breaks can arise from fork collapse in hu (petermann et al ., 2010), and thus, if unscheduled incorporation of h3 with k9 methylation was responsible, then an increase in h3k9me3 would be observed irrespective of whether the + 3.5 g4 motif was present . However, if localized g4-induced dna damage was responsible, then the appearance of h3k9me3 would be dependent on the + 3.5 g4 motif . We therefore examined h3k4me3 and h3k9me3 at the promoter of bu-1 in cells lacking the + 3.5 g4 motif . After 7 days in hu, h3k4me3 was reduced (figure 3b), but to a lesser extent than in wild - type cells (figure 3a), consistent with the reduced rate at which bu-1a variants are generated in cells lacking the + 3.5 g4 motif (figure 2b). However, we observed no associated increase in h3k9me3 (figure 3b). To monitor the extent to which hu induced dna damage in the two situations, we performed chip for phosphorylated h2ax (h2ax) (rogakou et al ., 1998) at the bu-1 promoter . H2ax was enriched 2.5-fold at the bu-1 promoter in wild - type cells after 7 days in hu, but not enriched in cells lacking the + 3.5 g4 motif grown under the same conditions (figure 3c). This favors a model in which heterochromatinization of bu-1 in hu is promoted by dna damage, likely from fork collapse associated with the + 3.5 g4 motif . We next examined the extent to which hu - induced changes in histone modifications were permanent by performing chip at the bu-1 promoter in the five stable bu-1a clones discussed above . This revealed that promoter h3k4me3 remained low, showing that loss of this mark was permanent (figure 3d). Thus, while h3k9me3 is induced by hu in cells containing the + 3.5 g4 motif, this mark is not essential to maintain the bu-1a state . This may be because it is installed only transiently during repair of hu - induced dna damage in the locus, or because cells in which h3k9me3 persists are growth disadvantaged and are lost from the population . The data thus far were consistent with a working hypothesis that reduced polymerase processivity increases the probability of g4 formation at the + 3.5 g4 motif through exposure of more single - stranded dna within the replisome, which, in turn, focuses replication stalling at this site . Implicit in this model is the idea that the + 3.5 g4 can form during replication but that it is usually rapidly resolved to maintain fork progression . We therefore reasoned that trapping the g4 structure using a g4-binding ligand also might induce instability of bu-1a expression in otherwise wild - type cells further, we predicted that g4 ligands and hu would act synergistically to destabilize expression of the locus . To test these ideas, we treated cells with the g4 ligand n - methyl mesoporphyrin ix (nmm) (nicoludis et al ., 2012). We first identified the maximum dose at which the cells retained normal viability and global replication dynamics . At 2 m nmm, the fork rate, as assessed by molecular combing, was 1.13 kb / min compared with 1.26 kb / min in wild - type cells, with no significant change in the inter - origin distance (figure s5). Nonetheless, fluctuation analysis for bu-1a loss in wild - type and bu-1 cells cultured for 7 days in 2 m nmm revealed instability in bu-1a expression in wild - type cells, but not cells lacking the + 3.5 g4 motif (figure 4a). Since 2 m nmm does not in itself significantly reduce global fork rates and the agent will only interact with the formed g4 structure, not with just the linear dna sequence (ren and chaires, 1999), this observation is consistent with transient formation of g4s during normal replication . We next asked whether combining nmm - induced g4 stabilization with hu - induced reduction in polymerase processivity led to a further destabilization of bu-1 expression . Interestingly, use of both drugs together resulted in significant toxicity, meaning that we had to reduce the dose of each drug by 50% in order to carry out the fluctuation analysis . As expected, growth of cells in hu at 75 m or nmm at 1 m individually had little effect on stability of bu-1a expression (figure 4b). However, the combination of nmm and hu at these doses resulted in a significant increase in bu-1a instability, revealing a marked synergy between hu - induced replication stress and g4 stabilization . We replaced the natural + 3.5 g4 motif with a series of four g4 motifs of varying in vitro thermal stabilities (schiavone et al ., 2014). All four motifs (g4 14) potentiated the formation of bu-1a variants upon treatment with hu (figure 5a). Interestingly, we observed no correlation between the degree of potentiation by the motifs and the in vitro melting temperature of the equivalent oligonucleotides (figure 5b). However, there was a significant trend toward greater potentiation of bu-1a loss being associated with longer non - g loops in the range of 1 to 9 bp (figure 5c, solid line). To explore this further, we also tested a single repeat of the g4 motif containing human ceb1 mini - satellite (piazza et al ., 2012), which has 18 bp between its first three and last run of dgs . This g4 motif, but not a mutated form that is incapable of forming a g4 structure in vitro (piazza et al ., 2012), also potentiated bu-1a instability after treatment with hu . However, this was not to a greater extent than the natural + 3.5 g4 dna with its central 9-bp loop, suggesting that there may be a limit after which lengthening the loop has no further effect . Finally, we asked whether we could detect genome - wide evidence of an interaction between hu and g4s . We therefore performed affymetrix expression microarray analysis on cells before and after culture in 150 m hu . Three parallel cultures of dt40 were treated with 150 m hu for 7 days, or mock treated, and then recovered into normal medium for 7 days, after which rna was prepared for array hybridization . Despite this relatively short treatment, a total of 2,937 of 12,920 unique genes exhibited a change in expression of> 0.25 log2 units with p <0.05, with an approximately equal number of genes being upregulated and downregulated (figure 6a). We previously have observed a similarly large number of dysregulated genes in cells deficient in the 5-3 g4-unwinding helicase fancj and in double mutants for the 3-5 helicases wrn and blm (sarkies et al ., further, we found a highly significant overlap in the identity of dysregulated genes in the two sets, the direction in which their expression changed, and the association of the dysregulated genes with g4 motifs (sarkies et al . We anticipated that if transcriptional dysregulation by hu was linked with g4s that there might be a significant similarity in the gene set altered by hu and the sets altered by loss of fancj and wrn / blm . Indeed, the overlap in the identities of the genes dysregulated in all three conditions was highly significant (figure 6b), as were the pairwise correlations in the direction of the change in expression (figure 6c). Nearly 68% of the 6,061 genes within the venn diagram in figure 6b have a g4 motif within 1 kb upstream of the tss and the end of the body of the gene in comparison with 59% of the 6,859 genes in the remainder of the array (p <1 10) (table s1). To ascertain whether the overlaps in the identity of dysregulated genes reflected the perturbation of common pathways in the three datasets, we analyzed the functional annotation terms associated with the genes in each set and in the overlap sets using david (https://david.ncifcrf.gov; huang et al . While treatment with hu resulted in dysregulation of genes with gene ontology (go) terms associated with cellular stress and nucleotide metabolism, a large number of miscellaneous go terms also were enriched to a similar degree (table s2). Significantly, despite the large number of genes overlapping in the three datasets, there was no evidence of their being members of common pathways (figure s6). This is consistent with much of the dysregulation of expression resulting from processes that are not related to a coordinated physiological response either to treatment with hu or ablation of fancj or wrn and blm helicases . Nonetheless, the degree of overlap in the dysregulated transcriptomes in these three conditions suggest that cells treated with hu and cells lacking fancj and wrn / blm face similar challenges . However, the enrichment of g4s in affected genes, while statistically significant, is still relatively modest, suggesting that other factors, such as secondary effects or other dna secondary structures, may be contributing as well . Numerous lines of evidence have linked replication stress with genetic instability (halazonetis et al ., 2008, zeman and cimprich, 2014). Imbalanced or depleted nucleotide pools during replication are an important cause of such stress and can arise from the expression of oncogenes uncoupling entry into s phase from upregulation of nucleotide supply (bester et al ., 2011). Importantly, the dna damage resulting from replication stressors like hu or aphidicolin that act directly on the replicative dna polymerases is not randomly distributed across the genome, but is instead focused on sites that often have features that make them potentially problematic to replicate even under ideal conditions (tsantoulis et al ., 2008). Many of these hotspots also correspond to classical fragile sites, in which chromosome breaks are observed after replication stress . Such sites have been linked to regions depleted in replication origins, meaning that single forks have to traverse long distances (letessier et al ., 2011). Thus, a combination of regions of low fork density and problematic structures may focus sites of fork collapse under conditions of global replication stress (wickramasinghe et al ., 2015). The mechanisms by which replication stress leads to epigenetic changes are less well explored . Alterations in chromatin composition and structure are common features of cancer cells (berdasco and esteller, 2010, hansen et al ., 2011, timp and feinberg, 2013) and are particularly associated with g4-dense breakpoint hotspots (de and michor, 2011). Although the cell line we used in this study, dt40, is itself transformed, we found no evidence of significant stress to the dna replication program under normal growth conditions . However, growth of the cells in low - dose hu recapitulated the key features of the acutely stressed replication observed in oncogene - expressing primary cells (bester et al . We have been able to explore directly the interaction between global replication stress induced by nucleotide depletion and a dna secondary structure to demonstrate how they conspire to exacerbate replication - dependent epigenetic instability . We have provided evidence that two parallel epigenetic perturbations contribute to permanent and transient epigenetic changes following an episode of replication stress . The first mechanism relates to the uncoupling of the activity of replicative helicase and polymerase (byun et al ., 2005, pacek and walter, 2004) during hu treatment . This has been shown to lead to interruption of the normal flow of histones from the parental to the nascent daughter strands, with the histone chaperone asf1 buffering the displaced h3/h4 (jasencakova et al ., 2010). Groth and colleagues suggested that their release from asf1 might lead to local alterations in epigenetic state of chromatin due to unscheduled incorporation of inappropriately marked histones (jasencakova and groth, 2010, jasencakova et al ., 2010). (2013) invoked this model to explain an increase in heterochromatin formation in cells deficient in fancj, suggesting that failure to unwind lagging - strand template g4 structures in fancj - deficient cells led to unscheduled deposition of histones bearing marks that would lead to h3k9me3 and heterochromatin formation . However, this model does not adequately explain the bidirectional changes in gene expression changes seen either in fancj cells or in wild - type cells treated with hu (sarkies et al . In contrast, the model we have developed previously, in which loss of processive replication at g4s leads to localized loss of parental histone mark recycling, could explain both derepression of loci, such as -globin (sarkies et al ., 2010), and loss of activation, as can be observed in the bu-1 locus (sarkies et al . However, since the mechanism by which h3k4me3 is maintained during replication is poorly understood, the precise mechanisms by which replication impediments disturb the maintenance of this mark remain to be fully elucidated this mark is induced alongside the loss of h3k4me3 when the + 3.5 g4 motif is present and cells are exposed to hu . Importantly, the appearance of this mark of heterochromatin is accompanied by h2ax phosphorylation, a marker of dna damage (rogakou et al ., 1998). G4 motifs have been linked to hotspots of genetic instability and translocation (de and michor, 2011). Further, the g4 ligand pyridostatin, which acts similarly to nmm, leads to localized h2ax accumulation at g4 motifs across the genome, suggesting the formation of dna breaks (rodriguez et al ., dna breaks have been shown to induce transcriptional repression (shanbhag et al ., 2010) and to induce h3k9me3 even in a normally euchromatic locus (ayrapetov et al ., 2014). Thus, the appearance of h2ax and h3k9me3 in the bu-1 locus only in hu - treated cells containing the + 3.5 g4 is consistent with collapse or incision of replication forks, already stressed by nucleotide depletion, that have stalled at the g4 . We therefore propose that nucleotide depletion can give rise to loss of parental h3k4me3 and the appearance of h3k9me3 by distinct mechanisms . H3k4me3 is lost stochastically as a result of interruption of parental histone recycling, a mechanism that is locally exacerbated by the presence of a g4 motif . In contrast, we propose that h3k9me3 may reflect protective transient heterochromatinization of the locus during repair of breaks resulting from hu - induced fork collapse at g4 structures (figure 7). In the case of loci like bu-1, in which hu - induced epigenetic instability is linked to g4 formation, an interesting question is whether hu results in a greater opportunity for g4 formation during replication or diminished g4 resolution . It is not currently possible to formally distinguish these possibilities and indeed it is likely that elements of both are true . Notably, the observation that the g4-binding ligand nmm can induce bu-1a expression instability at a dose that does not significantly impact on global replication dynamics provides strong evidence that g4 structures form readily during normal replication and that they are usually promptly resolved . The synergy between replication stress caused by nucleotide depletion and structured dna is of considerable potential importance to understanding the development of cancer . Epigenetic changes are prevalent in many cancer types, although their origin is unclear, and likely complex (berdasco and esteller, 2010, timp and feinberg, 2013). Recently, several instances of epigenetic instability in cancer have been linked to mutations in histone or dna - modifying enzymes . However, the widespread and often apparently random nature of epigenetic changes in tumors suggests that other processes also may be at work . We have suggested previously that delayed replication of g quadruplex structures could contribute to the epigenetic diversity of cancer (sarkies and sale, 2012). However, mutations in enzymes that may cause this form of epigenetic instability, for example rev1, fancj, wrn, and blm (sarkies et al ., 2010, sarkies et al ., 2012), are rarely observed in sporadic cancers . Replication stress, on the other hand, is emerging as an important feature of cancer cells, particularly in the early stages of their evolution (bester et al ., 2011, di micco et al ., 2006, thus, we suggest that some of the epigenetic changes seen in tumors may be explained by problems managing replication blocks . Consistent with this idea, both copy number variations and changes in dna methylation patterns in cancer have been linked to g4 motifs (de and michor, 2011). Finally, it is worth noting that hu is used extensively in treatment of hemoglobinopathies, such as sickle cell disease and thalassaemia, as it can re - induce expression of the fetal -globin gene, ameliorating the effects of the defective adult globins found in these disorders (platt et al ., 1984). Importantly, the effect of hu on -globin expression is unlikely to be specific, since chronic exposure to the drug leads to quite widespread changes in erythroid gene expression (flanagan et al ., 2012). Although the -globin locus in humans has no g4 motifs in the immediate vicinity of its promoter, its key transcriptional regulator, bcl11a (bauer et al ., 2013), has a high density of g4 motifs on both sides of its tss . It will, therefore, be interesting to explore whether the mechanisms we propose here could help explain the action of hu on fetal globin expression . Dt40 cells culture and the strategy for removing and replacing the + 3.5 g4 motif in the bu-1 locus have been described previously (schiavone et al ., 2014). Genetic manipulation of the + 3.5 g4 motif was performed in the bu-1a allele of cells in which the motif had been removed from the bu-1b allele to avoid the transvection - like effect between the alleles (schiavone et al ., 2014). Oligonucleotides are listed in table s3 . For fluctuation analysis, 150 m hu (sigma - aldrich, h8627) after 7 days, cells at a concentration between 0.2 and 1 10 were stained for 20 min at 37c with anti - bu-1a - phycoerythrin (1:100, santa cruz biotechnology clone 5k98, 70447). Bu-1a expression was assessed by flow cytometry using an lsrii cytometer (becton dickinson). Experiments with aphidicolin (sigma - aldrich, a0781) and nmm (frontier scientific, nmm580) were conducted in 96-well plates starting with ten cells expanded for 10 days . Bu-1a cells were isolated after hu treatment using a moflo sorting cytometer (dako - cytomation). Chip was performed as described previously (nelson et al ., 2006) with modifications . Following a 10-min incubation at room temperature with 1% (v / v) formaldehyde, glycine was added to 0.2 m for 5 min . The extracted nuclei were sonicated at 4c using a bioruptor water bath sonicator (diagenode) with 30 cycles of 30 s separated by 30-s intervals . Sheared chromatin samples were resuspended in dilution buffer (1.1% triton x-100, 1.2 mm edta, 16.7 mm tris [ph 8.0], 167 mm nacl supplemented with pmsf, and a protease inhibitor cocktail). For immunoprecipitation, lysates were incubated overnight with the following antibodies at 4c: histone h3 (1:100, cell signaling technology, 2650), h3k4me3 (1:100, cell signaling technology, 9727), h3k9/14ac (1:200, millipore, 17 - 615), h3k9me3 (1:200, abcam, ab8898), h2ax (1:50, abcam, ab2893), and the negative control normal rabbit igg (millipore). Following overnight incubation at 4c with tumbling and four washing steps, the qpcr was performed with power sybr green master mix (applied biosystems, 4367659) on an abi prism real - time cycler with the following cycle times: 50c for 2 min, 90c for 10 min, 45 cycles of 90c for 15 s plus 60c for 1 min . The cdna was made from 5 mg mrna with super rt (ht biotechnology) and oligodt primer in a final volume of 40 l . Dna molecular combing was conducted 3 days into culture with 150 m hu . It was performed and analyzed as previously described (guilbaud et al ., 2011). Rna was extracted from three independent wild - type cell populations treated for 7 days with hu and allowed to recover for another 7 days, as well as from three untreated parallel controls . Microarray analysis was performed using r (http://www.r-project.org/) and its bioconductor packages (gentleman et al ., 2004). Raw cel files were processed using the robust multichip average (rma) algorithm available in the affy package (gautier et al ., 2004). Genes that showed a change of> 0.25 log2 units relative to the mean wild - type intensity, with a p value of <0.05 (t test), were identified as exhibiting statistically significant transcriptional dysregulation . Custom written r scripts were used to identify and plot genes co - dysregulated between different mutants . Venn diagrams were generated with the limma package (smyth, 2004), and significance for the overlaps was calculated using fisher s hypergeometric distribution.
It has been proposed that solar devices based on thin film technologies and third - generation materials can greatly benefit from plasmonic and photonic structures that can assist with light trapping and absorption . One promising strategy is to replace the traditional transparent conducting oxide layer with a semitransparent metallic contact that exhibits a surface plasmon at visible wavelengths . Structured appropriately, for example, by perforating with an array of sub - wavelength holes, the metal film can provide the high transparency and conductivity needed in a transparent electrical contact while also actively assisting with photon management . A recent study demonstrated a high - performing organic photovoltaic cell with a metal contact patterned with an array of sub - wavelength holes, providing the contact with broad featureless transmission throughout the visible spectrum and robust coupling to the semiconductor as a function of incident light angle . Despite this progress, a key challenge is to probe and understand the details of the light - matter interactions between the incident photon, the surface plasmon modes of the structured contact, and the absorption properties of the active area . We have developed a set of small area, high - brightness spectroscopies that are appropriate for characterizing prototypes of light - harvesting devices utilizing plasmonic contact materials, for understanding the details of light - matter interactions in these systems . Our primary interest in developing a set of rapid, high - sensitivity characterization tools capable of probing the influence of the perforated metal nanostructure, including pore size and pitch, as well as film thickness on the optical transmission modes, and ultimately the effect on solar cell performance . The aim is both to enable rapid screening of test device structures and to design structures capable of providing specific physical insight into coupling between the device active area and the modes of the patterned metal film . Our strategy utilizes fourier transform (ft)-based measurements in the solar portion of the optical spectrum, for optimally combining spatial resolution, spectral resolution, speed, and dynamic range . Ft - based measurements are highly advantageous for characterizing nanoscale photovoltaic materials in the visible and near infrared (nir) portions of the electromagnetic spectrum, although they are more typically applied in the infrared . As compared to scanned techniques, ft photocurrent measurements benefit from the multiplex (fellgett) advantage because the detectors are not shot - noise - limited, unlike in optical measurements employing low - noise photodetectors (e.g. Raman scattering with a high sensitivity ccd or pmt). This is particularly important since, unlike dispersive optical measurements (e.g. Transmission, pl, and raman), photocurrent multi - channel detection with scanned sources is not typically possible . For measurements of photovoltaic phenomena, the throughput advantage (jacquinot) is much less relevant due to the lower required resolution, when compared to vibrational or high - resolution absorption measurements . This is particularly true when using a high - brightness light source and a small sample size such that the throughput becomes comparable to a scanned source . However, ft - based measurements still typically have more bandwidth capabilities, as there is no need to switch gratings between the visible and the nir . The key to the experiment is achieving solar (or higher) spectral irradiance levels in a spot size of <100 m in order to achieve high signal / noise external quantum efficiency (eqe) measurements on prototype devices . We have recently demonstrated the power of these types of measurements on individual single - walled carbon nanotube field effect transistors, but the benefit is equally applicable to other structures with active areas of the order of (100 m), such as photonic structures that are difficult and/or time - consuming to fabricate over large areas . Achieving a high irradiance in a small area requires the use of high radiance sources . The alternative increasing the power without increasing the size of the source or solid angle of radiation becomes impractical at these size scales, because most of the photon flux would be wasted . In the mid- to far - ir, similar advantages are gained using synchrotron - based sources for microspectroscopy . Here, we achieve these advantages in the solar spectral region with an inexpensive benchtop source a true - cw high - brightness laser - driven light source (energetiq eq-99). This differs significantly from a laser, in that the output radiates incoherently into 4 rather than being highly directional and coherent . We have also examined laser supercontiuum sources for the same purpose (e.g. Fianium sc-450), which are able to achieve even higher brightness levels due to the highly collimated output . However, for photocurrent and photovoltaic applications, we have found the laser - driven light source to be a much better choice for several reasons . Primarily, the pulsed nature of the laser supercontinuum was found to generate small but detectable electrical transients that added significant noise to low - eqe regions of the spectrum . Since a 200 m core multi - mode fiber is utilized to simplify alignment to the translating microscope optics, this negates the advantage a smaller emitter has in focusing capabilities . Additionally, we have found it difficult to stay in a low - fluence (solar - like) regime with the pulsed laser; focusing ps pulses with the full bandwidth of the laser has resulted in either saturation (resulting in signal non - linearity) or damage to samples and optical detectors, even with significant attenuation of the beam . We did not encounter the same issue for similar power levels in a true cw source . Lastly, the laser - driven light source unit is significantly cheaper than the laser supercontinuum (~5 difference). Similarly, other groups have found that despite the higher spectral power density of a supercontinuum source, the sensitivity of absorption - based measurements are higher with the ldls due to superior long - term stability . The laser - driven light source output is collimated by an off - axis parabolic mirror (1.5 diameter, 2 efl, na = 0.375) and coupled into a commercial ft spectrometer (bruker v80) using the backward input . The light is modulated using a broadband caf2 uv vis - nir beam splitter and the slowest rapid - scan setting available on our instrument (2.5 khz referenced to the hene line). The modulated light is sent to an output port of the spectrometer, where the remaining uv portion of the beam (<420 nm) is filtered out . The broadband light is focused into a 200 m core multimode fiber used to couple the light into an upright optical microscope (nikon fn-1) using a reflective collimating optic and a set of steering mirrors attached to an epi - illuminator . This microscope is designed for a stationary stage, such that electrical probes can be contacted with a sample via micromanipulators . As such, the microscope itself sits on a translating stage for sample visualization and beam alignment . The use of an optical fiber for coupling ensures that the beam stays aligned to the microscope even during translation . A 5050 beam splitter is used to simultaneously image and excite the device under test . For photoconductivity measurements, the photocurrent interferogram is sent through a trans - impedance amplifier (dl instruments, 1210) and an electronic band - pass filter (stanford research, sr650) before being digitized by the spectrometer bench electronics . To correct for the lamp spectrum and the transmission function of the bench and microscope optics, a set of photodiodes with known responsivity curves the resulting single - beam spectra from test devices are corrected by these reference spectra, which are collected at the same time as the unknown devices . The spot sizes at the output of the microscope are measured using a calibrated beam profiler (ophir - spiricon). For transmission spectra, the sample compartment of the spectrometer is used, resulting in a spot size of 0.75 mm at the focus . Samples are aligned to the beam using a xyz translating mount and the transmitted light is detected with a pyrolelectric detector (la - dtgs). We define arrays of sub - wavelength holes across areas of (300 m) by focused ion beam (fib) milling completely through 50 nm thick ag films sputter deposited on glass . Holes with average diameter of 200 nm are milled using a beam of ga ions (0.26 na), with dwell times ranging from 2 to 8 ms for hole separations (pitch) ranging from 240 to 560 nm, respectively . After defining the perforated ag electrode, the subsequent organic photovolatic device fabrication steps include: thermal evaporation of 9 nm thick of moo3 electron - blocking layer; spin coating (700 rpm for 60 s) the 170 nm thick semiconductor active layer, a 1:1 wt . Blend of poly(3-hexlythiophene): phenyl - c61-butyric acid methyl ester (p3ht: pcbm) (3 wt .% in chlorobenzene); and thermal evaporation of a 200 nm al film, which serves as an electron - collecting (and hole - blocking) electrode . We match the source, coupling, and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot . The critical quantity is the optical extent or tendue (g) of the system that will maximize the light throughput to the sample . It is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance, rather than the numerical aperture of the individual optical components . Because g is proportional to both the area of the source and the solid angle into which it propagates, we aim to match the collimating and focusing optics without introducing a bottleneck (e.g. The multimode optical fiber). The combination of the laser - driven light source and a parabolic mirror results in g ~ 3 10 mm sr, a value well matched to a 0.22 numerical aperture fiber with a 0.2 mm core size (~4.5 10 mm sr maximum g). The effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light . The numerical aperture of the objective determines the solid angle, but the spot size is not determined by the nominal magnification, which is referenced to a 200 mm tube lens . We assume that the reference specifying the magnification value is a 200 mm tube lens . Here, the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . As a result, the magnification of the objective is considerably smaller than the 10/50 values specified . For the 10 objective, we obtain nearly 1:1 imaging, with the core diameter of the multimode fiber limiting the ultimate spot size . For example, focusing the broadband ft - modulated light source with a 10 (0.3 numerical aperture) long - working distance objective yields a measured spot size of ~215 m (beam profile shown in figure 1(a)). We can achieve a smaller spot size using the 50 objective, for two reasons . First, the higher numerical aperture results in additional magnification, and second, the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size (thereby reducing the effective source size and the tendue out of the fiber). The net result is a reduction in spot size to ~40 m using the nominal 50 (0.45 numerical aperture) objective . As we discuss below, this small reduction in throughput still results in an overall higher spectral irradiance . These larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . Using a single - mode fiber and a monochromatic solid - state laser, we can achieve near diffraction - limited spots using these same optics . From the measured spot sizes and the focal length of the objectives, we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system . In all cases, the ntendue of the 10 objective is higher than that of the source and fiber, meaning that the limiting factor is not the small area of the detector (i.e. Sample). (a) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective . The spot size is approximately 215 m with the 10 and 40 m with the 50 . (b) spectral irradiance in the visible and near infrared with the 10 and 50 objectives compared to the solar air mass 1.5 standard (astm g-173). (the colour version of this figure is included in the online version of the journal .) With the combination of a high - radiance light source and microscope focusing optics, we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices . The spectral irradiance is shown in figure 1(b) for the output of the ldls microscopy system with the 10 and 50 objectives, along with the solar air mass 1.5 (astm g173 - 03) spectrum for comparison . The key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir, particularly in the visible region of the spectrum, where we achieve in excess of 10 times higher irradiances . These numbers can be further improved since a 50:50 beam splitter (optimized for the visible) is used to direct light into the microscope objective . Replacing the dielectric optic with a metallic one would double the output in the visible, but result in even more significant gains in the nir . However, for the purposes of this study, the irradiances achieved are more than sufficient for our measurements . The overall photon flux achieved is also quite high due to strong focusing of the light beam, exceeding 4.5 10 ms with high spatial resolution . We match the source, coupling, and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot . The critical quantity is the optical extent or tendue (g) of the system that will maximize the light throughput to the sample . It is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance, rather than the numerical aperture of the individual optical components . Because g is proportional to both the area of the source and the solid angle into which it propagates, we aim to match the collimating and focusing optics without introducing a bottleneck (e.g. The multimode optical fiber). The combination of the laser - driven light source and a parabolic mirror results in g ~ 3 10 mm sr, a value well matched to a 0.22 numerical aperture fiber with a 0.2 mm core size (~4.5 10 mm sr maximum g). The effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light . The numerical aperture of the objective determines the solid angle, but the spot size is not determined by the nominal magnification, which is referenced to a 200 mm tube lens . We assume that the reference specifying the magnification value is a 200 mm tube lens . Here, the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . As a result, the magnification of the objective is considerably smaller than the 10/50 values specified . For the 10 objective, we obtain nearly 1:1 imaging, with the core diameter of the multimode fiber limiting the ultimate spot size . For example, focusing the broadband ft - modulated light source with a 10 (0.3 numerical aperture) long - working distance objective yields a measured spot size of ~215 m (beam profile shown in figure 1(a)). We can achieve a smaller spot size using the 50 objective, for two reasons . First, the higher numerical aperture results in additional magnification, and second, the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size (thereby reducing the effective source size and the tendue out of the fiber). The net result is a reduction in spot size to ~40 m using the nominal 50 (0.45 numerical aperture) objective . As we discuss below, this small reduction in throughput still results in an overall higher spectral irradiance . These larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . Using a single - mode fiber and a monochromatic solid - state laser, we can achieve near diffraction - limited spots using these same optics . From the measured spot sizes and the focal length of the objectives, we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system . In all cases, the ntendue of the 10 objective is higher than that of the source and fiber, meaning that the limiting factor is not the small area of the detector (i.e. Sample). (a) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective . The spot size is approximately 215 m with the 10 and 40 m with the 50 . (b) spectral irradiance in the visible and near infrared with the 10 and 50 objectives compared to the solar air mass 1.5 standard (astm g-173). (the colour version of this figure is included in the online version of the journal .) With the combination of a high - radiance light source and microscope focusing optics, we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices . The spectral irradiance is shown in figure 1(b) for the output of the ldls microscopy system with the 10 and 50 objectives, along with the solar air mass 1.5 (astm g173 - 03) spectrum for comparison . The key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir, particularly in the visible region of the spectrum, where we achieve in excess of 10 times higher irradiances . These numbers can be further improved since a 50:50 beam splitter (optimized for the visible) is used to direct light into the microscope objective . Replacing the dielectric optic with a metallic one would double the output in the visible, but result in even more significant gains in the nir . However, for the purposes of this study, the irradiances achieved are more than sufficient for our measurements . The overall photon flux achieved is also quite high due to strong focusing of the light beam, exceeding 4.5 10 ms with high spatial resolution . In order to demonstrate the utility of a technique having simultaneously high spatial resolution, spectral resolution, and high sensitivity, we examined a set of prototype organic photovoltaic devices that employed a nanostructured plasmonic ag thin film as a transparent electrical contact . First, we discuss the photophysical properties of the perforated metal films with sub - wavelength apertures, in order to best match the contact s optical properties to the light absorption bands in the organic active area . We fabricated the nanostructured electrical contacts by depositing a thin ag film on glass and using a fib tool (fei helios dualbeam) to mill square arrays of uniformly sized holes across typical areas of (0.3 mm) (scanning electron micrograph, figure 2(a)). We can fully characterize even these small - area samples because of the high - brightness light source, therefore minimizing the associated fabrication time . The advantage of using the fib for fabrication is that the design can be varied (e.g. Adjusting hole size and/or diameter) without the need for any process re - optimization . The variation in an average hole size introduced by our fabrication approach does not significantly affect the optical properties of the array . The absolute optical transmission spectra of ag films fully perforated with hole arrays having pitches ranging from 240 to 560 nm on glass show a series of peaks and troughs resulting from the plasmon and diffraction modes in the array (figure 2(b). Spectra are offset for clarity). As predicted from momentum conservation arguments, these mode positions are nearly constant on a wavelength - to - pitch ratio scale . (a) scanning electron micrograph of a silver thin film perforated with a hole array having a pitch of 400 nm . (b) absolute optical transmission spectra of 100 nm thick ag films with a total area of (300 m) on glass vs. the wavelength / pitch ratio, for different pitch values . (the colour version of this figure is included in the online version of the journal .) The optical transmission properties of these perforated metal films are sensitive to the dielectric interfaces on either side, such that the modes of the film when assembled into solar cell will shift compared to those of the same metal film on glass . In order to understand the metal film transmission properties of our target device structure, we coat the perforated ag film (100 nm thick, 200 nm average hole diameter, and 400 nm average pitch) with a 220 nm thick polymer layer (microposit s1811) that mimics the dielectric constant of the organic semiconductor in the non - resonant regime of the spectrum, but does not exhibit significant absorption at visible wavelengths . To best imitate the device structure, we insert a 17 nm thick tio2 layer between the ag and polymer films, because such selective electronic transport layers are essential for organic device operation . We use finite difference time domain (fdtd) calculations (lumerical solutions, inc .) To understand the origin of the primary transmission resonances and their effect on the target photovoltaic device . We find reasonably good agreement between the measured (figure 3(a), black) and calculated (figure 3(a), red) transmission curves, with a primary maximum in the transmission spectrum at ~1020 nm and a minimum near ~930 nm . We believe the peak broadening in the nir results from hole size inhomogeneities . However, the hole pitch uniformity generates a resonance peak that is well matched to the calculation . (a) experimental (black line) and fdtd transmission spectrum (red) of a glass / perforated ag electrode (height = 100 nm, diameter = 200 nm, pitch = 400 nm)/tio2 (h = 17 nm)/polymer (220 nm)/air dielectric stack . Normalized field intensity distributions, \|e\|/\|e 0\| for light with (b) = 927 nm and (c) = 1020 nm . In (b) and (c), the glass spans from z = 0.2 m to z = 0 m, the ag mha spans from z = 0 m to z = 0.1 m (and the hole in the ag layer spans from x = 0.1 m to x = 0.1 m), the tio2 layer spans from z = 0.1 m to z = 0.117 m, and the polymer layer spans from z = 0.117 m to z = 0.337 m . (the colour version of this figure is included in the online version of the journal .) Our calculations allow us to examine the light intensities of corresponding spatial modes in order to identify the mechanism for light dissipation or transport . In figure 3(b) and (c), we plot the normalized field intensity \|e\|/\|e 0\| in the xz plane (where incident light propagates in the + z direction, downwards in the figure). This calculation shows that at 927 nm (figure 3(b)), near the transmission minimum, the field intensity is delocalized throughout the polymer active area, with a minimum under the hole open area . This calculated spatial distribution is consistent with that of a wood s anomaly (evanescent mode), which redirects the light propagation due to diffraction . In contrast, the normalized field intensity profile \|e\|/\|e 0\| for a wavelength near the transmission maximum (1020 nm) shows feature characteristics of the near - field enhancement due to surface plasmon modes in the perforated metal film, resulting in hot spots of electric field intensity near the edges of the contacts along the metal - polymer layer . Though one benefit of these plasmon modes is enhanced light transmission through sub - wavelength apertures, there is also an opportunity for modification of the absorption and radiative properties of the active layer via the purcell effect . Though this subject is beyond the scope of the current manuscript, it does imply that the benefits of perforated metal films as electrical contacts potentially extend beyond their role as passive, light - transmitting elements . Because the primary features in the transmission spectrum of the perforated metal film result from light - matter interactions at the metal / polymer interface, we can leverage this structure for design of a solar cell transparent electrical contact . In this demonstration, we employ a 35 nm thick ag film (perforated with a square array of 200 nm diameter holes on a 410 nm pitch) to couple light into a 160 nm thick active layer of blended organic p3ht: pcbm . The device also includes both hole transporting (10 nm thick moo3) and electron transporting (220 nm thick al) on either side of the organic active layer (schematic in figure 4(a) and cross - sectional sem image in figure 4(b). (a) device schematic and (b) sem cross - section of an organic photovoltaic device with a perforated ag film as transparent contact . The high extinction due to absorption by the active layer occurs around 500 nm and is indicated by the arrow . (d) dark (dot - dash) and illuminated (lines) j v curves for fully fabricated devices using the ag / polymer materials shown in (c). Device hole array pitches are: 320 nm (green), 400 nm (red), and 480 nm (blue). Also shown is the performance of a similar device having an unpatterned 50 nm thick ag electrical contact (dark: dashed, illuminated: black). (the colour version of this figure is included in the online version of the journal .) The optical transmission spectra of organic devices having metal contacts with a hole array of pitches 320, 400, and 480 nm (figure 4(c), measured prior to forming the al electrical contact) contain features in the nir region that are similar to that of the non - resonant model system (figure 3(a)), but show dramatic differences in the spectral regions where the p3ht: pcbm blend absorbs . The primary polymer absorption manifests as dramatic dip around 500 nm (indicated with an arrow in figure 4(c)). The magnitude of the transmission (<10%) is similar to that of the electrode evanescent mode near 900 nm, and is consistent with an active area thickness of ~170 nm . Although the primary propagating mode of the perforated ag film (> 700 nm) is far from the organic material absorption peak (at 500 nm), higher order surface plasmon modes (visible at shorter wavelengths in figure 3(a)) can nevertheless couple light efficiently into the device structure in order to generate photocurrent . For example, the current - voltage (j v) characteristics of fully assembled devices under illumination by 1 sun simulated am 1.5 g radiation display average photocurrent (j sc) in the range of j sc ~ 4 ma / cm (nearly independent of hole array pitch), open circuit voltage, v oc = 0.58 v, and power conversion efficiencies around ~1% (figure 4(d)). Although conventional transparent contact materials (e.g. Indium tin oxide) have achieved power conversion efficiencies around ~4% in p3ht: pcbm devices, there are opportunities for considerable improvement by better matching the electrode s propagating mode to the absorption resonance in the organic layer . Potential approaches to achieving performance improvements include scaling the hole arrays to smaller pitches, or leveraging recent breakthroughs in utilizing nir absorbing polymers which are better matched to the size of the perforated metal contacts described in this work . The high - brightness photocurrent measurement is ideal for acquiring high - sensitivity eqe spectra of these small - area devices (300 m) (figure 5). Although in these measurements we are not required to match the lamp spectrum to the solar spectrum, we nevertheless target an overall spectral irradiance comparable to that of solar illumination in order to avoid high - fluence recombination processes . The achievable spot size under low magnification is 215 m, well matched to our device area of (300 m) and to the solar irradiance in the active region of the device . These measured eqe spectra result from averaging for approximately one minute in total, because the entire spectrum (from 420 to 2500 nm) is collected in a single shot . Ft photocurrent spectra of (300 m) area devices on a (a) linear and (b) log scale for organic photovoltaic devices with perforated metal film transparent contacts (hole array pitches of 320 nm (green), 400 nm (red), and 480 nm (blue), along with a reference device (black) utilizing an ito transparent contact . On a log scale, weak absorption by ct states can be visualized with over six orders of magnitude dynamic range . (the colour version of this figure is included in the online version of the journal .) The integrated eqe of the organic devices using perforated metal films as transparent electrical contact (figure 5(a), green, red, and blue colors) is approximately five times lower than a similar organic device having a traditional ito transparent contact (figure 5(a), black), due primarily to low - light transmission through the contact in the wavelength range where the organic material absorbs strongly . Unlike the j v measurement (figure 4(d)), the eqe spectrum more clearly demonstrates the effect of changing hole array pitch on the device performance . The primary absorption changes shape as a function of pitch and relative to the ito contact (figure 5(a)), with the most apparent difference being a flattening of the absorption spectra moving towards the uv region, compared to the conventional ito device . The high sensitivity of our high - brightness ftpc measurement facilitates detailed investigations of the nir spectral regions, where although the polymer shows weak absorption, the effect of coupling to surface plasmon modes is expected to be the strongest . Previous high - sensitivity measurements on organic photovoltaic devices have shown that weak absorption characteristics of charge transfer (ct) states contribute to the overall photocurrent signal . The response of these ct states are readily seen on a log scale of the eqe spectra (figure 5(b)) in both the ito and hole array contact devices . Although the primary absorption features of the polymer are considerably weaker than the control devices, the ct absorption is not . In fact, structure in the ct absorption can be seen, leading to fluctuations (particularly in the 480 nm pitch device) that exceed those of the control . Work is still on - going to separate the relative contributions of the propagating (plasmonic) and evanescent (wave - guiding) modes to the change in the overall eqe spectrum, and this will be the subject of future work . Here, we stress that these types of studies are enabled by our ability to look at photocurrent spectra with more than six orders of magnitude of sensitivity with high spatial resolution . This allows us to quickly prototype and characterize photovoltaic devices employing plasmonic and photonic structures . We demonstrate that high - sensitivity photocurrent spectroscopy can be achieved on small - area organic photovoltaic devices with greater than six orders of magnitude dynamic range in the eqe spectra . These methods can be used to understand the interactions between the plasmonic modes of sub - wavelength metal hole arrays and an organic absorber . There are several factors that must be considered when evaluating performance of these devices, including scattering of light perpendicular to the direction of incidence, enhancement of absorption and radiative rates in near fields via the purcell effect, and photonic confinement of the propagating modes in the fully assembled device stack.
Heat shock proteins (hsps) are a group of proteins that repress the denaturation of molecules by various stressful circumstances such as exercise, gravity, heat, oxygen, ca etc . Hsp family has a structurally common chaperone subset controlling the form of proteins folding (misfolding, facilitating, or reconstructuring), which maintains homeostasis of proteins to stressful circumstances (dimauro et al ., 2016). Heat - induced heat shock factor 1 controls transcriptional upregulation of hsps (brinkmeier and ohlendieck, 2014). Small hsp (shsp), hsp70, and hsp90 exist in the cytoplasm and glucose regulated proteins (grp) 78 (a homologue of hsp70), bip, hsp47 are located in mitochondria to transmit each of specific functional proteins to mitochondria . Tcp1 (cytoskeleton forming chaperones) is associated with neogenesis of actin and tubulin and shsp are involved in intracellular dynamics (polymerization and depolymerization). A transcript (unfolded protein response element, ure; ccaaan9 ccacg) binds to the sequence of the chaperone gene . Hsp families are made up by range from 10 to more than 100 kda in molecular size, and are located in various cellular compartments . The expressional locations of homologue or cognate members of hsps (such as hsc70 vs hsp70 or hsp90 vs hsp90) are arbitrary according to the accumulated evidences indicating cell or tissue restricted expressions in vivo systems . Accumulated studies show that hsp families are located in various sites within cell; hsp10, hsp60, and hsp75 are located in mitochondria, however, others are present in the cytoplasm, cytosol, endoplasmic reticulum and nucleus in physiological conditions (xu, 2002). The following general description involving molecular chaperones shows the outline regarding the maintenance of physiological homeostasis of living body . Based on comprehensive studies of hsps, this review offers important information regarding the mechanism related with the characteristic procedure of signal pathway according to the specifically cared substrate of each hsp, structural characteristics, specifically expressed location and those roles . Molecular weight of shsp varied from 15 to 43 kda are also called heat shock protein (hspbs) and have a chaperoning function in the process of embryonic development . Hspbs are also interacting with cytoskeleton proteins to maintain the homeostasis of cytoskeleton proteins by preventing those from damages, which results in conservation of the cell function . Hspb1, hspb6, hspb7, and hspb12 are involved in the development of respiratory morphology such as cardiac muscles and the lethal myopathy in diaphragm and cardiomyopathy is caused with the lack of those hspbs (juo et al ., 2016; ke et al ., 2011; rosenfeld et al ., hsp10 (chaperonin) is considered as a suppressor of maternal immune response via releasing from fetal placental unit (noonan et al ., 1979). This includes 101 amino acids and is used as a plausible biomarker in endometrial cancer (dub et al ., 2007). Hsp 10 in mitochondria plays a role in protein folding supplied by adenosine triphosphate (atp) (table 1) (meyer et al ., 2003). It has been reported that hsp10 is a by - product during the process of neoplastic cell proliferation and is considered as a growth factor in the cell (quinn and morton, 1992). Hsp10 is also known as an obligatory autocrine growth factor in tumor cells (quinn et al ., 1990). Molecular size 1530 kda hsps are related with shsp and it has been know that there are 9 shsp in mammals (mounier and arrigo, 2002): hsp27 (denoted hsp25 in mice), a- and b - crystallins, hsp20, hspb2, hspb3, cvhsp or hspb7, hsp22 or hspb8, and hspb9 . Human shsp has 105205 amino acids and -crystallin domain as a homologous 80 sequenced residues (ingolia and craig, 1982). This domain shows highly conserved structure and 38%60% of amino acid identity (mounier and arrigo, 2002). Alpha - crystallin structure is influenced by factors such as ph, temperature, calcium ions, and ionic strength (mounier and arrigo, 2002). Each of the shsp is phosphorylated via specific kinase to involve teleological signal pathway (fig . Hsp16.5 has been structurally studied that it has dimeric -crystallin domain as a basic structural domain unit and this common domain is possibly related with the basic function as a molecular chaperone (bertz et al ., 2010). (2006) reported a function of shsp on myosin enzymatic activity that b - crystallin cares atpase activity of myosin and the comparing rate shows 58% in relation to control (8%, without b - crystallin) under 43c for 30 min . Alpha b - crystallin maintaining atpase activity of myosin prevent aggregation of the protein and this is probably related with myofibrilogenesis without myosin unfolding that induces muscular performance under stress condition or vigorous exercise (melkani et al ., 2006). Forty - three kda sized hsps has various amino acidic sequences that results in different n - terminal length . Those hsps also comprise conserved -crystallin core domains and c - terminal extension domain including highly conserved i - x - i / v motif (ghosh et al ., 2005). Hsp47 acting as a chaperone for procollagen has been also found to be involved in atherosclerosis . Heat shock and oxidized low density lipoprotein stimulate the expression of hsp47 mrna in smooth muscle cells . These findings identify hsp47 as a novel constituent of human coronary atheroma, and selective upregulation by stress raises the possibility that hsp47 may be a determinant of plaque stability . Collagen, an extracellular matrix substrate, requires hsp47 to be transported within the cell and folded from three helices . Hsp47 located in endoplasmic reticulum and functions to support collagen folding and its release to extracellular environments . Hsp60 forms a large (970 kda) hetero - oligomeric protein complex called the tcp1 ring complex (containing tcp1 and several other proteins), which is essential for protein assembly . The hsp60 family has been shown to be involved in the development of many diseases, such as adjuvant arthritis in rats, rheumatoid arthritis in humans, insulin - dependent diabetes mellitus in mice, and systemic sclerosis in humans . Hsp70 is in the cytosol and has family of grp78 which plays role in helping protein folding assembly and refolding, transporting and blocking protein degradation in endoplasmic reticulum . Hsp70 and hsp90 expression levels are variant according to different individual athletic abilities and seem to be upregulated in response to heat acclimation (banfi et al ., 2004; mcclung et al ., 2008; shastry et al ., 2002). As a chaperone, hsp70 plays a role in the assembly and transport of newly synthesized proteins within cells, as well as in the removal of denatured proteins . Downhill (eccentric contraction exercise induced muscle damage by increased delayed onset muscle soreness) in hot conditions elevates the largest expression of hsp72 and hsp90 mrna level (tuttle et al ., in related with myocyte formation, hsp90 plays a key role in myosin folding and sarcomere formation (du et al ., 2008; etard et al ., 2007; hawkins et al ., 2008; srikakulam et al hsp90 consists of n - terminal atp binding domain, substrate interacting middle domain, and c - terminal dimerization domain (jackson, 2013). By atp binding to the n - terminal atp - binding domain, hsp90 regulates myosin thick filament formation and muscle myofibrilogenesis (hawkins et al ., 2008). Hsp90 binds steroid receptors, protein kinases, intermediate filaments, microtubules, and actin microfilaments in a specific manner . Hsp90 is an essential component of the glucocorticoid receptor, assembled in a complex of several proteins . Hsp90 is divided into hsp90 and hsp90 . Hsp40 and hsp70 are cochaperones with the hsp90s . Hsp90s are located in cytoplasm and endoplasmic reticulum to play roles in folding newly made proteins . Hsp100 is located in the cytoplasm and cochaperones with hsp40, hsp70, and hsp90 . Hsp110 is in the cytosol and nucleus (dimauro et al ., 2016). Hsp110 and hsp70 super family commonly have the presence of a loop structure and hsp110 helps immune response (zuo et al ., 2016). Hsp110 is also in working with hsp70 or grp78 to fold proteins and counter stress for cell survival (gething and sambrook, 1992; hartl, 1996). (2016) vaccinated with purified hsp110 results in reduced cancer metastasis thus large hsps amplify inflammatory signals in the cellular environment, suggesting potential endogenous immuno - stimulation during injury such as infection (zuo et al ., 2016). There are comparatively very rare studies regarding large hsps especially in the effect of exercise on the expression of large hsp and its function . Over several decades, hsps have been known as a potent factor playing a role in maintaining the homeostasis of living body . Versatile functions of hsps according to those wide ranges of molecular weight that care those each substrate have been increasingly attentive with other research subjects such as exercise, carcinogenesis, muscles, etc . The broad ranges of hsps are organelle - specifically expressed within the body to play physiological roles via interacting with various signal pathways . Provoking these pathways in a positive way (e.g., physical exercise) enhance hsps signal pathway and maintain / improve vital function of hsps in the living body.
Adenoid cystic carcinoma was first described by billroth in 1859 and called " cylindroma " due to its characteristic histologic appearance1 . In 1953, adenoid cystic carcinoma is a malignant salivary gland tumour characterized by a deceptive histologic pattern, indolent, locally invasive growth with high propensity for perineural invasion, local recurrence and distant metastasis . These uncommon neoplasms account for fewer than 1% of all head and neck malignancies and fewer than 10% of all salivary neoplasms . They make up 15%-30% of submandibular gland tumours, 30% of minor salivary gland tumours, and 2%-15% of parotid gland tumors1 . It is defined by the world health organization as " a basaloid tumour consisting of epithelial and myoepithelial cells in various morphological configurations, including tubular, cribriform and solid patterns . The present case report is unique as it shows presentation of adenoid cystic carcinoma as a solitary ulcer on the floor of the mouth rather than the classical nodular swelling . A 56-year - old male patient presented with a chief complaint of an ulcer in the left floor of the mouth for one week . He gave a history of pain of respect on the left side of the jaw which was continuous, dull, throbbing, and radiating to the ear of the same side . The patient had under gone extraction for tooth #18 but the pain still persisted . Thereafter, he noticed an ulcer on the left floor of the mouth which was initially small and increased to the present size . There was no history of bleeding or discharge, but he did have difficulty eating and speaking . Intraoral examination of the soft tissues, the buccal mucosa, labial mucosa, tongue, and palate showed no abnormalities, but there was a solitary ulcer on the left floor of the mouth . Examination of gingival status revealed his oral hygiene status to be poor, with severe stains and calculus deposits . On hard tissue examination, healing socket was present in respect to tooth #19 . On local examination of the lesion, inspectory findings revealed a solitary ulcer presenting as a mucosal tear involving the mucosa and connective tissue extending mesially from left side of the lingual frenum and distally to an area corresponding to the extracted tooth #19 region . It was oval in shape, 32 cm at its widest point, and 3 cm deep . The margins were everted, the edges of the ulcer were sloping, and the floor was not evident . The colour was the same as that of the adjacent mucosa, but erythematous at the periphery of the ulcer . 1) on palpation of the site, the size and the extent of the ulcer were confirmed . The margins and the base of the ulcer were mildly indurated in a posterior aspect . Based on the history given by the patient and the clinical features, a provisional diagnosis of carcinoma of the floor of the mouth t4an0m0, chronic generalized periodontitis, partially edentulous in respect to tooth #7, #5, #3, #13, #14, #15, #24, #19, #25, and #31 was made . Lesions presenting as solitary ulcers on the floor of the mouth were considered in the differential diagnosis . Traumatic ulcers are most common on the tongue, lips, mucobuccal fold, gingiva and palate . They present as solitary ulcers with raised reddish borders and white necrotic floors with associated history of trauma . On the floor of the mouth, they are usually due to calculus or sharp denture margins . Major recurrent apthous ulcers are mostly solitary, deep, and painful, with smooth margins and a reddish halo and may persist for months with history of reoccurrences . Habit associated lesions seen in quid chewers may also form ulcerations at the region of quid placement . Odontogenic infections may be associated with an ulcer that may serve as a cloacal opening of a sinus draining a chronic alveolar abscess . Other conditions such as acute necrotic ulcerative gingivostomatitis or gangrenous stomatitis usually present as necrotic sloughing ulcerative lesions diffusely involving the gingiva . Solitary ulcers are also seen in nonodontogenic systemic diseases such as uncontrolled diabetes mellitus, blood dyscrasias (leukemia, sickle cell anemia), gastrointestinal and immunocompromised individuals and autoimmune conditions (pemphigus, pemphigoid, erythema multiforme, epidermolysis bullosa). The ulcers in such conditions are well demarcated, painful, and shallow with an erythematous halo and a grey necrotic floor, usually in the marginal and interdental gingiva . Salivary gland disorders such as adenoid cystic carcinoma, mucoepidermoid carcinoma, mucous adenocarcinoma, warthin tumor and necrotising sialometaplasia are seen as solitary palatal ulcers . An orthopantomogram and a mandibular crossectional occlusal radiograph were taken and showed no abnormality in the region of interest. (fig . 2) a magnetic resonance image was taken, and the axial sections showed a t1-weighted image of a mass of medium intensity extending from the midline of the mandible to the premolar region on the left side. (fig . 3) an incisional biopsy was performed and the histopathologic report showed the presence of uniform basaloid cells arranged in the form of solid islands, along with a cribriform pattern at some places . Based on the clinical features and the histopathologic report, a final diagnosis of adenoid cystic carcinoma t4an0m0 was made. (fig . Adenoid cystic carcinoma is a malignant tumour with a deceptively benign histological appearance, characterized by indolent locally invasive growth with a high propensity for local recurrence and distant metastasis . According to the literature gender predilection is an inconsistent feature in the literature; however, it is slightly more common in women than in men4,5 . Although the palate is the most commonly involved intraoral site, other commonly involved areas are the major and minor salivary gland regions, the floor of the mouth, the tongue and the gingiva (in decreasing order). Adenoid cystic carcinoma usually presents as a slowly growing, firm, unilobular mass in the glands . However, in our case it presented as a solitary ulcer on the floor of the mouth . The clinical course is characterized by an initial period of slow and indolent growth that is usually asymptomatic, although bone invasion or perineural spread can cause pain or hypoesthesia5 . Adenoid cystic carcinoma of the salivary glands is a malignant epithelial tumour with bidirectional differentiation towards luminal (ductal) and abluminal (myoepithelial and basal) cells . The tumour is composed of basaloid cells with small, angulated, and hyper chromatic nuclei and scant cytoplasm arranged into three prognostically significant patterns: cribriform, tubular, and solid . The combination of surgery and postoperative radiation therapy has improved locoregional control of the disease . Despite this achievement, late local recurrence and distant metastasis rates remain high and may occur decades after initial diagnosis5 . In its most frequently seen histological pattern, the majority of the cells are small and darkly stained with scant cytoplasm . The cells are arranged in nests or sheets that are fenestrated by round or oval spaces, creating the characteristic " cribriform " design . Occasionally, the tumours have a predominantly solid cellular growth with a basaloid or anaplastic appearance that has few, if any, fenestrations . Tubular structures with minimal stratification of the lining epithelium are often mixed with the classic cribriform and solid areas6 . Several authors have suggested that a solid histological pattern indicates a more serious prognosis than a cystic pattern . Stewart7 first noted the increased aggression suggested by the solid variant, although he credits the initial observation to patey and thakray8 . Little information exists on cytogenetic abnormalities in salivary gland neoplasms, but in adenoid cystic carcinoma, anomalies in the terminal part of the 6q and 9p chromosomes have been reported . Recent studies have demonstrated a high incidence of loss of heterozygosity at chromosome 6q23 - 359 . The differential diagnosis of adenoid cystic carcinoma in cludes tumours that also exhibit tubular and cribriform structures, such as polymorphous low - grade adenocarcinoma, tumours with basaloid cellular morphology, such as basal cell adenoma, and basal cell adenocarcinoma and tumours with a dual population of ductal and myoepithelial cells, such as pleo morphic adenoma8 . Possible treatments of adenoid cystic carcinoma include four different modalities: surgical therapy, radiotherapy, chemotherapy and combined therapy . Avery et al.11 recommend postoperative radiation since radiation often produces tumour regression and relieves symptoms . Postoperative radiotherapy is also indicated when the tumour location is close to the base of the cranium with the presence of neck lymph node metastasis and perineural invasion, with a solid histological subtype, and for recurrent tumours11 . Adenoid cystic carcinoma shows a limited response to chemotherapy, and it is believed that this lack of a response is due to its slow growth rate . However, a study by alcedo et al.12 opened up a new possibility in the pharmacological treatment of this tumour by demonstrating its response to imatinib mesylate, a potent inhibitor of kit gene tyrosine kinase, which is an enzyme involved in the pathogenesis of the tumour . However, more studies are needed to confirm its effectiveness . Distant metastasis can occur even decades after the primary tumour has been treated and after having achieved adequate locoregional control . Furthermore in the setting of inoperable, incompletely resected or recurrent tumours, outcomes after conventional therapy are dismal and remain a therapeutic challenge . Some authors have applied other forms of radiotherapy, particularly neutron irradiation, aiming to improve treatment results13 . However, adenoid cystic carcinoma remains incurable because further improvements in local - regional control are not likely to impact survival due to the high number of distant failures . The present case differs from usual cases of adenoid cystic carcnioma with respect to site and presentation, making it a rare entity . Thus, the aim of this article is to emphasise that though the literature states that adenoid cystic carcinoma ulcerates the superficial lesional mucosa, it can also present as an ulcerative lesion.
India leads the world with the largest number of patients with diabetes earning the dubious distinction of being termed the diabetes capital of the world . According to the diabetes atlas 2015 published by the international diabetes federation, the number of people with diabetes in india is currently around 69.2 million and is expected to rise to 123.5 million by 2040 . Although there are large regional variations in the prevalence of diabetes, it has more than quadrupled in the past 20 years from <1%3% to 10%15% in urban and 3%5% in rural areas . Diabetes registries can be used as an important epidemiological tool: to monitor the prevalence and incidence of diabetes, provide a sampling frame for epidemiologic and clinical studies, provide information to health service providers and planners on risk factors and complications, and assist in the overall monitoring of diabetes control program . Diabetes registries are used in many countries for population management of diabetes, outcomes management, and development of clinician decision support system, for example, national diabetes register (ndr) in sweden, new york glycated hemoglobin (hba1c) registry, and singapore diabetes registry . Hospital - based diabetes registries have been established in developing countries such as thailand and malaysia . In india, the first diabetes registry was set up in goa as a public private partnership, with the aim of population - based disease management . It is essential for similar registry in all states to implement and monitor activities under the national program for prevention and control of cancer, diabetes, cardiovascular diseases, and stroke . To make the registry sustainable and replicable, it is necessary to implement it within the existing system of primary health care . A community - based cohort study showed the incidence of diabetes in rural puducherry to be 21.5 for 1000 person - year follow - up . In this context, a pilot project was set up to establish a diabetes registry in the primary health care setting of puducherry . Here, we share the design and implementation of the registry and propose a management information system for diabetes registry in puducherry district . Union territory of puducherry has one of the best health care delivery services in the country . Primary health care is delivered through a network of 27 primary health centers (12 urban and 15 rural) and 52 subcenters, besides 8 hospitals . Patients with diabetes registered in the chronic disease clinics run at phcs receive free insulin injections and oral hypoglycemic drugs along with regular blood sugar monitoring . This was a facility - based prospective registry study done at six randomly selected phcs in urban puducherry district . Patients with a diagnosis of diabetes mellitus (dm), enrolled in chronic disease clinics at the selected phcs were the participants . All known patients with diabetes, both type i and ii, receiving treatment from primary health - care facilities were included in the registry . The project was approved by the institute scientific advisory committee and ethics committee and registered with the clinical trials registry of india (ref/2012/04/003467). Research assistant was recruited to assist in data collection, data management, and final compilation of the registry . Manuals for training of medical officers and health - care workers on data collection and details on diabetes, risk factors, complications, and lifestyle modifications were prepared and issued to all the phcs . The study was planned in two phases: development of diabetes registry and development of management information system for diabetes [figure 1]. Case report forms (crfs) were developed for collecting information required for the diabetes registry in terms of demographic characteristics, risk factors, complications, coexisting chronic conditions, lifestyle and medical management, and clinical outcomes phase i. the data elements captured in crfs for the registry are shown in figure 2 . The follow - up card for obtaining data for registry maintenance design of the diabetes registry project data elements captured for the diabetes registry and source of data the research assistant underwent intensive training on all aspects of the study interview techniques, data recording, anthropometry, data entry, communication skills, organization of health systems, functioning of phc, etc . Training of medical officers and health workers from the selected phcs was done through a sensitization workshop, in coordination with the state government health services . Logistics were planned out, and roles and responsibilities were identified during the workshop . A pilot study was carried out in urban health center of a tertiary care institute . The crfs were modified, and data entry format in epi info [centers for disease control and prevention (cdc) in atlanta, georgia (usa)] was finalized . Union territory of puducherry has one of the best health care delivery services in the country . Primary health care is delivered through a network of 27 primary health centers (12 urban and 15 rural) and 52 subcenters, besides 8 hospitals . Patients with diabetes registered in the chronic disease clinics run at phcs receive free insulin injections and oral hypoglycemic drugs along with regular blood sugar monitoring . This was a facility - based prospective registry study done at six randomly selected phcs in urban puducherry district . Patients with a diagnosis of diabetes mellitus (dm), enrolled in chronic disease clinics at the selected phcs were the participants . All known patients with diabetes, both type i and ii, receiving treatment from primary health - care facilities were included in the registry . The project was approved by the institute scientific advisory committee and ethics committee and registered with the clinical trials registry of india (ref/2012/04/003467). Research assistant was recruited to assist in data collection, data management, and final compilation of the registry . Manuals for training of medical officers and health - care workers on data collection and details on diabetes, risk factors, complications, and lifestyle modifications were prepared and issued to all the phcs . The study was planned in two phases: development of diabetes registry and development of management information system for diabetes [figure 1]. Case report forms (crfs) were developed for collecting information required for the diabetes registry in terms of demographic characteristics, risk factors, complications, coexisting chronic conditions, lifestyle and medical management, and clinical outcomes phase i. the data elements captured in crfs for the registry are shown in figure 2 . The follow - up card for obtaining data for registry maintenance design of the diabetes registry project data elements captured for the diabetes registry and source of data the research assistant underwent intensive training on all aspects of the study interview techniques, data recording, anthropometry, data entry, communication skills, organization of health systems, functioning of phc, etc . Training of medical officers and health workers from the selected phcs was done through a sensitization workshop, in coordination with the state government health services . Logistics were planned out, and roles and responsibilities were identified during the workshop . A pilot study was carried out in urban health center of a tertiary care institute . The crfs were modified, and data entry format in epi info [centers for disease control and prevention (cdc) in atlanta, georgia (usa)] was finalized . Banners intimating the process, timings, and requirements were put up, and notices were issued to patients with diabetes indicating the same . Registration was scheduled for 5 days a week on fixed timing, for a period of 12 months . Health workers facilitated the distribution of registration cards and created awareness and sufficient publicity for registration among the patients . During the chronic diseases clinics, patients with a diagnosis of dm, enrolled in chronic disease clinics at these phcs, were enlisted in the registry . Consent from the participants for use of clinical data was sought after they are explained about the registry and its purpose . A unique i d number was created for each patient based on phc code (3 digits), presence of comorbidity (dm or diabetic and hypertensive [dh]), and patient identification (3 digits). Details on their demographic characteristics, information on diabetes and coexisting chronic conditions, and clinical outcomes were recorded . The medical officer in charge also assisted in quality control by verifying a proportion of randomly chosen participants . Discussions were held with a few health workers and medical officers before finalizing the register . This was done to maintain brevity without compromising on data quality and utility of register at the clinics . Data in the compiled registry can be categorized as (1) demographics, (2) risk factors, (3) disease and treatment details, (4) glycemic control, and (5) microvascular end - organ disease data . Around 2177 patients with diabetes six patients had type 1 dm; 332 patients received insulin from the phcs . On an average, the research assistant spent nearly 1015 min per patient to review all the details in the patient notebook, conduct interview regarding risk factors and treatment - seeking behavior, and perform anthropometry . Time taken to complete the registration process at each phc was variable and depended mainly on the number of clinic days and efficiency of the existing system (ranging from 6 to 12 weeks at each center); completion of registry took around 14 months . Second phase involves setting up a management information system based on monthly reporting of a number of patients with diabetes, glycemic control, referrals, and compliance to treatment from all these centers . This requires the use of register by the staff - in - charge of the clinics on a regular basis for monitoring patient care and follow - up . The proposed flow of information from the phcs to the nodal office (in the directorate of public health) would constitute the management information system for diabetes . Based on the registry compiled, patient level indicators in terms of a number of patients who achieved glycemic control, compliance to treatment, existing level of complications, proportion of patients screened for complications in the past year, etc . Based on monthly reporting of activities, process and outcome measures can be designed to monitor the services provided . The target for a process indicator for each patient would be availability of investigation reports during the stipulated interval (1 year). Process targets such as proportion of patients with hba1c, low - density lipoprotein (ldl), microalbuminuria, or foot examination done in the past 1 year would be designed . Outcome targets could be set based on desired clinical outcomes, for example, proportion of patients with glycemic control, ldl <130 mg / dl, and blood pressure <130/80 mm hg . Banners intimating the process, timings, and requirements were put up, and notices were issued to patients with diabetes indicating the same . Registration was scheduled for 5 days a week on fixed timing, for a period of 12 months . Health workers facilitated the distribution of registration cards and created awareness and sufficient publicity for registration among the patients . During the chronic diseases clinics, patients with a diagnosis of dm, enrolled in chronic disease clinics at these phcs, were enlisted in the registry . Consent from the participants for use of clinical data was sought after they are explained about the registry and its purpose . A unique i d number was created for each patient based on phc code (3 digits), presence of comorbidity (dm or diabetic and hypertensive [dh]), and patient identification (3 digits). Details on their demographic characteristics, information on diabetes and coexisting chronic conditions, and clinical outcomes were recorded . The medical officer in charge also assisted in quality control by verifying a proportion of randomly chosen participants . Banners intimating the process, timings, and requirements were put up, and notices were issued to patients with diabetes indicating the same . Registration was scheduled for 5 days a week on fixed timing, for a period of 12 months . Health workers facilitated the distribution of registration cards and created awareness and sufficient publicity for registration among the patients . During the chronic diseases clinics, all patients with diabetes were approached and explained the purpose of this registry . Patients with a diagnosis of dm, enrolled in chronic disease clinics at these phcs, were enlisted in the registry . Consent from the participants for use of clinical data was sought after they are explained about the registry and its purpose . A unique i d number was created for each patient based on phc code (3 digits), presence of comorbidity (dm or diabetic and hypertensive [dh]), and patient identification (3 digits). Details on their demographic characteristics, information on diabetes and coexisting chronic conditions, and clinical outcomes were recorded . The medical officer in charge also assisted in quality control by verifying a proportion of randomly chosen participants . Discussions were held with a few health workers and medical officers before finalizing the register . This was done to maintain brevity without compromising on data quality and utility of register at the clinics . Data in the compiled registry can be categorized as (1) demographics, (2) risk factors, (3) disease and treatment details, (4) glycemic control, and (5) microvascular end - organ disease data . Around 2177 patients with diabetes six patients had type 1 dm; 332 patients received insulin from the phcs . On an average, the research assistant spent nearly 1015 min per patient to review all the details in the patient notebook, conduct interview regarding risk factors and treatment - seeking behavior, and perform anthropometry . Time taken to complete the registration process at each phc was variable and depended mainly on the number of clinic days and efficiency of the existing system (ranging from 6 to 12 weeks at each center); completion of registry took around 14 months . Second phase involves setting up a management information system based on monthly reporting of a number of patients with diabetes, glycemic control, referrals, and compliance to treatment from all these centers . This requires the use of register by the staff - in - charge of the clinics on a regular basis for monitoring patient care and follow - up . The proposed flow of information from the phcs to the nodal office (in the directorate of public health) would constitute the management information system for diabetes . Based on the registry compiled, patient level indicators in terms of a number of patients who achieved glycemic control, compliance to treatment, existing level of complications, proportion of patients screened for complications in the past year, etc . Based on monthly reporting of activities, process and outcome measures can be designed to monitor the services provided . The target for a process indicator for each patient would be availability of investigation reports during the stipulated interval (1 year). Process targets such as proportion of patients with hba1c, low - density lipoprotein (ldl), microalbuminuria, or foot examination done in the past 1 year would be designed . Outcome targets could be set based on desired clinical outcomes, for example, proportion of patients with glycemic control, ldl <130 mg / dl, and blood pressure <130/80 mm hg . Any delivery system seeking to manage their patients with diabetes using a disease management program that is internally integrated within their primary care delivery system must develop a registry for diabetes as an essential element of the program . While setting up a population - based register, we need to notify the public health authorities, publicize the registry, establish arrangements for access to data, data security, and accountability (in terms of reporting and feedback). Efforts to achieve improved outcomes for diabetes require an organized population - based approach to diabetes management using all of the components of the chronic illness care model that identifies the six essential elements of a health - care system the community, the health system, self - management support, delivery system design, decision support, and clinical information systems . The uk, australia, new zealand, and canada have taken the lead in adopting models of chronic care . Health information technologies such as electronic health records and registers can be considered as an input or building block of health systems . There is a great potential of these interventions in facilitating decision - making both at the individual and community level . The swedish ndr was initiated in 1996 with 41,000 patients as a tool for local quality control and benchmarking against the national treatment aims . One aim of the ndr was that all patients with diabetes in sweden should ideally be reported yearly, based on registered annual data from actual patient visits in primary health care . Another aim was to provide local centers with data regarding the quality indicators of diabetes care, also making a comparison possible with the national or regional ndr data . Ndr is probably among the largest ndrs in the world, with repeated annual surveys . Nearly 40% of all patients with diabetes in sweden in 2005 were registered in the ndr . A web - based interactive gis system, saudi national diabetes registry, was designed to serve as an electronic medical file for patients with diabetes retrieving data from medical files by trained registrars . A total of 84,942 patients were registered from 2000 to 2012, growing by 10% annually . The singapore diabetes registry was built to enhance the continuity of care for patients with diabetes and facilitate greater efficiency in outcome measurement . The chronic disease management system provides reports of clinical outcomes in a systematic and efficient manner for quality improvement and evidenced - based population management . These include process indicators consisting of the rates of hba1c, ldl - cholesterol (ldl - c), and nephropathy tests, and intermediate outcome indicators of the proportion of patients with poor hba1c (> 9%) and optimal ldl - c (<2.6 the malaysian diabetes registry was set up in 2008 with the objective of creating an accessible diabetes information system, open for health clinics and hospitals, and to provide data for public health action ., the first diabetes registry was set up in goa as a public private partnership, with the aim of population - based disease management . A state - wide campaign over 4 years has screened and revealed about 44,000 patients with diabetes in the state . This registry would help the state health services in regular monitoring, identify people at risk of diabetic complications, and aid in reducing the gap between evidence - based recommendations and clinical outcomes . Similar innovative initiatives under the changing diabetes barometer of novo nordisk education foundation have targeted states such as bihar and gujarat . A total of 12,140 subjects were screened at five primary health centers in gujarat found 13.1% diabetic and 11.3% prediabetes population . Diabetes registry involves collecting and sharing of quality data, analyzing for variations, identifying current practices, comparing with existing guidelines for care, giving feedback, and providing learning opportunities for the health systems . At the patient level, this registry can facilitate needs assessment for specialist services, recall facility for complications screening, and aid in reducing the gap between evidence - based recommendations for care and clinical outcomes . At the health systems level, this can facilitate integration with other services (such as ophthalmology and podiatry). The registry can be used as a tool for the assessment of quality of care through system inputs (number of clinic visits and investigations), process measures (eye screening and foot status), and clinical outcomes (glycemic control, micro and macrovascular complications). This can also provide useful information to health service providers and planners on risk factors and complications, and hence the data can be utilized for the planning of preventive services and better resource management [figure 3]. This could help change behaviors, enhance the quality of care, increase the value of the resources spent, and thus contributing toward a virtuous improvement cycle . Potential applications of diabetes registry in this project in puducherry, this pilot project was started with the aim of creating a population - based diabetes registry . In the first phase, known patients with diabetes availing services at the selected phcs were enrolled . This can be expanded to the entire population for registering all known patients with diabetes through house - to - house enumeration . The proposed design and methodology of implementation can be useful to plan such registries for chronic diseases under the national health program . This study has documented the methodological details, and the learning experiences gained while developing a diabetes registry at the primary health care level and the scope for upscaling to a management information system for diabetes and a state - wide registry . John's medical college and research institute, bengaluru, with training supported by nhlbi - uh, global health initiative for chronic diseases . John's medical college and research institute, bengaluru, with training supported by nhlbi - uh, global health initiative for chronic diseases.
Subclinical hypothyroidism (sh), described as a condition characterized by elevated serum level of thyroid stimulating hormone (tsh) with normal levels of free thyroxine (ft4) and free triiodothyronine (ft3), is a common disorder having a prevalence of about 7.58.5% in females and 2.84.4% in males, worldwide . Several studies have established overt hypothyroidism to be associated with abnormalities of lipid metabolism such as elevated levels of total cholesterol (tc), low - density lipoprotein - cholesterol (ldl - c), and triglycerides (tg), thereby predisposing to cardiovascular diseases . However, the association of sh with abnormalities in lipid parameters have been studied with conflicting results . Some studies have also established a link between sh and metabolic syndrome while others have failed to do so . It has also been indicated that small changes in thyroid hormone levels within the reference range may influence the severity of atherosclerosis . Currently, serum lipid ratios such as tc / high - density lipoprotein - cholesterol (tc / hdl - c), tg / hdl - c, and ldl - c / hdl - c have been shown to be better predictors of cardiovascular risk compared to conventional lipid profile . Small dense ldl (sdldl), a subtype of ldl has been described as the main determinant of the atherogenicity of ldl - c . Atherogenic index of plasma (aip) has been defined as the logarithmically transformed ratio of tg to hdl - c . Aip, which is a good surrogate marker of sdldl, has been indicated as a superior predictor of cardiovascular risk than the previously used lipid parameters . However, research on the complex interrelationship between sh and lipid ratios and aip has been extremely rare . Thus, though the effect of overt hypothyroidism on abnormalities in lipid parameters has already been established, the relationship between sh and lipid metabolism needs further study . Furthermore, there is insufficient information regarding the effect of sh on lipid ratios and aip . This is particularly true in the indian scenario where conflicting results have been obtained, especially in the female population where sh is more prevalent . Therefore, this study was conducted to understand the association of sh with abnormalities in the serum lipid profile and aip in apparently healthy adult females . The study also aims to find out whether any correlation exists between aip and serum tsh levels among sh cases . The study was a retrospective, time - bound, hospital - based, case control study carried out by detailed analysis of patient records in the department of biochemistry at a medical college in southern india . The study was approved by the institutional research committee and ethics committee, which follow the guidelines set by the helsinki declaration (reference number - rc/14/123). Data collection was done for 2 months from may 1, 2015, to june 30, 2015 . Study subjects were taken from the records of apparently healthy adult females coming for a master health check to our hospital from june 1, 2014, to may 30, 2015 . Adult females with high tsh (> 4.2 iu / ml) but normal ft3 (2.04.4 pg / ml) and ft4 (0.931.7 ng / dl) levels were included in the sh group . Adult females having normal tsh (0.274.2 iu / ml), ft3 (2.04.4 pg / ml), and ft4 (0.93 - 1.7 ng / dl) levels were included in the euthyroid (eu) group . Individuals with a history of overt hypothyroidism or levothyroxine replacement therapy, history of radioiodine treatment or thyroid surgery, history of alcohol abuse and smoking, history of drugs such as estrogens, diabetic individuals, subjects with renal, hepatic or other systemic diseases, and known history of dyslipidemia, were excluded from the study . Totally, 504 female subjects came for master health check during the period that had been selected . Of these, 300 patient records could be studied during the 2 months when the study was carried out . Based on the ft3, ft4, and tsh values, the subjects were classified into sh and eu groups according to the inclusion criteria . Based on age - matching between the sh and eu groups, seventy consecutive patients in each group, according to their date of presentation were taken for statistical analysis . Detailed epidemiological information along with a detailed history was taken from the hospital records of the patients who came for a master health check to our hospital . Specific emphasis was given to age, history of smoking and alcohol intake, history of overt hypothyroidism or levothyroxine replacement therapy, history of thyroid surgery, history of hypertension, diabetes or dyslipidemia, history of liver, hepatic or any other systemic disease and history of medications such as oral contraceptive pills . Next, the reports of the subjects were collected with special emphasis on serum fasting glucose, liver function tests, renal function tests, complete blood count with peripheral smear, and routine examination of urine . For the study, the following results were noted: serum tsh, ft3, ft4, tc, tg, hdl - c, and ldl - c . For each subject included in sh group and eu group, the lipid ratios were calculated as tc / hdl - c, tg / hdl - c, and ldl - c / hdl - c . The routine serum parameters in all the study subjects had been measured using automated routine chemistry analyzer (roche cobas integra 400 plus, roche diagnostics limited, switzerland) with commercial kits (roche cobas c packs, roche diagnostics limited, switzerland) according to manufacturer's protocol . Thyroid profile of all the study subjects had been measured using automated electrochemiluminescence immunoassay analyzer (roche cobas e411, roche diagnostics limited, switzerland) with commercial kits (cobas cassettes containing working solutions) according to manufacturer's protocol . The data were analyzed using spss statistical software (v 16; ibm corporation, armonk, ny, usa). To describe continuous variables, mean and standard deviation was used . Since the data were not normally distributed, the difference in lipid parameters, lipid ratios, and aip between sh group and eu group, were compared using mann whitney u - test and a p <0.05 was considered statistically significant . In the sh group, aip and tsh logistic regression was performed to ascertain the effects of age, tc, tg, hdl, ldl, tg / hdl, tc / hdl, ldl / hdl, and aip on sh . Variables which are significant at <0.1 in simple regression were considered for multiple regression analysis and because of multicollinearity, only one of the following variables were included in the model at a time: tg, tg / hdl, and aip . The data were analyzed using spss statistical software (v 16; ibm corporation, armonk, ny, usa). To describe continuous variables, mean and standard deviation was used . Since the data were not normally distributed, the difference in lipid parameters, lipid ratios, and aip between sh group and eu group, were compared using mann whitney u - test and a p <0.05 was considered statistically significant . In the sh group, aip and tsh were correlated using spearman's correlation . Logistic regression was performed to ascertain the effects of age, tc, tg, hdl, ldl, tg / hdl, tc / hdl, ldl / hdl, and aip on sh . Variables which are significant at <0.1 in simple regression were considered for multiple regression analysis and because of multicollinearity, only one of the following variables were included in the model at a time: tg, tg / hdl, and aip . There was no statistically significant difference (p> 0.05) among the mean ages of sh and eu groups . However, there was a statistically significant (p <0.05) difference between the means of ft3, ft4, and tsh of the sh and eu groups . Comparison of baseline characteristics of the study subjects the data in table 2 represent a comparison of lipid profile parameters of eu group and sh group . It includes mean and standard deviations of tc, tg, hdl - c, and ldl - c of eu and sh groups . The differences between the means of tc, tg, and ldl - c were found to be statistically significant while that for hdl - c was found to be insignificant (p = 0.712). The data presented in table 3 shows the comparison of lipid ratios such as tc / hdl - c, tg / hdl - c, and ldl - c / hdl - c between sh and eu groups . The difference in the mean tg / hdl - c between the two groups was found to be significant (p <0.005). Comparison of lipid profile parameters between the study groups comparison of lipid ratios between the study groups figure 1 represents the comparison between the mean aip of sh group and eu group . The mean aip of eu group was 0.078, and that of sh group was 0.211 . When the means were compared using mann whitney u - test, the difference was found to be statistically significant (p = 0.002). The spearman's correlation coefficient r is 0.174 (p = 0.149) indicating that there is no significant correlation between aip and tsh in the sh group . Eu: euthyroid group, sh: subclinical hypothyroidism group, aip: atherogenic index of plasma . * difference of the means of aip between eu and sh groups is statistically significant (p = 0.002, <0.05) correlation between atherogenic index of plasma and thyroid stimulating hormone in subclinical hypothyroidism group . No significant correlation, spearman's correlation coefficient r is 0.174 (p = 0.149) table 4 represents the results of logistic regression analysis . Aip has a significantly higher risk (or = 22.38) than tg (or = 1.01) or tg / hdl (or = 1.42). The link between dyslipidemia and sh has been explored in a large number of studies of varying methodologies in different parts of the world . However, studies on the abnormalities in lipid ratios and aip which are better markers of cardiovascular risk than conventional lipid profile are rare . With this background control study, where we retrospectively studied patient records to investigate the abnormalities in conventional lipid profile, lipid ratios, and aip in sh . The study also aimed to find out if there was any correlation between tsh and aip levels in sh . In our study the mean age of sh patients in our study was 42.5 similar to other studies done in tamil nadu (india), greece, and turkey but higher than studies done in kashmir (india), italy, and turkey . The differences can be explained by the different study populations used in the different studies which differed on ethnicity, gender preponderance, race, etc ., however, tsh levels were significantly higher in sh as compared to eu similar to other studies . In our study, we also reported that the mean ft3 and ft4 was significantly lower in sh as compared to eu though in both groups the values were within normal reference ranges . This is in contrast to the other study done in tamil nadu where no significant difference was noted . The difference could be explained by our larger sample size whereby the study in tamil nadu had a sample size of 30 whereas our study had a sample size of 70 . Regarding lipid profile parameters, our study showed a significantly lower tc and ldl - c level and a significantly higher tg level in sh as compared to eu . Several other studies conducted in india and abroad have also reported similarly higher tg levels, but tc and ldl - c were also reported to be higher in sh as opposed to our study . Since our study was based on retrospective analysis of patient records, it can be suggested that a prospective study could lead to a better agreement . On the other hand, a contrasting result was obtained in national health and nutrition examination survey iii, a large study of 215 subjects with sh and 8013 euthyroid individuals, where no significant difference in lipid parameters between sh and euthyroid individuals was reported, when adjusted for age, race, sex, and the use of lipid - lowering agents . In overt hypothyroidism, the molecular basis of this dyslipidemic pattern has been elucidated . In overt hypothyroidism, due to the reduced activity of ldl - c receptors, the catabolism of ldl - c, and intermediate density lipoprotein - cholesterol are affected, consequently increasing serum tc and ldl - c . Moreover, in overt hypothyroidism, the activity of the enzyme lipoprotein lipase is lowered, thereby inhibiting the removal of tg - rich lipoproteins leading to a high serum tg level . In our study, the mean hdl - c was lower in sh than in eu, but the difference was not statistically significant . This result is in contrast to the study done in tamil nadu where the hdl - c was significantly lower in sh compared to eu . In our study, the lower than desirable hdl - c levels in both sh and eu can be explained by the lack of exercise and sedentary lifestyle among the study participants . In the indian context, this poses a serious health risk and needs to be investigated further by large population - based studies as well as experimental studies to elucidate the molecular etiopathogenetic links . Several studies have also shown that levothyroxine therapy has led to significant improvement in dyslipidemia in sh patients, but we have not studied this aspect . In contrast, in overt hypothyroidism, elevation of serum hdl - c levels may be seen due to increased concentration of hdl2 particles . Considering our results about the lipid ratios, our study showed a significantly higher tg / hdl - c in sh as compared to eu, but no significant difference was seen in tc / hdl - c and ldl - c / hdl - c levels . In a study, it was proved that 6-month treatment with levothyroxine led to normalization of tsh, and this improved the adverse lipid profile of elderly women with sh . This effect was reflected in the lipid ratios and thereby concentrations of tc / hdl - c, tc / ldl - c, lp(a) and apob were significantly reduced but apoai and hdl - c were also reduced but not significantly . However, the difference in the means of lipid ratios between sh and eu have not been studied . Thus, though controversies exist regarding the association of sh with cardiovascular risk, to the best of our knowledge, no other study has investigated the status of lipid ratios in sh . Thus, to fill this knowledge lacuna, we decided to include lipid ratios in our study . In our study, aip was significantly higher among sh subjects than eu subjects . The mean value of aip in sh subjects was found to be 0.211 which falls in the at risk category (aip> 0.11) for cardiovascular diseases . Aip has already been established as strongly correlating with serum sdldl level, which is the main atherogenic fraction of ldl . On the other hand, sh has been shown to be associated with highly atherogenic pattern b of ldl - c subfractions . It has been reported that as a marker of lipoprotein particle size, aip adds predictive value beyond that of the individual lipids, and/or tc / hdl - c ratio . This is corroborated in our multiple regression analysis, where aip emerged as the single most significant factor associated with sh among the study parameters . This could also indicate that sh is associated with high aip and thereby a highly atherogenic lipid profile . Nevertheless, despite extensive literature search in other metabolic disease states such as metabolic syndrome, hyperuricemia, and diabetes mellitus, aip was found to be a good predictor of cardiovascular risk . In overt hypothyroidism also aip levels have not been studied, but conflicting results were seen in studies investigating the impact of overt hypothyroidism on ldl - c subfractions, particularly sdldl . Though a recent study conducted on newly diagnosed hypothyroid patients showed an association between hypothyroidism and elevated sdldl, other studies could not conclusively prove such an association . All these results reiterate the need for studies on the association of aip and abnormal lipid ratios with sh . In our study, we have attempted to bridge this gap and from our results, it could be speculated that though serum ldl - c was lower in sh subjects, their pattern is significantly atherogenic with a high level of sdldl . We failed to find any correlation between aip and tsh levels in sh subjects thereby indicating that though sh patients were at a greater risk of cardiovascular diseases, the risk did not proportionately increase with an increase in tsh levels . Several studies have reported that dyslipidemic changes were not apparent when serum tsh levels were below 10 miu / l but above this level, dyslipidemia was more overt . An indian study reported that there was no association between lipid abnormalities and sh when the value of serum tsh was <10 miu / l . On the other hand, patients with tsh concentrations mu / l had significantly greater levels of tc and ldl - c as well as lower hdl - c compared with controls . Based on these findings, it would not be wrong to speculate that since a majority of our patients had a tsh level <10 miu / l, aip could not be correlated with tsh levels in sh patients . Ours is a retrospective, hospital - based, time - bound study where hospital records of study subjects were analyzed . Large, well - planned, community - based, prospective studies need to be conducted to gain a better understanding of dyslipidemia in sh, particularly the study of aip . Thus, the positive association of dyslipidemia and sh indicates a need for regular screening of sh patients for dyslipidemia to enable early diagnosis and treatment of the condition . Even in patients who have a normal conventional lipid profile, lipid ratios, and aip the high prevalence of low hdl levels reiterates the need for counseling of patients regarding healthy dietary habits and regular exercise . High aip among the sh population too underlines the need for large - scale, community - based programs for health awareness and lifestyle modification . However, this is a cross - sectional study and so, whether sh leads to high aip could not be determined . Moreover, the molecular basis for this association can only be elucidated by experimental studies with animal models . This study helped to bridge the knowledge gap as well as paucity of indian data available regarding the complex interplay between thyroid hormones and atherogenic lipid parameters, particularly in sh . The study also emphasizes the importance of aip in the detection of atherogenic risk in sh patients, even in the presence of minor dyslipidemia . Abnormalities found in conventional lipid profile as well as in lipid ratios and aip imply sh to be a risk factor for dyslipidemia and atherosclerosis . An atherogenic lipid profile is a prequel to cardiovascular and cerebrovascular diseases, and hence, its association with sh in general and calculation of aip, in particular, could provide avenues for early diagnosis and rigorous management of dyslipidemia in hypothyroidism.
The ability of mammals to respond to an inadequate o2 supply, commonly termed hypoxia, is crucial for their survival . Although a proper response to changed o2 tensions triggers adaptation, a number of pathological conditions or failures in the o2 response are associated with various diseases such as anemia, myocardial infarction, thrombosis, atherosclerosis, or cancer . When exposed to hypoxia or even anoxic conditions, mammalian organisms initiate a variety of responses in different organs, aiming to increase the delivery of oxygen to the tissues . In addition to the switch from an aerobic to an anaerobic metabolism and the suppression of energy - using reactions, the carotid body chemoreceptor cells stimulate the brain stem center controlling the respiratory and cardiovascular systems to enhance ventilation, heart rate, and blood pressure (reviewed by prabhakar1). In addition, neuroepithelial cells in the lung contribute to adjusting pulmonary perfusion and gas exchange . Moreover, organs and cells switch their gene expression profile: the kidneys produce erythropoietin, which increases red blood cell production in the bone marrow, and vascular cells produce vascular endothelial growth factor to promote angiogenesis and flow of enhanced blood volume (reviewed by semenza2). In addition to the expression of erythropoietin and vascular endothelial growth factor, the expression of more than 500 genes, products of which are involved in glycolysis, angiogenesis, erythropoiesis, cell death, and differentiation, is also changed on exposure to hypoxia (reviewed by wenger and stiehl3 and semenza4). In mammals, the hypoxia - dependent changes on the level of gene expression are mainly mediated by the -subunits of hypoxia - inducible transcription factors (hifs). Hif -subunits are tightly regulated, and post - translational hydroxylations in response to hypoxia appear to be of major importance . In addition to hypoxia, hif -subunits were also found to respond to various growth and coagulation factors, hormones, cytokines, or stress factors already under normoxia . Indeed, different kinases, among them glycogen synthase kinase 3 (gsk-3), have been identified to directly phosphorylate hif- proteins . This review discusses the regulation of hif- by gsk-3 and compares it with hydroxylase - dependent hif- protein regulation . In their active form, hifs are heterodimeric transcription factors consisting of an - and -subunit . The hif -subunit represents the stable nuclear subunit primarily represented by the ubiquitously found arnt (arylhydrocarbon receptor - nuclear translocator) protein; however, arnt2 or artn3, although to a lesser extent, also appear to be able to take part in the formation of hif dimers (reviewed by semenza5). So far, three -subunit proteins, hif-1, hif-2 (also known as epas,6 hlf,7 hrf,8 or mop29), and hif-3 have been identified . Together, the different hif - and -subunits may give rise to the formation of several combinations of hif dimers.5,10 hif-1 and hif-2 are the best - studied hif- isoforms . Although they share structural and functional similarities, it appears that differences in the cell - type expression pattern, the target genes, the embryonic deletion phenotypes, and the effects on tumorigenesis exist between hif-1 and hif-2.1114 the function of hif-3, from which several splice variants exist in humans,15,16 is largely unknown, although some human hif-3 variants and a mouse splice variant termed inhibitory pas protein (ipas) appear to act as negative regulators of the hypoxic response.1619 similar to the arnt proteins, the hif -proteins belong to the basic helix - loop - helix pas (per - arnt - sim) protein family . In particular, hif-1 and hif-2 show the highest degree of sequence identity in the basic helix- loop - helix (85%), pas - a (68%), and pas - b (73%) domains . Both also contain two nuclear localization sequences responsible for translocation to the nucleus under hypoxia; they are localized in the n terminus (amino acids 1733 in hif-1 and amino acids 150 in hif-2) and in the c terminus (amino acids 718721 in hif-1 and amino acids 689870 in hif-2).20,21 with the exception of hif-3, which does not contain a c - terminal transactivation domain (c - tad),22,23 hif -subunits also contain n- and c - terminal transcriptional activation domains (n - tad and c - tad). A unique oxygen - dependent degradation domain (oddd, amino acids 401603 in hif-1 and amino acids 517682 in hif-2) overlaps n - tad . The residues between n - tad and c - tad represent an inhibitory domain (amino acids 604785 in hif-1 and amino acids hif -subunit activation under hypoxia is mainly the result of an increased protein stability and coactivator recruitment, although transcriptional and translational mechanisms also were shown to be involved in hif -subunit activation.22,2630 as a result, hif- proteins accumulate, translocate to the nucleus, and dimerize with hif- to form a functional transcription factor.31 thus, in the presence of oxygen (ie, normoxia), hif- proteins become degraded . This is primarily achieved by oxygen - dependent hydroxylations at the oddd.32 under normoxia, prolyl hydroxylase domain proteins (phds),33,34 in particular phd2, hydroxylate two crucial residues in the oddd of hif -subunits (p402 and p564 in hif-1 and p405 and p531 in hif-2).25,32,35 prolyl hydroxylation is required for binding the von hippel - lindau protein (vhl),36,37 which represents the substrate recognition subunit of an e3 ubiquitin protein ligase consisting of elongin c, elongin b, ring box 1, cullin 2, and an e2 ubiquitin - conjugating enzyme (figure 1). The prolyl hydroxylation and ubiquitination can be further promoted by the binding of phd2 to os938 and that of hif-1, vhl, and elongin c to ssat2, respectively.39,40 in addition to prolyl hydroxylation, a conserved asparagine residue (n803 in hif-1 and n852 in hif-2) in the c - tad is hydroxylated by the factor - inhibiting hif in an oxygen - dependent manner . This hydroxylation prevents interaction with the coactivator proteins cbp / p300.4144 thus, the major posttranslational modification appears to be the oxygen - dependent hydroxylation.36,37 in addition to hydroxylation, hif- transcriptional activity and protein stability appear also to be dynamically regulated by other posttranslational modifications such as acetylation, s - nitrosylation, sumoylation, and phosphorylation (for review, see dimova and kietzmann45). Phosphorylation appears to be of special importance under normoxic conditions, mediating the response of hif- to various growth and coagulation factors, hormones, cytokines, or stress factors (reviewed by dimova et al46) under normoxia . Indeed, a panel of protein kinases is reported to be involved in hif-1 phosphorylation, either directly (table 1) or indirectly.4752 although the individual action of certain kinases on hif-1 regulation was mainly studied in vitro (table 1), the in vivo mechanisms are likely much more complex . At least the extent to which the kinases can be involved in hif- phosphorylation may vary according to the signal, cell type, or tissue . Given the different developmental and/or differentiation status of a cell or tissue, the expression of various growth factors, their receptors, and respective signaling components and thus, it seems not to be surprising that phosphorylation of hif- by different kinases or after modulation of signaling pathways may be a highly cell type - specific event . Although direct proof is currently lacking, it is plausible that the phosphorylation pattern of hif- in a certain cell may be explained by different layers of regulations that affect kinases depending on the cellular context . In addition to being activated by a variety of extracellular signals, the pi3k / akt cascade appeared also to be regulated by hypoxia, thus integrating hypoxia signaling with extracellular signals affecting multiple cellular processes such as apoptosis, metabolism, cell proliferation, and cell growth (for review, see braccini et al53). The pi3k / akt pathway is considered to control hif-1 within the cell via regulation of hif-1 protein synthesis and stability . However, it appeared that hif- proteins are not directly phosphorylated by pkb / akt but, rather, by a pkb / akt target . The pkb / akt targets hdm2,54,55 mammalian target of rapamycin (mtor),56 and gsk-357 were shown to contribute to changes in hif- protein levels; however, only gsk-3 was shown to do this directly, ie, by phosphorylating hif- proteins . Gsk-3 is a serine / threonine kinase that was first identified as a negative regulator of glycogen synthesis; inhibition is achieved through phosphorylation of glycogen synthase.58,59 since its initial discovery, gsk-3 has been found to be involved in numerous signaling pathways initiated by diverse stimuli and to contribute to the regulation of cell proliferation, stem cell renewal, apoptosis, and development, which are processes often associated with hypoxia . Because of these multiple involvements, dysregulation of gsk-3 has been implicated in the pathogenesis of human diseases, including type 2 diabetes, bipolar disorders, inflammation, alzheimer s disease, and cancer (reviewed by frame and cohen,60 grimes and jope,61 and woodgett62). Two isoforms, gsk-3 (51 kda) and gsk-3 (47 kda), have been identified in mammals . Despite their homology in the catalytic domain (98%), they significantly differ in their n- and c - terminal parts63,64 and do not have entirely overlapping roles in metabolism (reviewed in force and woodgett65). Moreover, gsk-3 (gsk-3) homozygous knockout mice showed an embryonic lethal phenotype around day 16 because of hepatic apoptosis or a cardiac pattern defect,66,67 whereas homozygous gsk-3 (gsk-3) knockout mice are viable and fertile.68,69 gsk-3 is a target of pkb / akt, which can phosphorylate both gsk-3 isoforms (serine 21 of gsk-3 and serine 9 of gsk-3), leading to an inhibition of gsk-3 activity.70 interestingly, these serine residues can also be phosphorylated by other kinases such as erk1/2,71 p70 ribosomal s6 kinase 1,72 camp (cyclic adenosine monophosphate)-dependent protein kinase a (pka),73 and protein kinase c (pkc).74 in contrast, an autophosphorylation event leading to phosphorylation of tyrosine 279 in gsk-3 and of tyrosine 216 in gsk-3 increases gsk-3 activity.75,76 neurons seem to possess a spliced gsk-3 variant called gsk-32 that contains a 13 amino acid residue insert within the kinase domain, leading to reduced kinase activity.77,78 although gsk-3 is mostly known in the insulin field as a regulator of glycogen synthesis, it has been shown that early hypoxia enhanced pi3k / akt activity and increased hif-1 protein levels.57 similarly, hypoxia was capable of inhibiting gsk-3 by phosphorylation in different cell types, such as pc-12 (rat pheochromocytoma cell line) cells,79 ht1080 (human fibrosarcoma cell line) cells,80 and hepg2 (human liver hepatocellular cell line) cells,57 as well as in vivo.81 although this effect was not observed in other cell types, including some breast cancer cell lines,82 pc-3 prostate cancer cells,83 and 3t3 cells,84 it was considered to have a cell type - specific component . However, the findings that gsk-3 inhibition57 and small interfering rna - mediated depletion induced hif-1, whereas gsk-3 overexpression reduced hif-1 protein levels,85 suggested that hif-1 is a direct target of gsk-3. Indeed, the oddd86 and three sites, s-551, t-555, and s-589, located within the oddd overlapping the n - tad of hif-1 were found to be directly phosphorylated by gsk-3.85 another study reported five sites, t-498, s-502, s-505, t-506, and s-510, within the n - tad of hif-1 as gsk-3 phosphorylation sites.87 the disparity of the different phosphorylation sites is difficult to explain, but the different oxygen concentrations (8% o2 compared with 2% o2) used in these studies may contribute to the differences . It is possible that different oxygen levels may induce variable signaling pathways that have unequal effects on hif-1 and its ability to act as a substrate for gsk-3. Another possibility could be the different cell types (hepg2 compared with sk - ov-3 [human ovarian cancer cells]) that were used in the studies . Despite the differences in the phosphorylation sites, both studies demonstrated that the regulation of hif-1 by gsk-3 is independent of o2, hydroxylation, and recruitment of the vhl - containing e3 ubiquitin ligase . Experiments with vhl - deficient cells showed that gsk-3-dependent hif-1 degradation occurred independent of vhl, indicating that the phosphorylation of hif-1 by gsk-3 target hif-1 for proteasomal degradation in an oxygen - independent manner.85 this suggested involvement and recruitment of another so - far - unknown e3 ubiquitin ligase to gsk-3-phosphorylated hif-1. Indeed, two groups demonstrated that the f - box and wd protein fbw7 (also known as hcdc4 in yeast, hsel10 in caenorhabditis elegans, or ago in drosophila) acted as the substrate - recognition component of a multisubunit e3 ubiquitin ligase, which was crucial for the proteasomal degradation of gsk-3 phosphorylated hif-1.85,87 in this e3 ligase, fbw7 interacts with skp1 (s - phase kinase - associated protein 1), cul1 (cullin 1), and rbx1, forming the so - called scf complex . Similar to vhl, fbw7 is considered to serve as a tumor suppressor, and three fbw7 isoforms (fbw7, fbw7, and fbw7) are known to be produced by alternative splicing . They are found in the nucleoplasm, cytoplasm, and nucleolus, respectively.88 in addition to hif-1, fbw7 was shown to be involved in the degradation of various oncogenic proteins, including cyclin e,89 c - myc,90,91 c - jun,92,93 and notch.94 several studies have shown that loss of the fbw7 gene is associated with malignant transformation, especially in ovarian cells and t cells,95 in breast cancer cells,96 and later also in human colorectal cancers,97 which leads then to chromosomal instability and some types of malignancy . Furthermore, investigation of more than 1,500 human tumors revealed that approximately 6% of those tumors showed mutations in the fbw7 coding region . Specifically, cholangiocarcinomas (35%), t - cell acute lymphocytic leukemia (31%), and endometrial (9%), colon (9%), and stomach (6%) cancer98 had the highest mutation rates . Strikingly, nearly half (43%) of these were missense mutations that resulted in amino acid substitutions within the wd40 domain (arg465 and arg479), which are shared by all three fbw7 isoforms, suggesting that all fbw isoforms might collectively contribute to the tumor - suppressor function.98 with respect to hif-1, all three fbw7 isoforms were able to induce hif-1 degradation, and the loss of the fbw7 wd domain abolishes gsk-3-initiated degradation, leading to higher hif-1 levels, which has been found to be associated with several tumors.99101 the finding that hif subunits can be targeted for degradation by two different e3 substrate recognition proteins indicates that the system is highly dynamic . The human genome encodes nearly 100 deubiquitylating enzymes that are predicted to be active and that oppose the function of around 600 e3 ligases.102,103 similar to e3s, deubiquitylating enzymes have a central role in cell cycle regulation and dna damage response and, depending on the context, can act either as a tumor promoter or suppressor (see references in love et al104). With respect to vhl, two different deubiqiutinating enzymes, vdu1 (usp33) and vdu2 (usp20), were suggested to oppose the vhl - e3 ubiquitin ligase.105,106 later, it was shown that vdu2 but not vdu1 can interact with hif-1.107 experiments with cycloheximide and hypoxia showed that the half - life of hif-1 was significantly increased upon overexpression of vdu2, whereas a catalytic inactive vdu2 c154a mutant had no effect . In addition, it was shown that only vdu2, not vdu1, deubiquitinated hif-1, resulting in the stabilization of hif-1 protein107 (figure 1). Experiments with gsk-3- and fbw7-deficient cells revealed that gsk-3- and fbw7-dependent hif-1 degradation can be antagonized by ubiquitin - specific protease 28 (figure 1).99 these findings suggest that the gsk-3-dependent degradation of hif-1 is not limited by the presence of oxygen and is therefore independent of vhl . Together, these results demonstrate that hif-1 protein stability is regulated in a dynamic manner involving different ubiquitin ligases and deubiquitinases . As such, the hydroxylation- and vhl - dependent ubiquitination and degradation of hif-1 under normoxia is opposed by the deubiquitinase vdu2 . In contrast, the oxygen - independent but phosphorylation - dependent ubiquitination of hif-1 is counteracted by ubiquitin - specific protease 28-mediated deubiquitination . The latter process allows the integration of the hif system into the cellular response to various physiologic and pathophysiologic signals independent of the oxygen tension . The finding that gsk-3 is involved in the degradation of hif-1 indicated similarities with the destruction of -catenin in the canonical wnt signaling pathway . In this pathway, gsk-3 and -catenin are part of a destructive complex in which binding of gsk-3 and -catenin promotes phosphorylation of -catenin by gsk-3, which requires priming phosphorylation by casein kinase 1, -isoform . The phosphorylated -catenin is recognized by the f - box / wd protein -trcp and subsequently ubiquitylated and targeted for proteasomal degradation (for review, see cohen and frame108 and metcalfe and bienz109). When this phosphorylation event is blocked, -catenin accumulates and binds to the t - cell - specific transcription factor / lymphoid enhancer - binding factor 1 family of transcriptional activators to activate numerous target genes (reviewed by reya and clevers110) contributing to embryonic development and adult tissue homeostasis (reviewed by clevers111). Similarly, gsk-3-mediated phosphorylation of hif-1 recruits fbw7, and thus targets hif-1 for ubiquitylation and proteasomal degradation.99 those very similar scenarios imply interference or interconnection of both the wnt/-catenin and hypoxia / hif-1 signaling on the level of gsk-3 . Actually, crosstalk between the hypoxia and/or hif-1 and wnt/-catenin pathway was reported and appears to be quite complex because of controversial and likely cell / tissue / differentiation - stage specific data.112117 indeed, it was reported that hypoxia and/or hif-1 can inhibit wnt/-catenin signaling . Several mechanisms, such as binding of hif-1 to hard1 (human arrest - defective-1 protein) with subsequent interference with acetylation of -catenin,112 blocking processing and secretion of wnt proteins,113 down - regulating -catenin via p53-dependent activation of siah-1 (seven in absentia homolog 1),118 or direct interaction between hif-1 and -catenin119,120 were proposed to contribute to these effects . In contrast, hypoxia was also shown to activate wnt/-catenin signaling in undifferentiated cells and in vivo.115,117 in hypoxic embryonic stem cells, this occurred via hif-1-mediated expression of lymphoid enhancer - binding factor 1 and t - cell - specific transcription factor, followed subsequently by increased interaction of -catenin with lymphoid enhancer - binding factor 1/t - cell - specific transcription factor, and thus activating wnt/-catenin targets.115 in addition, hypoxia was able to activate -catenin via gsk-3 inactivation116,121 in different human cell lines such as ht-29 (human colorectal adenocarcinoma cell line) and hepg2; this activation contributed to an endothelial mesenchymal transition program, leading to significantly increased invasiveness,121 and in renal tubular cells, this process impaired wound healing.116 together, the reported findings indicate that complex interconnections between hypoxia and/or the hif-1 and wnt/-catenin pathway exist and that cell-, tissue-, and differentiation - specific aspects contribute to their functional consequences . Hypoxia and hifs play important roles in many critical aspects of physiological and pathological processes . Most solid cancers contain hypoxic areas, and clinical data demonstrate that overexpression of hif-1 is associated with an increased risk for patient mortality . In line, downregulation of hifs interferes with tumor growth, vascularization, invasion, and metastasis, as well as radiation and chemotherapy . Activation of multiple oncogenic pathways including growth factor signaling coupled with enhanced kinase signaling is a common event in tumors, thus making it likely that kinases are involved in the modulation of hif- function . Because regulation of hif- protein stability is critical for its activation, identification of kinases contributing to hif- stability may provide a link explaining normoxic hif- stabilization by extracellular stimuli . In light of this, dysregulation of gsk-3 is thought to underlie the pathogenesis of various diseases that are also associated with hypoxia and changed hif- levels, such as type 2 diabetes mellitus, alzheimer s disease, mood disorders, cardiovascular diseases, and cancer.122 thus, given that gsk-3 upstream regulation leads to inhibition of gsk-3 and hif-1 accumulation, this raises the question whether it is an option to target gsk-3 in those diseases and disregard the adverse effects . Although research from the last decade has demonstrated that a number of kinase pathways contribute to hif-1 regulation, data for hif-2 or hif-3 are limited . Taking into consideration the overlapping, but different, roles of the hif- proteins, more knowledge about the phosphorylation - dependent regulation of hif-2 and hif-3 is necessary to better understand both already - observed general and different effects.
The pars distalis of the pituitary is composed of both granular and agranular cell types, the former largely hormone - producing cells, the latter forming a reticular and canalicular network in and around the granulated cells . Among the nongranulated cells are folliculo - stellate (fs) cells, which have numerous long cytoplasmic processes that insinuate among the endocrine cells . Neighboring fs cells are joined by well - developed junctional complexes forming an interconnected network of channels extending throughout the anterior pituitary, but particularly, the pars distalis [2, 3]. Fs cells resemble polarized epithelial cells, with their apical surfaces containing microvilli and lining the lumen of the follicular cavities [2, 3]. Of relevance to the current study, there is evidence that fs cells play a role in the absorption of ions and water from the luminal spaces [46], which could involve the activity of an apical na / h exchanger . Many aspects of metabolism, growth, stress, immunity, and reproduction are under the direct influence of granular cell secretions of the pars distalis; however, the contribution of fs cells to intrapituitary communication and the mode(s) by which this occurs are less well understood . Fs cells play a regulatory role both by secretion of paracrine factors, including activin, follistatin, and vascular endothelial growth factor [7, 8], and by intercellular communication via ca signals transmitted through gap junctions, which has been suggested to contribute to synchronization of hormone secretion by endocrine cells [9, 10]. A potential additional means of intrapituitary communication that could play a role in coordination of both fs cell and endocrine cell activities is the network of channels formed by the fs cells . Previous studies showed that genetic ablation of the na / h exchanger isoform 2 (nhe2, gene symbol, slc9a2), which is expressed at high levels in stomach, causes loss of gastric acid secretion, decreased viability of parietal cells, and severe metaplasia and hyperplasia of the gastric mucosa [12, 13]. Histological abnormalities were not observed in the kidney or intestinal tract of nhe2-null (nhe2) mice; however, they exhibited defects in the absorption of na and hco3 in both the renal nephron and colonic crypts [14, 15], where nhe2 is also expressed, indicating that nhe2 can function as an absorptive na / h exchanger . In the current study, it was shown that pituitaries from mice lacking nhe2 exhibited significant histopathological changes, particularly in the pars distalis and pars intermedia, both of which are derivatives of the primitive gut ectoderm . There was a greater accumulation of cytoplasmic lipid in fs cells and parenchymal cells, an undulating and reduplicated basement membrane, and decreased thickness of the pars intermedia, with a reduction of projections into the pars nervosa . However, the most striking change was a dilated fs cell canalicular system with large cyst - like spaces throughout the pars distalis, which is consistent with the loss of an absorptive ion transport mechanism in the luminal membrane of fs cells . Interestingly, the cystic fibrosis transmembrane conductance regulator (cftr), a cl channel that is defective in cystic fibrosis, is expressed in the pituitary gland and likely mediates the camp - stimulated anion currents detected in cultured fs cells [4, 5]. A recent study showed that treatment with forskolin, which stimulates cftr - mediated cl secretion, affects secretion of growth hormone from pituitary slices in culture and suggested that loss of cftr activity in the pituitary might be responsible, in part, for the reduced growth rates in both humans and pigs with cystic fibrosis . The current results support the view that fs cells play a major role in regulating the ionic composition and volume of the interstitial and follicular fluid and suggest that nhe2 serves as an absorptive na / h exchanger on the luminal surface of fs cells that counters fluid accumulation resulting from cftr - mediated secretion of anions . Nhe2 and age- and gender - matched wild - type (wt) mice of the original 129/svj and black swiss background were used for these studies . All mice were housed in humidity and temperature controlled rooms, on a 12-hour light / dark cycle, with access to standard mouse chow and water ad libitum . Mice varied in age from 17 days to over 1 year old at euthanasia, with approximately equal numbers of males and females . Animals were separated into two age groups, age 1: 17 days to 2.5 months, age 2: 4 months1 yr, and 37 mice were used . The number (n) of mice in any given analysis or bar graph is given in the legend . Animal protocols were approved by the university of cincinnati institutional animal care and use committee, and animal handlers were trained in an american association of assessment and accreditation of laboratory animal care facility . Genotypes were determined by pcr analysis of dna isolated from tail biopsies using a set of 3 primers that simultaneously amplified wild - type and nhe2-null alleles . Forward (5-catctctatcacaagttgcccacaatcgtg-3) and reverse (5-gtgactgcatcgttgagcagagactcg-3) primers from the 5 and 3 ends of exon 2 amplified a 450-base pair product from the wild - type allele . A reverse primer (5-gacaatagcaggcatgctgg-3) complementary to sequences within the neomycin resistance gene cassette, which was inserted into exon 2, and the forward primer described above amplified a 221-base pair product from the targeted allele . Using total rna from colon and pooled pituitaries of 3 - 4 month - old mice and a 3.1 kb rat nhe2 cdna probe spanning nucleotides 4493561 (accession number l11236). In situ hybridization using s - labeled antisense (as) and sense (s) probes corresponding to codons 684813 and detected using autoradiographic emulsion . Pituitaries were removed immediately after euthanasia and immersed in 2% paraformaldehyde/2.5% glutaraldehyde or 4% paraformaldehyde in phosphate - buffered saline (pbs) for at least 24 hrs . Tissues were postfixed in 1% osmium tetroxide in either millonig's buffer or pbs for 2 hrs, dehydrated stepwise in increasing concentrations of ethanol, with two changes of propylene oxide and one change each of 1: 1 and 1: 3 propylene oxide: spurr (electron microscopy sciences, hatfield, pa). Tissues were left overnight in fresh 100% resin and flat - embedded in cut - off beem capsules (electron microscopy sciences, hatfield, pa) the following day in fresh resin . Sections, 1.5 m thick, were stained with toluidine blue for light microscopy . Pituitaries were also preserved in 4% paraformaldehyde, dehydrated in ethanol and xylene, blocked in paraffin, and serially sectioned at 5 m . Some of these slides were stained with hematoxylin and eosin (h&e) and others were prepared for in situ hybridization . Pituitaries fixed for fine structural detail and opportunely oriented were thin - sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy . Light microscopic morphology and morphometry were conducted on h&e stained sections as well as on plastic sections stained with toluidine blue . The vd (percentage) of cytoplasmic elements was calculated from the number of intersections of a 320 point grid lying over any element/(320 minus the points over nontissue elements) 100 . The 320-point grid was printed directly onto digital light micrographs acquired from toluidine blue stained sections at a magnification of 100x . Intersections of that grid lying over the following cytoplasmic and nuclear features were counted for pars distalis: (1) fs cell canalicular space, (2) obvious fs cell cytoplasm and nuclei (mainly where the cells were adjacent to canalicular lumens), (3) nuclei and cytoplasm of somatotrophs and mammotrophs (as a single group of cells containing large granules) and other endocrine cells (those with smaller granules), (4) lipid droplet inclusions, (5) blood vessels and vascular luminal contents (endothelium, red cells, and vascular space), and (6) negative areas (those which were not tissue content, such as grid bars, edges, folds, and tears). The number of cells per light micrograph was approximately 200; metaphase cells were counted and the percentage of cells in mitosis calculated . Although it was not possible to absolutely identify the cell types in 1.5 m thick sections at the light microscopic level, morphological consistencies among cell types allowed for a reasonable separation of the large granule cells, granule cells with smaller granules, fs cells, and capillaries . Fs cells are somewhat under - represented in light micrographs because the thinness is just at the limits of the light microscope . Basement membrane redundancy was quantified by drawing a line perpendicular to the endothelial cell basal lamina up through the basement membranes to the closest fs cell basal lamina . Measurements of the thickness of the pars intermedia were obtained similarly, using both paraffin and plastic sections at 10x magnification, drawing a line perpendicularly to the interface with pars nervosa up to the pars distalis, sometimes ending at the remnants of rathke's pouch . Data were analyzed using sas 9.1 (sas institute, cary, nc) and sigmaplot 2000 . Means and standard errors of the means were determined by genotype, gender, and age using the general linear model for vd parameters . Mean thickness of the pars intermedia, standard errors, and unpaired t - tests were obtained using sigmaplot . For data shown in bar graphs or tables, n is the number of animals . Data points from wt mice were pooled since no significant differences were found in morphometry within age comparisons . Images were obtained using a spot i camera and nikon inverted microscope or scanned from electron microscopy prints . Images were contrast enhanced, and, when needed, the grey tones were evened, and the bandaid, dust, and scratches filters were conservatively applied with photoshop 7 (adobe.com), without altering the integrity of the histological data . Previous studies of nhe2 mice revealed functional defects directly related to loss of ion transport activity in stomach, kidney, and colon [12, 15, 16]. Although some disturbances in fertility of nhe2 mice were suggested, gross appearance, growth curves, and overall health of nhe2 mice were seemingly normal . However, in a routine histological survey of h&e - stained tissues, we observed cyst - like structures in the pars distalis of nhe2 pituitary glands (figure 1). These first became noticeable by 5 weeks of age and by 10 weeks were well developed . Such cystic changes could be due to increased secretion of ions and fluid into canalicular spaces or to reduced absorption . To determine whether the cysts were a direct effect of the loss of nhe2, an absorptive na / h exchanger [15, 16], in pituitary rather than an indirect effect of pathology in other organs, the expression of nhe2 in the pituitary gland was analyzed by blot hybridization and in situ hybridization . Northern blot analysis of pituitary and colon rna (figure 2) showed that nhe2 mrna is expressed in the pituitary, but at much lower levels than in colon, the tissue in which the highest levels of nhe2 are expressed . The relative levels of expression suggest that expression of nhe2 mrna might be restricted to a limited area of the pituitary or to a small subset of pituitary cells . In situ hybridization (figure 3) showed three distinct areas in coronal and transverse sections from both wt and nhe2 mice, comprising the more or less concentrically arranged view of these anatomical divisions, with pars distalis forming the outer layer and pars intermedia in a semicircular array around the centrally located pars nervosa (figure 3). In situ hybridization using the antisense probe demonstrated similar grain distribution patterns for both wt and nhe2 pituitaries, though the latter signal was greatly reduced in intensity . The antisense probe was complementary to the part of the mrna that encoded amino acids 684813 of the protein . This region is present in the mutant mrna, which contains a frame - shift mutation just beyond codon 172; it is expressed at low levels in some tissues, likely accounting for the reduced signals detected . Hybridization with a control sense probe labeled the entire pituitary uniformly, and at very low levels, with no specific signal in any region (data not shown). With the antisense probe, grain deposition was the highest in the pars distalis, with lower levels in the pars intermedia . In pars distalis, the pattern of distribution of the grains was not clearly specific to either fs cells or to granule cells; however, this may be due to the interdigitation of fs cell cytoplasmic projections around the granule cells . Cystic changes were not observed in pars intermedia or pars nervosa of nhe2 or wt mice . The pars intermedia of nhe2 mice showed significant thinning (figure 3, cf . 3(i) and 3(j)) compared to that of wt mice (nhe2: 96 8 m, n = 6; wt: 146 8.4 m, n = 5; p = .003). Finger - like projections of the pars intermedia into the pars nervosa (black arrows in figures 3(e) and 3(g)) appeared to be reduced in nhe2 mice as well . These data show that pars intermedia is also affected, although the loss of nhe2 did not cause cystic changes as in pars distalis, at least in the time frame studied . Fs cells and their junctional complexes formed the canaliculi . On tissue sections they occupied a very small percent of the pars distalis in wt mice, a volume density (vd) of less than 2% . Canalicular lumens were generally collapsed in wt mouse pituitary but on occasion canaliculi contained small amounts of membrane - like debris . In contrast, in nhe2 mice there was a considerable increase in the combined vd of fs cells and canalicular space (figure 4) to a vd of more than 10% (nhe2 null: 12.05% 2.9; wt: 1.45% 0.35). The integrity of the canalicular lumens seemed not to be compromised, but it became apparent that with increasing age the combined vd of fs cells and follicular space (figure 4) increased in the pars distalis . Analysis of pituitary sections from a single 56-day old achlorhydric mouse that lacked the gastric h, k - atpase subunit revealed no histopathology . The vd of large granule pituitary cells (somatotrophs + mammotrophs) was greater in nhe2 pituitaries (figure 4) compared to wt . However, no differences were detected in the actual morphology of the large granule cells . Both vd of fs cells (figure 5) and dilation of canaliculi gradually increased in nhe2 mice compared to wt (figure 5). And while the vd of fs cell cytoplasm and nucleus increased to about 4% in the aged nhe2 mice (p = .04) it still represented only a small portion of the pars distalis as a whole . The increase in the vd of fs cells appeared to be fs cell hypertrophy rather than hyperplasia; this backed up the fact that there was no statistically significant increase in the number of mitotic cells in the nhe2 mice (nhe2: 0.022 0.17; wt: 0.003 0.02, p = .48). While the vd of folliculo - stellate cells increased in nhe2 mice, in several other respects, fs cell morphology and ultrastructure were similar to those of wt fs cells (figure 5). Connections among fs cells surrounding the dilatations were seemingly occlusive, and there was no evidence for expansion or edema in the lateral (intercellular) spaces . Junctional complexes were prominent and extensive and, like desmosomes, were most common just beneath the canalicular lumens . Canalicular cystic dilatations were quite variable in size and were clearly demarcated by contiguous fs cells . Nhe2 mice had fs cell canaliculi lined with unremarkable microvilli and an occasional primary cilium and sometimes contained membrane - like fragments and other cellular debris, and recognizable apoptotic bodies . There were occasional phagolysosmal structures within fs cells, particularly in the nhe2 mice . Ultrastructure of the fs cell cytoplasm and nucleoplasm was not obviously changed in organization (figure 5), and intercellular interdigitations among fs cells, appearance of the rough endoplasmic reticulum, polyribosomes, mitochondria, golgi bodies, nuclei, and other organelles appeared to be unchanged from wt as well . An increase in small lipid droplets (figure 6) was found predominantly in fs cell cytoplasm, and also in other cell types, including granulated cells . The vd of lipid was about 3-fold greater in the aging nhe2 mice than in wt mice, but in both groups the vd of lipid increased as age increased . Values for vd of lipid for each group were compared with the genotype - matched younger age group (resp . ), but statistical significance was reached in the nhe2 animals only . Lipid droplets were typically less than a few m in diameter and were found either as part of or adjacent to inclusions (phagolysosomes or autophagosomes) (figure 6(a)). Microvilli on the apical membranes of fs cells of nhe2 mice (projecting into the canalicular space) were not noticeably different from wt (figure 7). They appeared similar to the flexible microvilli that were found in numerous other tissues, such as bile canaliculi in the liver and canalicular microvilli of parietal cells in the stomach (about 1 m in length). The actin filaments seen on cross - section were more or less evenly distributed within the microvillus, if not peripherally, and were definitely not bundled centrally as one typically sees in intestinal and renal brush border . The microvilli were not very densely packed along the fs cell apical membrane in either wt or nhe2 fs cells (figure 7) though, on occasion, areas of densely packed microvilli were found in the nhe2 fs cells displaying a phenotype not unlike that of parietal cell canaliculi . Similar to the membrane - like debris occasionally found in canalicular lumens, membrane - like debris was sometimes found within the apical cytoplasm of fs cells of nhe2 mice (figure 7). Basal membranes of fs cells adjacent to capillary endothelium abutted two basement membranes: one an fs cell product and the other produced by the capillary endothelium . In wt mice reduplicated and redundant basement membranes were common in the nhe2 mice, older animals showing greater changes . Redundancies were sometimes focal, and on occasion as many as 8 redundancies were counted, with a mean number of basement membranes in the nhe2 samples significantly greater than in wt (wt: n = 3, 1.7 0.19; nhe2: n = 4, 2.2 0.14, p = .036). Subjectively, areas of redundant and duplicated membrane appeared more likely to be attributable to the fs cells than to the capillary endothelium . An increase in other extracellular matrix proteins may have occurred as well, and occasionally collagen was seen . The vd of blood vessels in the pars distalis was not significantly different among the groups of old, young, null, or wt mice . There were many desmosomal - mitochondrial connections, which spanned adjacent fs cell membranes at the site of desmosomes (figure 9). These comprise mitochondria with a portion of outer lamella abutting the keratin filaments which radiate from the desmosome into the cytoplasm . Such desmosomal - mitochondrial juxtapositions often were present on both sides of the desmosome (i.e., adjacent to desmosomes in adjacent cells), other times adjacent to the desmosome on one side only . Since so many of these juxtapositions occurred, they are likely more paired than not, at least in wt mice, and section orientation has left one mitochondrion unseen (table 1). The order and incidence of junctional complexes, desmosomes, and desmosomal - mitochondrial associations in a tally made from electron micrographs revealed a significant reduction from wt in the number of desmosomes which were associated with 2 mitochondria (table 1). Junctional complexes, intercellular spaces, membrane interdigitations, microvilli, and pinocytotic vesicles / caveolae are not different in nhe2 than in wt samples, as counted in this study . A count of caveolae per membrane profile per cell showed no significant difference between wt and nhe2 (wt: 0.69 0.16; nhe2: 0.23 0.10; p = .2). The most prominent histological finding consequent to gene - targeted ablation of the nhe2 na / h exchanger was the dilation of the canaliculi formed by fs cells in the pars distalis of the anterior pituitary . The cyst - like structures were lined by fs cells, with their luminal surfaces containing microvilli, and a morphology clearly indicating that they were derived from the network of interconnected follicular cavities . The cysts became apparent at about 5 weeks of age and were very prominent by 8 and 10 weeks . The absence of cysts in pituitary sections from an achlorhydric mouse lacking the gastric h, k - atpase subunit indicated that the cysts in the nhe2 pituitaries were a direct response to the loss of nhe2 in the pituitary rather than an indirect response to achlorhydria . Given the known absorptive functions of nhe2 [14, 15], the expression of nhe2 mrna in pars distalis and at lesser levels in pars intermedia, both of which are reported to contain fs cells, and the previously demonstrated transcellular fluxes of ions and water across fs cells [4, 5], the data suggest that nhe2 serves as an important absorptive na / h exchanger on the luminal surface of fs cells of the anterior pituitary . Nhe2 mrna expression, as indicated by northern blot analysis, was very low in the pituitary gland when compared with its expression in colon, the tissue with the highest expression in rats and mice [11, 12]. The apparent low level of expression may be due in part to the relatively low abundance of fs cells compared to endocrine cells, as indicated by measurements of fs cell vd; however, other factors might also be involved . Under normal conditions, the luminal membrane of the follicles, where nhe2 is presumably expressed, is a small percentage of the total plasma membrane of fs cells . In addition, the gradual appearance and expansion of the canaliculi in nhe2-null pituitary glands suggest that the absorptive process is far less robust than in colonic crypts and kidney, where large quantities of ions and water are absorbed on a continuous basis . Rather, the pituitary phenotype developing with the loss of nhe2 suggests that absorption mediated by low levels of nhe2 may counter relatively low levels of fluid accumulation in the follicular cavities . Attempts to identify the membrane location of nhe2 protein in the pituitary gland using antibodies that have been used for immunolocalization in apical membranes of salivary acinar and duct cells were unsuccessful . Thus, at the present time the membrane location of nhe2 can only be inferred from the histological phenotype and from its expression on apical membranes in epithelial tissues where it is expressed at much higher levels . Earlier studies showed that fs cells in culture can function like typical epithelial cells involved in transepithelial ion and fluid transport . When grown as primary cultures, fs cells derived from the pars distalis and pars tuberalis and from the pars intermedia formed domes, indicating that they absorbed ions and water from the culture fluid . Studies using cultured cells mounted in ussing chambers showed that application of -adrenergic agonists to the serosal surface led to currents consistent with cftr - mediated anion secretion, whereas application of amiloride to the mucosal surface, at concentrations known to inhibit the epithelial na channel, led to a reduction in transepithelial currents, with a further reduction in response following inhibition of the na, k - atpase with ouabain . These data indicated that fs cells contained an apical anion channel and apical transporters that were able to absorb na, with accompanying anions, from the luminal spaces . Aquaporin 4 has also been shown to be expressed in fs cells, suggesting that it contributes to water transport associated with ion movements . The identities of specific ion transporters involved in transcellular ion fluxes in fs cells have not been determined; however, a recent study showed that cftr is expressed in the pituitary and the current data indicate that nhe2 plays a critical role in this process . Nhe2 is one of two absorptive na / h exchangers in apical membranes of renal and intestinal epithelial cells [14, 15]. Nhe3 mediates high levels of nacl absorption via coupling with either cl / formate exchange or cl / hco3 exchange [21, 22]. However, when operating alone, na / h exchange mediates na and hco3 absorption . Nhe2 has an unusual ph sensitivity that would appear to make it suitable for a role in nahco3 absorption . Its activity is very low at normal extracellular ph, but it exhibits 50% activity at ph 8.0, and its activity increases further as the extracellular ph increases . In the kidney, nhe2 plays a role in na and hco3 absorption and is particularly important under conditions of increased delivery of hco3 to the distal nephron . In colon, nhe2 is expressed primarily in the crypts, where the luminal ph is relatively alkaline in the presence of physiological concentrations of short - chain fatty acids, again consistent with a function in nahco3 absorption . As far as we are aware, the ph and hco3 content of follicular fluid is not known, but it is possible that the sensitivity of nhe2 to extracellular ph makes it more suitable for its role in the pituitary . Cystic dilatations were not observed in the pars intermedia of nhe2 mice despite the presence of fs cells and nhe2 expression in this segment of wt pituitary glands . It is possible that secretory processes that expand the canaliculi, which may involve cftr - mediated anion secretion, are less active in this segment than in the pars distalis or that a compensatory absorptive mechanism prevents their expansion . For example, an ion transport mechanism that can compensate for the loss of nhe2 has been shown to be activated in colonic crypts of nhe2 mice . For example, de bold et al . Reported the presence of two types of interconnected channels in the pars intermedia, which they noted was highly avascular and presumed that fluid and solutes in the channel systems can reach the general circulation . If this were the case, it could provide drainage, thus preventing cyst formation . It is difficult to draw clear parallels between the pituitary and stomach phenotypes of nhe2-null mice . Nhe2 was originally proposed to function on the basolateral membrane of the parietal cell, operating in concert with the ae2 cl / hco3 exchanger to mediate na and cl uptake that is required for stimulated acid secretion . However, it is clear that the few mature nhe2 parietal cells were able to secrete high levels of acid and there is evidence that nhe4 is the basolateral na / h exchanger required for acid secretion . An alternative possibility is that nhe2 is expressed in canalicular membranes, where it could operate in concert with the slc26a6 cl / hco3 exchanger and other transporters to dehydrate secretory membranes during transitions to the resting state . Such a function would be similar to the absorptive function proposed in fs cells and consistent with its apical expression in other epithelial tissues [14, 15, 19, 28]. The cystic dilations in the fs cell canaliculi in nhe2 mice did not readily conform to those of known human or animal pituitary disease, where most reported cystic changes were within pituitary neoplasms or were cysts containing mucins or colloid - type inclusions [29, 30]. Metaplastic transformation of fs cells, described in human pituitary, was not found in the nhe2 mice . While neither colloid contents nor mucins were found with any regularity within the dilated canaliculi of nhe2 (or wt) pituitaries, membrane and nuclear debris were seen on occasion . They were greater in quantity in nhe2 pituitaries than in wt, and likely the residue from an occasional expulsion of previously phagocytosed apoptotic debris into the canalicular lumen . The amount of cellular debris, apoptotic bodies, or phagolysosomal vacuoles did not suggest excessive fs cell death in the nhe2 mice . This is in contrast to conspicuous parietal cell death which occurred in the stomach of nhe2 mice . Fs cell microvilli most closely resembled the morphology of bile canaliculi in the liver, but nhe2 fs cells occasionally formed apical invaginations containing clusters of densely packed microvilli (figure 7(b)) resembling those of parietal cell canaliculi . It is unclear whether these rare instances of densely packed microvilli correspond to an absorptive state or to a secretory state, as is the case for parietal cells, but given the invagination of the canaliculus into the cell a secretory state seems more likely . Ultrastructure was also affected on the basal side of fs cells and comprised duplicated and/or redundant basement membranes . Fs cells produce their own basement membranes, of the continuous type, distinct from, and parallel to, basement membranes of the capillary bed . The ability of these cells to produce matrix proteins and basement membrane structures has been described using a transgenic mouse model . Redundancies in basement membranes occurred primarily beneath fs cells and were short and fragmented . Duplicated basement membranes have been described in bile duct and other tissues [3436] but were not reported in stomach or other affected tissues of the nhe2 mouse [12, 13]. It is possible that physical strain related to canalicular dilation could induce an increase in matrix proteins or that perturbations of cellular homeostasis related to the ion transport defect are involved . Fs cells displayed numerous instances of close physical associations between mitochondria and the intracellular filaments of desmosomes . In wt mice, over half of the desmosomes exhibited associations with two mitochondria, one from each cell contributing to the desmosomal junction . Nhe2 fs cells exhibited a significant decrease in the percentage of desmosomes associated with 2 mitochondria, from 52% in wt to 7% in mutant mice . Desmosomes undergo frequent remodeling, so if mitochondria are involved in this process, it would suggest less remodeling in nhe2 fs cells . The frequent associations between mitochondria and desmosomes have led to speculations of their providing for energy requirements and calcium delivery, but the function is not known . These desmosome - mitochondria associations [3841] lend further credence to the dynamic nature of the apical membrane of fs cells . Lipid droplet accumulation in the cytoplasm of cells is a well - recognized indicator of cell stress and pathology [4245]. Therefore the significant increase in cytoplasmic lipid in fs and granule cells of the pars distalis of nhe2 mice may be an indication of cell stress resulting from the loss of nhe2 . This observation and the increased vd of large granule cells indicate that these endocrine cells are negatively affected by the fs cell defect . The function of the canalicular network is unclear; however, there is compelling evidence that fs cells themselves form an extensive cell network in addition to the canalicular network and that intrapituitary communication occurs via ca signaling through gap junctions [9, 10, 4648]. This mechanism, which allows propagation of ca signals over long distances within the anterior pituitary, has been suggested to provide a means by which fs cells synchronize secretory activity of endocrine cells [9, 10]. It is clearly important, and perhaps predominant; however, it has also been noted that the anastomosing channels formed by fs cells might provide a means of synchronizing hormone secretion . One can speculate that the two networks operate separately, with fs cell ca signaling through gap junctions providing rapid coordination of endocrine activity and fs cell control of the canalicular fluid composition via ion secretion and absorption providing longer - term regulation . Alternatively, anion secretion and ca signaling might act in concert if they were stimulated together or if one activity triggered the other . Additional studies will be needed to identify all of the transporters involved in ion secretion and absorption by fs cells and to determine their relevance to human diseases involving transepithelial ion transport defects . In this regard, it has long been reported that pituitary function is impaired in cystic fibrosis, the most common genetic disease in humans, and recent work showed that cftr is expressed in pituitary and that the absence of its activity impairs hormone secretion (, and references therein). In conclusion, the loss of nhe2 produced anterior pituitary pathology, including a gross dilation of fs cell canaliculi, increase in the vd of fs cells, a significant accumulation of small lipid droplets in fs cell and granule cell cytoplasm, a reduction in desmosomal - mitochondrial complexes, reduplication of the fs cell basement membranes, an increase in the vd of somatotrophs and mammotrophs, and a thinning of the pars intermedia . The absence of apparent effects on growth and development suggests that dilation of the canaliculi resulting from the loss of nhe2 is a relatively benign defect . Nevertheless, a fertility defect remains unexplained, and the increase in both vd and lipid droplets in large granule cells showed that endocrine cells are affected . The dilation of the canaliculi suggests that nhe2 is the major absorptive mechanism that counters the accumulation of canalicular fluid resulting from ion secretion by fs cells . Thus, if ion secretion serves an important function in the pituitary, as suggested by studies on the pig cystic fibrosis model and human infants with cystic fibrosis, then one might expect that disease conditions or drugs that impair ion secretion or homeostatic regulation of the volume and ionic composition of the canalicular fluid would affect pituitary function and human health.
Autonomic neuropathy (an) has long been recognized as a significant health risk and leads to reduced quality of life (qol), increased mortality and morbidity and increased health care costs (1 - 2 - 3). Cardiovascular autonomic neuropathy (can) is an end - stage condition and is characterized by structural deficits due to loss of parasympathetic or sympathetic neurons that innervate the heart and blood vessels, resulting in abnormalities in heart rate (hr) control and vascular dynamics (3). Can is associated with multiple cardiovascular disease states, including low ejection fraction (4, 5), atrial fibrillation (6), ventricular arrhythmias (7), cardiomyopathies (8, 9), symptomatic systolic heart failure (10) and myocardial ischemia (11), as well as many of the traditional and nontraditional risk factors for heart disease (12). In patients diagnosed with diabetes and coronary artery disease serial an testing of a large population (774) of patients with diabetes demonstrated that 26% progressively worsened over time, despite clinical surveillance, while only 3% improved (13). A meta - analysis of 15 studies of patients diagnosed with diabetes and can demonstrated a relative mortality risk of 3.65 over those without can (3). The disease process of an is poorly understood by many clinicians and the early stages of disease are often overlooked . The two branches of the autonomic nervous system (ans), parasympathetic and sympathetic (p&s), act together to maintain sympathovagal balance (sb) (14). Establishing and maintaining appropriate sb slows the progression of an, reducing morbidity and mortality and improving outcomes (3, 15, 16). Eta1-adrenergic agonists (beta - blockers) are known to reduce sympathetic activity (17) and thereby alter sb . Is known to be cardioprotective and is associated with greater longevity in geriatric and heart disease patients (16, 18). Propranolol and metoprolol have been studied previously for their effect on diabetic autonomic nerve dysfunction, and these drugs affect only sympathetic inhibition (19, 20). The third - generation adrenergic blocker, carvedilol, has both beta - blocker and alpha - blocker effects . The alpha - blocker acts as a vasodilator (21), and an antioxidant and endothelin biosynthesis suppressor (22). Thus, carvedilol has a broader affect on the ans, which may explain why it further reduces mortality (23). Our hypotheses are that the beta - blocker, metoprolol, would inhibit sympathetic tone less than the combination alpha- and beta - blocker, carvedilol . Second, we hypothesize that carvedilol will also result in an increase in parasympathetic activity . Patients who had undergone serial autonomic p&s testing (anx-3.0; ansar medical technologies, inc ., the registry consists of 147 arrhythmia - free patients diagnosed with noninsulin - dependent (type 2) diabetes mellitus from eight ambulatory clinics located in new york, pennsylvania, new jersey, maryland and virginia . The patient selection method and demographics, and the three noninvasive autonomic measurement methods used for screening, are described in the companion article (24). (+) beta - blocker, are those patients who were beta - blocker nave at the beginning of the study and were started on beta - blocker during the study . ()beta - blocker, consists of patients who started the study period on beta - blocker and had beta - blocker discontinued during the study . Eg3, (o)beta - blocker, the control group, are patients who were on stable doses of beta - blockers that did not change throughout the study . Eg3 patients were case - matched to the study population patients with respect to comorbid conditions such as hypertension (htn) and cardiovascular disease (cvd), as well as age, gender, height and weight . Each experimental patient in the registry performed a baseline p&s study (test n) the day of the prescribed change to their beta - blocker . To remain in the registry, there needed to be documentation of the patient s compliance according to grouping, for at least 3 months, after which the patient performed a follow - up p&s study (test n+1). All graphs consist of two curves: the broken lines represent the responses to carvedilol, and the solid lines represent the responses to metoprolol . In all figures, the responses are normalized to 1.0 in the baseline test to highlight the changes () in response to beta - blocker titration . The figure data present the average change () in a parameter from baseline to follow - up (test n+1). Absolute values for the data in the figures are presented in the tables of the companion article (24). The average age, height and weight of the cohort are 61.8 12 yrs, 166.7 27 lb and 64.8 6 in, respectively, and serial tests are separated by 4.1 (1.2) months (24). Figure 1 displays the normalized, average hemodynamic responses from the two tests for each of the three subpopulations . Similarly, figures 2 through 4 display the results from the three autonomic measurement methods: (1) p&s method results, (2) spectral - domain hrv - alone results and (3) time - domain hrv - alone results, respectively . (a) and (b) represent the experimental groups (1 and 2, respectively) and (c) represents the control group . (a) and (b) represent the experimental groups (1 and 2, respectively) and (c) represents the control group . (please see text for details .) In response to increasing either agent, the mean hr (mhr) and mean systolic bp (sbp) decreased . In response to increasing metoprolol, the mhr decreases from 78 to 72 bpm and the average mhr response to increasing carvedilol decreases from 76 to 70 bpm (both statistically significant, p<0.001). The average changes in sbp were not significant in response to increasing either agent, staying at 125 mmhg for metoprolol and changing from 122 to 121 mmhg for carvedilol . The metoprolol - nave subpopulation demonstrates an average increase in dbp from 73 to 74 mmhg, and the carvedilol - nave subpopulation demonstrates no change . In response to discontinuing either agent, all hemodynamic measures increase there is a statistically significant increase in mhr in response to discontinuing carvedilol (from 73 to 77 bpm) as compared with metoprolol (68 to 69 bpm; p<0.001). The average sbp increase to discontinuing either agent (from 115 to 116 mmhg and 122 to 123 mmhg for metoprolol and carvedilol, respectively) is also statistically significant (p<0.001), but not clinically significant . The average dbp increases in response to discontinuing either agent are not significant (from 67 to 69 mmhg for metoprolol and 69 to 70 mmhg for carvedilol). In response to continuing either agent, none of the hemodynamic responses are significant . The normalized data (to highlight the respective changes in hemodynamic responses) are displayed in figure 1 . From figure 2, the results of the p&s method show that when either beta - blocker is introduced (fig . However, only the (greater) decrease for carvedilol is significant (p<0.001). A significant increase in parasympathetic activity (fig . 2a, middle) is measured (p<0.001) in response to either agent, with the average parasympathetic increase in response to carvedilol greater than that for metoprolol . A significant decrease in sb (fig . 2b, top) to discontinuing either agent increases significantly (p<0.001 for carvedilol response and p<0.010 for metoprolol response), and the average parasympathetic responses decrease significantly (p<0.001; fig . 2b, middle). For both p and s, the change in response to carvedilol is greater than that for metoprolol the average sb response to discontinuing carvedilol increases, whereas the average sb response to discontinuing metoprolol decreases . For those patients continuing beta - blocker therapy without any adjustment (fig . 2c), there is very little change in p&s method results: p = 0.747 for sympathetic activity, p = 0.301 for parasympathetic activity and p = 0.275 for sb, overall . The results of the spectral domain hrv - alone analysis [low frequency (lf), high frequency [hf] and lf / hf ratio) are presented in figure 3 . The total spectral power (tsp) normalized lf (lfnu) increases in response to discontinuing carvedilol, and decreases in response to discontinuing metoprolol (fig . 3b, middle; p<0.010) in response to carvedilol, and increases in response to metoprolol . Ratio decreases in response to carvedilol, and increases in response to metoprolol (fig . Tsp decreases in response to discontinuing either agent, with only the metoprolol change being significant (fig . These data show that average tsp changes to either agent are statistically significant (see fig . 4, top row). In response to increasing beta - blocker, tsp increases in response to carvedilol (p<0.001) and decreases in response to metoprolol (p<0.010) (see fig . 4a, top). While the absolute value of the change in tsp in response to carvedilol is much greater than that for metoprolol (240.9 vs 42.2 ms, respectively), the relative changes with respect to baseline (test n) are similar . For patients who continued beta - blocker without adjustment (fig . 3c), carvedilol further decreases the lf measure significantly (lfnu = 0.34, p<0.010) and increases the hf measure significantly (hfnu = + 0.77, p<0.010), whereas only metoprolol decreases the hf measure significantly (hfnu = 0.08, p<0.010). Spectral - domain hrv - alone responses to beta - blocker therapy at baseline and at follow - up . (a) and (b) represent the experimental groups (1 and 2, respectively) and (c) represents the control group . The time - domain hrv - alone responses to the introduction of a beta - blocker (fig . 4) were mixed and statistically significant in response to either agent (p<0.001). Rangehr (not shown graphically, see (24, table 4)) and e / i ratio increase in response to carvedilol and decrease in response to metoprolol . 4a, bottom). The average response to introducing carvedilol (p<0.001) is greater than that to metoprolol (p<0.010). Rangehr decreases significantly in response to either agent (data not shown, see (24, table 4); p<0.001), while the e / i ratio increase is statistically significant for carvedilol (fig . 4b, bottom) to discontinuing either agent are statistically significant (p<0.001 for both). Total spectral power and time - domain hrv - alone responses to beta - blocker therapy at baseline and at follow - up . (a) and (b) represent the experimental groups (1 and 2, respectively) and (c) represents the control group . Our study concurs with, and expands upon, previously reported effects of beta - blockers in restoration of autonomic balance (20). More specifically however, our study shows that carvedilol, which is both an alpha- and beta - adrenergic blocker, demonstrates a greater response than metoprolol, which is a pure beta - blocker, and demonstrates clear differences on the effects on sympathovagal balance (sb). This may be due to the fact that carvedilol contains more adrenergic blockade as compared with metoprolol . Whether hrv - alone is in the form of time - domain measures or frequency - domain measures, hrv - alone is shown in this study to not be a reliable or consistent measure to differentiate parasympathetic activity independent from sympathetic activity (see the discussion in (24)). The subpopulation of patients who continued current beta - blocker therapy without any adjustment can be considered the control group . As shown in figure 2c, no significant changes in average hemodynamic responses are demonstrated (note the scale of the ordinate). Similarly, as shown in figure 3c, no significant changes in average parasympathetic or sympathetic responses are demonstrated . The changes in the p&s method are consistent with the hemodynamic changes observed for the same group (p = 0.055). As seen in figure 4c, the hrv - alone, spectral analysis method showed statistically significant responses to the continuation of beta - blockers . Carvedilol further decreased the lf measure (assumed to represent sympathetic tone) and increased the hf measure (assumed to represent parasympathetic tone), whereas metoprolol decreased hf and produced no change to the lf measurements . The spectral analysis of hrv - alone changes are not consistent with the hemodynamic changes observed for the same group (p = 0.880). It is known that the sympathetic nervous system exerts major effects on mhr and the baroreceptor reflex response, which in turn contributes to the regulation of bp . While beta - blockers cause decreases in mhr, sbp and sympathetic activity (see figs . 2 and 3), the net desired effect is an overall reduction in relative sympathetic activity . The data demonstrate that, on average, metoprolol has less of a sympatholytic effect that carvedilol (see fig . These results are corroborated by the sb responses (sb = s / p from the p&s method) seen in figure 3a, bottom . 3a) responses to the introduction of metoprolol are greater than that to carvedilol due to overall reduction in relative sympathetic activity, a significantly greater increase in tsp (fig . 4a, top) in response to carvedilol with respect to metoprolol is demonstrated . Since tsp is based on the arithmetic sum of lf and hf (25), and since the lf measure decreases, and the hf measure increases, the change in tsp indicates a relatively greater increase in the hf with carvedilol than with metoprolol . Therefore, the change in tsp would indicate a net, relative increase in parasympathetic activity . Similarly, increase in e / i ratio, rangehr and hrv, and the greater sdnn increase in response to carvedilol over metoprolol is significant . These results suggest that carvedilol not only reduces overall sympathetic dominance, but concomitantly increases parasympathetic activity, while metoprolol only reduces relative sympathetic dominance . After weaning from beta blockers, an increase in sympathetic activity and decrease in parasympathetic activity increases in mhr, bp and sympathetic activity increase significantly more after weaning from carvedilol than weaning from metoprolol . Significant increases in the sb response to withdrawing carvedilol indicate a shift in the autonomic balance toward a more sympathetic dominant state, as our results reflect . The spectral domain hrv - alone (lf and hf) responses to discontinuing beta - blockers are mixed . Lf increases in response to discontinuing carvedilol, which is consistent with a significant increase in sympathetic activity as compared with the decrease in parasympathetic activity . However, lf decreases in response to discontinuing metoprolol . This dichotomy may also be due to the dual alpha- and beta - adrenergic activity of carvedilol having greater sympathetic affect . Given that lf is defined as a measure of sympathetic activity as modulated by parasympathetic activity (25), the effect of discontinuing metoprolol on the lf measure may indicate a weaker sympathetic rebound and a relatively stronger decrease in parasympathetic activity . In other words, sb (the measure of relative sympathetic to parasympathetic activity) increases when carvedilol is discontinued and decreases when metoprolol is discontinued . This theory is supported by the absolute p&s values of these changes (see (24), table 2). The subpopulation of patients who continued current beta - blocker therapy without any adjustment can be considered the control group . As shown in figure 2c, no significant changes in average hemodynamic responses are demonstrated (note the scale of the ordinate). Similarly, as shown in figure 3c, no significant changes in average parasympathetic or sympathetic responses are demonstrated . The changes in the p&s method are consistent with the hemodynamic changes observed for the same group (p = 0.055). As seen in figure 4c, the hrv - alone, spectral analysis method showed statistically significant responses to the continuation of beta - blockers . Carvedilol further decreased the lf measure (assumed to represent sympathetic tone) and increased the hf measure (assumed to represent parasympathetic tone), whereas metoprolol decreased hf and produced no change to the lf measurements . The spectral analysis of hrv - alone changes are not consistent with the hemodynamic changes observed for the same group (p = 0.880). It is known that the sympathetic nervous system exerts major effects on mhr and the baroreceptor reflex response, which in turn contributes to the regulation of bp . While beta - blockers cause decreases in mhr, sbp and sympathetic activity (see figs . 2 and 3), the net desired effect is an overall reduction in relative sympathetic activity . The data demonstrate that, on average, metoprolol has less of a sympatholytic effect that carvedilol (see fig . These results are corroborated by the sb responses (sb = s / p from the p&s method) seen in figure 3a, bottom . 3a) responses to the introduction of metoprolol are greater than that to carvedilol due to overall reduction in relative sympathetic activity, a significantly greater increase in tsp (fig . 4a, top) in response to carvedilol with respect to metoprolol is demonstrated . Since tsp is based on the arithmetic sum of lf and hf (25), and since the lf measure decreases, and the hf measure increases, the change in tsp indicates a relatively greater increase in the hf with carvedilol than with metoprolol . Therefore, the change in tsp would indicate a net, relative increase in parasympathetic activity . Similarly, increase in e / i ratio, rangehr and hrv, and the greater sdnn increase in response to carvedilol over metoprolol is significant . These results suggest that carvedilol not only reduces overall sympathetic dominance, but concomitantly increases parasympathetic activity, while metoprolol only reduces relative sympathetic dominance . After weaning from beta blockers, an increase in sympathetic activity and decrease in parasympathetic activity is expected . Increases in mhr, bp and sympathetic activity increase significantly more after weaning from carvedilol than weaning from metoprolol . Significant increases in the sb response to withdrawing carvedilol indicate a shift in the autonomic balance toward a more sympathetic dominant state, as our results reflect . The spectral domain hrv - alone (lf and hf) responses to discontinuing beta - blockers are mixed . Lf increases in response to discontinuing carvedilol, which is consistent with a significant increase in sympathetic activity as compared with the decrease in parasympathetic activity . However, lf decreases in response to discontinuing metoprolol . This dichotomy may also be due to the dual alpha- and beta - adrenergic activity of carvedilol having greater sympathetic affect . Given that lf is defined as a measure of sympathetic activity as modulated by parasympathetic activity (25), the effect of discontinuing metoprolol on the lf measure may indicate a weaker sympathetic rebound and a relatively stronger decrease in parasympathetic activity . In other words, sb (the measure of relative sympathetic to parasympathetic activity) increases when carvedilol is discontinued and decreases when metoprolol is discontinued . This theory is supported by the absolute p&s values of these changes (see (24), table 2). Noninvasive measures of hemodynamic and autonomic responses to changes in beta - blocker therapy were collected . Overall, the hemodynamic results are consistent with the known effects of changes in specific beta - blocker vs alpha- and beta - blocker therapy (21 - 22 - 23). The combination of alpha- and beta - blocker decreases sympathetic tone and increases parasympathetic tone, whereas a pure beta - blocker precipitates a reflex increase in alpha - adrenergic activity, leading to less of an increase in parasympathetic tone . The p&s method results are consistent with the hemodynamic results and with the expected autonomic effects of changes in beta - blocker therapy known to underlie the hemodynamic responses (13). As shown, hrv - alone measures are not always consistent with the hemodynamic responses, and are only consistent with expected variations in hr and perhaps total autonomic activity . Adrenergic - blockade is shown to reduce sympathetic activity . By providing both alpha- and beta - blockade, carvedilol has a more profound effect in reducing sympathetic activity as compared with the pure beta - blockade of metoprolol . Our results also suggest that carvedilol not only reduces overall sympathetic dominance, but concomitantly increases parasympathetic activity . Adrenergic antagonists may restore autonomic balance and decrease functional disturbances that can lead to early an (26), which leads to increased morbidity and earlier mortality (3, 27). A simple measure of balance (sb) done prospectively may add significantly to the ability to identify and predict people at risk, and the adoption of multifactorial preventive measures . Both agents block sympathetic input to the heart, reducing cardiac sympathetic tone, thereby lowering hr . Carvedilol also blocks sympathetic input to the vasculature, reducing sympathetic tone in the blood vessels, leading to vasodilation . The importance of autonomic assessment is emphasized by the current reports from the diad study (28) and the accord study (29, 30). Diad demonstrates that abnormal hrv is associated with a prediction of greater increased risk of major adverse cardiovascular events (maces). The risk is greater than all traditional risk factors, including perfusion scanning (28). The development of ad is a multifactorial process, and maintaining sb may improve longevity and decrease morbidity and mortality . Thus, it is likely that combination therapies directed at the various components of the pathogenic pathway will be needed to reduce the development of autonomic nerve fiber dysfunction . With intensification of glycemic control, accord demonstrates a 22% increase in sudden deaths associated with ad . Establishing and maintaining normal parasympathetic and sympathetic balance may reduce morbidity risk and minimize mortality risk . The importance of autonomic assessment is emphasized by the current reports from the diad study (28) and the accord study (29, 30). Diad demonstrates that abnormal hrv is associated with a prediction of greater increased risk of major adverse cardiovascular events (maces). The risk is greater than all traditional risk factors, including perfusion scanning (28). The development of ad is a multifactorial process, and maintaining sb may improve longevity and decrease morbidity and mortality . Thus, it is likely that combination therapies directed at the various components of the pathogenic pathway will be needed to reduce the development of autonomic nerve fiber dysfunction . With intensification of glycemic control, accord demonstrates a 22% increase in sudden deaths associated with ad . Establishing and maintaining normal parasympathetic and sympathetic balance may reduce morbidity risk and minimize mortality risk.
Location of the study area - the administrative district of barcelos is located north of am . To the east, it borders the state of roraima; to the southeast and south, it borders the district of santa isabel do rio negro and, to the north, it borders venezuela (latitude 058'1 " south of the equator and longitude 6256 " west of greenwich). The city of barcelos, where this study was conducted, is located on the right bank of the negro river, 490 km from manaus, the capital of am (fig . 2). 2location of barcelos, state of amazonas, brazil, 2010, and number of blood samples collected by teams (a - f) and city district for screen tests . Survey and samples - the new serological survey was conducted in the main settlement of barcelos and covered the entire resident population that was willing to participate in the survey after providing informed consent . For minors, the main settlement of the municipality was divided into six areas (a - f), which were to be covered by the municipality's health agents under supervision by two laboratory technicians at the municipal hospital . The teams were trained by the project coordinator (jr coura, a physician and infectologist) and were monitored throughout the survey by a postgraduate student (mhp marquez). Each team was composed of two health agents; the two laboratory technicians each supervised three of the teams and all six teams were monitored by the postgraduate student . In total, 4,880 blood samples were collected on filter paper in accordance with the technique of souza and camargo (1966) for the screening test: 770 samples were collected by team a (so sebastio), 925 by team b (so lzaro), 1,000 by team c (so francisco), 728 by team d (nazar), 817 by team e (vila linhares) and 640 by team f (aparecida). All the blood samples on the filter paper were eluted in 250 l of phosphate - buffered saline (ph 7.2) and were tested using the iif test, as recommended by fife and muschel (1959) and souza and camargo (1966), as adapted by ostermayer et al . (2011) for the detection of anti- t. cruzi antibodies . The iif - chagas kit produced by biomanguinhos was used . In this kit, the antigens used are cultured epimastigote forms of t. cruzi in liver infusion tryptose medium . The tests were performed in the serology sector of the parasitic diseases laboratory of the oswaldo cruz institute, oswaldo cruz foundation, state of rio de janeiro (rj), brazil . The slides were prepared in accordance with the classical techniques for iif and were examined under an immunofluorescence microscope by at least two observers . The slide readings were considered to be negative (unreactive, absence of fluorescence), doubtful (weakly reactive, low intensity of fluorescence) or positive (strongly reactive, high intensity of fluorescence). Confirmatory serological test - to perform confirmatory reactions, venous blood (5 ml) was collected by the laboratory technicians from 315 patients (60%) who presented positive (137 patients; 62%) or doubtful (178 patients; 59%) iif reactions to t. cruzi infection based on the serological screening of blood on the filter paper . After separation, the serum was frozen, packed on dry ice and sent from barcelos to our laboratory in rj . These serum samples were subjected to three serological reactions: iif (chagas kit, biomanguinhos) with a dilution of 1:40, with the positive samples tittered as much as 1:1,280, conventional elisa (dmed kit) and recombinant elisa (wiener lab . Samples were considered to be seronegative if two or three of the tests were negative and seropositive if two or three of the tests were positive . Serological screening test - the screening test performed on the blood placed on the filter paper using immunofluorescence, 221 (4.5%) of the 4,880 samples were considered to be positive (strongly reactive) and 302 (6.2%) were considered to be doubtful (weakly positive) (table ii). Of the 4,880 samples examined, 2,741 (56.2%) were from females and 2,139 (43.8%) were from males . Among the 221 samples that were considered to be positive, 104 (3.8%) were from females and 117 (5.5%) were from males . The numbers of blood samples collected on filter paper, along with the percentage of positivity for t. cruzi infection, varied according to the number of people living in the area and their degree of exposure to infection in rural areas, especially in relation to piassava harvesters and their families . This characteristic has already been presented in previous studies by our group (coura et al . 1995a, b, 2002a, b, 1999, albajar et al . 2010, coura & junqueira 2012, junqueira et al . 2013). Table ii age groups (years) tested n positives n (%) doubtful n (%) <5 495 9 (1.8) 17 (3.4)5 - 962618 (1.9)30 (4.8)10 - 1462212 (2.9)30 (4.8)15 - 1952424 (4.6)35 (6.7)20 - 2987246 (5.3)56 (6.4)30 - 3957834 (5.9)41 (7.1)40 - 4941324 (5.8)39 (9.4)50 - 5929319 (6.5)18 (6.1)60 - 6922717 (7.5)17 (7.5)70 - 791389(6.5)10 (7.2)> 79554 (7.3)2 (3.6)unknown 37 5 (13.5)7 (18.9) 4,880 221 (4.5) 302 (6.2) a: yanomami indians . A: yanomami indians . The prevalence of serological positivity in samples collected on filter paper gradually increased after the age of 15 years, precisely when adolescents (especially males) started to be involved in piassava harvesting and after the age of 20 years among women, when as wives would accompany their husbands to the piassava plantations (fig . The unknown age group (yanomami indians who do not know their age) is the most exposed to wild triatomines in the forest; therefore, they had the highest percentage of iif - positive results (13.5%), as shown in fig . 3percentage of positive immunefluorescence by age group in the screen tests in barcelos, state of amazonas, brazil, 2010 . Confirmatory serological test - among the 137 serum samples that were considered to be positive based on immunofluorescence testing, 37 (27%) were considered to be seronegative based on the confirmatory serological test, 67 (48.9%) were considered to be serodivergent, as only one of the three reactions was positive, and only 33 (24.1%) were confirmed as seropositive, with two or three positive reactions (table iii). Of the 178 serum samples for which the immunofluorescence was considered doubtful, 89 (50%) were seronegative in all three confirmatory reactions, 79 (44.4%) were considered to be serodivergent, with only one positive reaction out of the three reactions, and only 10 (5.6%) were considered to be seropositive, with two or three positive reactions (table iii). In summary, of the 137 samples that were positive in the screening serological tests, only 33 (24.1%) were positive in the confirmatory tests, while only 10 (5.6%) of the 178 samples that were doubtful in the screening test were positive in the confirmatory test, as shown in table iii . The numbers of serum samples and the titres of igg confirmed by immunofluorescence as positive are shown in fig . 4 . These data most likely reflect the low level of parasitaemia and the particular t. cruzi strain circulating in the area (tci), which likely provide low stimulation for antibody genesis . Table iiiconfirmatory serological tests of 137 samples positives and 178 doubtful by indirect immunofluorescence (iif) in blood from filter paper evaluated by iif and elisa conventional and or recombinantserological evaluation positives iif from filter paper n (%) doubtful iif from filter paper n (%) seronegatives 37 (27) 89 (50)serodivergents 67 (48.9)79 (44.4)seropositives33 (24.1)10 (5.6) total 137 (100) 178 (100) fig . 4titres of igg of immunofluorescence in serum from confirmatory serological tests in barcelos, state of amazonas, brazil, 2011 . The high prevalence found in the preliminary serological screening surveys that we conducted in 1991, 1993 and 1997 (coura et al . 1999) certainly occurred due to cross - reactions with a variety of diseases that are endemic in this area, including malaria, american cutaneous leishmaniasis, tuberculosis, leprosy and trypanosoma rangeli infection . Additionally, the low titres of anti- t. cruzi antibodies demonstrated in the present study favoured occurrences of cross - reactions with other infections . The prevalence of t. cruzi infection in barcelos was observed to vary according to the intensity of exposure . In a group of 244 highly exposed individuals, we found that 11% were serologically positive (iif and tesa - blot). However, among the 46 individuals in this group who underwent xenodiagnosis and polymerase chain reaction (pcr) analysis, we only isolated t. cruzi in 19% of the individuals through xenodiagnosis and the pcr was only positive in these cases (coura et al . (1998a, b) demonstrated only two lineages of t. cruzi in the area (tci and z3). Although no studies have provided evidence, we can suppose that these lineages of t. cruzi are poorly immunogenic for antibody generation . Finally, we recommend that the serological diagnosis of t. cruzi infection in the amazon region should be made using at least two different techniques, e.g., immunofluorescence and elisa and confirmed by western blot analysis when possible.
All procedures were performed under the guidelines and with the approval of the ethics committee in animal experimentation at the federal university of minas gerais (ufmg), protocol 174/2011 . One hundred and seventyeight regional lymph nodes (162 inguinal, 14 axillary, and 2 location not available) were assessed from 97 female dogs with malignant mammary tumors . The intact or spayed animals were subjected only to the surgical treatment of their mammary tumors, without adjuvant treatment, at the hospital of the veterinary school of the federal university of minas gerais (ufmg) between 2008 and 2013 . Before surgery, all animals had complete clinical examinations that included hematology and serum biochemistry . Moreover, the dogs underwent thoracic radiography to rule out distant metastasis at the time of diagnosis and abdominal ultrasound examination only when serum biochemical changes or increased abdominal size were observed . The surgical technique (e.g., simple tumor removal, simple mastectomy, regional mastectomy, and radical mastectomy) was chosen according to the number of lesions and sites, considering the lymphatic drainage and established prognostic factors such as lesion size and existence of skin or muscular adherences, as previously described.34 the present study included 3 (3.1%) simple mastectomies, 46 (47.4%) regional mastectomies, and 43 (44.4%) radical mastectomies; the type of surgery was unknown for 5 dogs . All of the tumors were completely excised, and the muscle fascia was not removed in any cases . The inguinal lymph nodes were resected en bloc, along with the inguinal mammary gland, whenever this gland was removed, or in the same manner as the axillary lymph nodes when they were enlarged, adherent, or firm . The surgical specimens (mammary tumors and lymph nodes) were obtained from archives of the pathology department of the veterinary clinic and surgery at the veterinary school of ufmg and of the laboratory of comparative pathology at the institute of biological sciences (icb / ufmg) and were analyzed by 3 veterinary pathologists (mra, ef, and gdc). All of the sections were included in different cassettes and were analyzed together, evaluating the long axis of the lymph node . The tumor samples and regional lymph nodes were fixed in 10% buffered formalin and routinely prepared and stained with hematoxylineosin (h&e). Three veterinary pathologists (mra, ef, and gdc) histologically examined a minimum of 3 sections (3 m) of the mammary tumors . When tumors were> 5 cm, a minimum of 5 sections were examined . All tumors were classified according to the veterinary histological classification.33, 37, 38 one section (3 m) of each lymph node was cut and stained with h&e . Three veterinary pathologists (mra, ef and gdc) reviewed all of the lymph nodes . In addition, the maximum width and length (the 2 greatest dimensions) of all metastatic foci in each positive lymph node were measured, and the larger of the 2 measurements was used as the maximum diameter of the metastatic focus . To confirm the absence of metastasis, a consecutive section (3 m) of the same paraffin block obtained for h&e analysis of each lymph node negative for metastasis was cut for ihc analysis . The slides were deparaffinized, rehydrated in graded alcohol, and subjected to heatinduced antigen retrieval with antigen retrieval solution1 (water bath at 98c; ph 6.0). The sections were stained with antibodies specific for cytokeratin (ck) ae1/ae31 (1:100, clone ae1ae3), vimentin1 (1:100, clone v9), or p632 (1:80, clone 4a4) and were incubated for 1 hour at ambient temperature . The cytokeratin stain was performed for all h&e lymph node slides that were scored as negative for metastasis . The vimentin and p63 stains were performed only for h&e lymph nodes slides negative for metastasis and with primary diagnoses of carcinosarcoma and malignant adenomyoepithelioma, respectively . The ihc was used only to confirm the absence of metastasis and cases positive for metastasis by ihc were excluded from pathologic analyses . After incubation, the antigen was immunodetected using the dako advance (hrp) visualization method1 with diaminobenzidine (dab substrate system)1 as the chromogen . Sections were counterstained with mayer's hematoxylin, dehydrated, and mounted in a synthetic medium . Neoplastic cells in the lymphoid parenchyma or distributed in the lymphatic sinuses (i.e., subcapsular, medullary, or both) in h&estained sections were considered metastases as previously described.21 when> 1 lymph node in the same patient was affected by several metastatic foci, the largest metastatic focus was measured (maximum diameter) for the classification of metastasis as macrometastases, micrometastases, or itc . Images of all metastatic foci were obtained on an olympus bx41 microscope using a spot insight color capture system with different objectives depending on the size of the metastatic focus . The animals were organized into 5 groups according to a classification of metastasis adapted from human medicine: a (animals without metastasis); b (animals with macrometastases: lymph node metastases> 2 mm; fig 1a); c (animals with micrometastases: foci of neoplastic cells with diameters ranging from 0.2 to 2 mm; fig 1b); d (animals with itc: foci of neoplastic cells <0.2 mm; fig 1c); and e (animals with nonmeasurable metastasis). Group e included animals with lymph nodes that had considerable metastatic involvement but in which it was not possible to measure metastasis (i.e., lymph nodes partially sectioned or lymph nodes with metastatic burden distributed diffusely without defined limits). (a) neoplastic proliferations characterized by numerous structures resembling acini contain significant fibrosis replacing part of the lymphoid parenchyma (macrometastases). (b) neoplastic proliferation characterized by structures resembling acini localized in the subcapsular sinus (micrometastases). (c) multiple deposits of isolated epithelial neoplastic cells (arrows) in the subcapsular sinus . The size of the largest metastatic deposit, measuring 0.06 mm, was used to classify lymph node metastasis as isolated tumor cells (itc). In addition, all animals with metastasis were stratified according to the number of lymph nodes involved, the number of metastatic foci, the maximum diameter of metastasis, and the area of metastasis (maximum width maximum length). Moreover, when> 1 metastatic focus was identified in 1 lymph nodes in the same patient, individual metastatic areas were summed to calculate the total metastatic area (tma).8 for all of these analyses, a cutoff (mean plus 1 standard deviation [sd]) was established for stratification into groups . The number of lymph nodes involved, the maximum diameter of the lymph node metastases and the number of metastatic foci from each animal were used to obtain the averages for each group . All of the metastatic foci were measured to obtain an average metastatic area in animals that had> 1 metastatic focus . The animals with malignant mammary tumors were assessed, and the endpoints were defined according to the type of analyses: histologic tumor types (827 days of followup; 84 animals), presence or absence of lymph node metastasis (1112 days of followup; 84 animals), number of lymph nodes involved (778 days of followup; 84 animals), classification of metastasis (778 days of followup; 84 animals), area of metastasis (285 days of followup; 33 animals), number of metastatic foci (537 days of followup; 33 animals), maximum diameter of metastasis (285 days of followup; 33 animals), and tma (778 days of followup; 24 animals). Survival time was defined as the period (days) between surgical tumor removal and date of death from disease . Animals that were alive and that died of unknown causes or causes unrelated to the tumor were censored . Among the most common causes was visceral (calazar) leishmaniasis (leishmania chagasi), which is endemic in the city of belo horizonte, minas gerais, brazil, where the research was conducted . Other causes of death were external causes (road accidents), orthopedic surgery, and cardiorespiratory and neurological conditions . Meier estimator, and statistical significance was examined using a logrank test, after a normality test, with p <.05 considered significant . Survival data were evaluated according to the histologic tumor types, presence or absence of lymph node metastasis, number of lymph nodes involved, classification of lymph node metastasis, area of metastasis, number of metastatic foci, maximum diameter of metastasis, and tma . One hundred and seventyeight regional lymph nodes (162 inguinal, 14 axillary, and 2 location not available) were assessed from 97 female dogs with malignant mammary tumors . The intact or spayed animals were subjected only to the surgical treatment of their mammary tumors, without adjuvant treatment, at the hospital of the veterinary school of the federal university of minas gerais (ufmg) between 2008 and 2013 . Before surgery, all animals had complete clinical examinations that included hematology and serum biochemistry . Feed, but not water, was withheld for 36 hours before surgery . Moreover, the dogs underwent thoracic radiography to rule out distant metastasis at the time of diagnosis and abdominal ultrasound examination only when serum biochemical changes or increased abdominal size were observed . The surgical technique (e.g., simple tumor removal, simple mastectomy, regional mastectomy, and radical mastectomy) was chosen according to the number of lesions and sites, considering the lymphatic drainage and established prognostic factors such as lesion size and existence of skin or muscular adherences, as previously described.34 the present study included 3 (3.1%) simple mastectomies, 46 (47.4%) regional mastectomies, and 43 (44.4%) radical mastectomies; the type of surgery was unknown for 5 dogs . All of the tumors were completely excised, and the muscle fascia was not removed in any cases . The inguinal lymph nodes were resected en bloc, along with the inguinal mammary gland, whenever this gland was removed, or in the same manner as the axillary lymph nodes when they were enlarged, adherent, or firm . The surgical specimens (mammary tumors and lymph nodes) were obtained from archives of the pathology department of the veterinary clinic and surgery at the veterinary school of ufmg and of the laboratory of comparative pathology at the institute of biological sciences (icb / ufmg) and were analyzed by 3 veterinary pathologists (mra, ef, and gdc). All of the sections were included in different cassettes and were analyzed together, evaluating the long axis of the lymph node . The tumor samples and regional lymph nodes were fixed in 10% buffered formalin and routinely prepared and stained with hematoxylineosin (h&e). Three veterinary pathologists (mra, ef, and gdc) histologically examined a minimum of 3 sections (3 m) of the mammary tumors . When tumors were> 5 cm, a minimum of 5 sections were examined . All tumors were classified according to the veterinary histological classification.33, 37, 38 one section (3 m) of each lymph node was cut and stained with h&e . Three veterinary pathologists (mra, ef and gdc) reviewed all of the lymph nodes . In addition, the maximum width and length (the 2 greatest dimensions) of all metastatic foci in each positive lymph node were measured, and the larger of the 2 measurements was used as the maximum diameter of the metastatic focus . To confirm the absence of metastasis, a consecutive section (3 m) of the same paraffin block obtained for h&e analysis of each lymph node negative for metastasis was cut for ihc analysis . The slides were deparaffinized, rehydrated in graded alcohol, and subjected to heatinduced antigen retrieval with antigen retrieval solution1 (water bath at 98c; ph 6.0). The sections were stained with antibodies specific for cytokeratin (ck) ae1/ae31 (1:100, clone ae1ae3), vimentin1 (1:100, clone v9), or p632 (1:80, clone 4a4) and were incubated for 1 hour at ambient temperature . The cytokeratin stain was performed for all h&e lymph node slides that were scored as negative for metastasis . The vimentin and p63 stains were performed only for h&e lymph nodes slides negative for metastasis and with primary diagnoses of carcinosarcoma and malignant adenomyoepithelioma, respectively . The ihc was used only to confirm the absence of metastasis and cases positive for metastasis by ihc were excluded from pathologic analyses . After incubation, the antigen was immunodetected using the dako advance (hrp) visualization method1 with diaminobenzidine (dab substrate system)1 as the chromogen . Sections were counterstained with mayer's hematoxylin, dehydrated, and mounted in a synthetic medium . Neoplastic cells in the lymphoid parenchyma or distributed in the lymphatic sinuses (i.e., subcapsular, medullary, or both) in h&estained sections were considered metastases as previously described.21 when> 1 lymph node in the same patient was affected by several metastatic foci, the largest metastatic focus was measured (maximum diameter) for the classification of metastasis as macrometastases, micrometastases, or itc . Images of all metastatic foci were obtained on an olympus bx41 microscope using a spot insight color capture system with different objectives depending on the size of the metastatic focus . The animals were organized into 5 groups according to a classification of metastasis adapted from human medicine: a (animals without metastasis); b (animals with macrometastases: lymph node metastases> 2 mm; fig 1a); c (animals with micrometastases: foci of neoplastic cells with diameters ranging from 0.2 to 2 mm; fig 1b); d (animals with itc: foci of neoplastic cells <0.2 mm; fig 1c); and e (animals with nonmeasurable metastasis). Group e included animals with lymph nodes that had considerable metastatic involvement but in which it was not possible to measure metastasis (i.e., lymph nodes partially sectioned or lymph nodes with metastatic burden distributed diffusely without defined limits). (a) neoplastic proliferations characterized by numerous structures resembling acini contain significant fibrosis replacing part of the lymphoid parenchyma (macrometastases). (b) neoplastic proliferation characterized by structures resembling acini localized in the subcapsular sinus (micrometastases). (c) multiple deposits of isolated epithelial neoplastic cells (arrows) in the subcapsular sinus . The size of the largest metastatic deposit, measuring 0.06 mm, was used to classify lymph node metastasis as isolated tumor cells (itc). In addition, all animals with metastasis were stratified according to the number of lymph nodes involved, the number of metastatic foci, the maximum diameter of metastasis, and the area of metastasis (maximum width maximum length). Moreover, when> 1 metastatic focus was identified in 1 lymph nodes in the same patient, individual metastatic areas were summed to calculate the total metastatic area (tma).8 for all of these analyses, a cutoff (mean plus 1 standard deviation [sd]) was established for stratification into groups . The number of lymph nodes involved, the maximum diameter of the lymph node metastases and the number of metastatic foci from each animal were used to obtain the averages for each group . All of the metastatic foci were measured to obtain an average metastatic area in animals that had> 1 metastatic focus . The animals with malignant mammary tumors were assessed, and the endpoints were defined according to the type of analyses: histologic tumor types (827 days of followup; 84 animals), presence or absence of lymph node metastasis (1112 days of followup; 84 animals), number of lymph nodes involved (778 days of followup; 84 animals), classification of metastasis (778 days of followup; 84 animals), area of metastasis (285 days of followup; 33 animals), number of metastatic foci (537 days of followup; 33 animals), maximum diameter of metastasis (285 days of followup; 33 animals), and tma (778 days of followup; 24 animals). Survival time was defined as the period (days) between surgical tumor removal and date of death from disease . Animals that were alive and that died of unknown causes or causes unrelated to the tumor were censored . Among the most common causes was visceral (calazar) leishmaniasis (leishmania chagasi), which is endemic in the city of belo horizonte, minas gerais, brazil, where the research was conducted . Other causes of death were external causes (road accidents), orthopedic surgery, and cardiorespiratory and neurological conditions . Survival curves were calculated using the kaplan meier estimator, and statistical significance was examined using a logrank test, after a normality test, with p <.05 considered significant . Survival data were evaluated according to the histologic tumor types, presence or absence of lymph node metastasis, number of lymph nodes involved, classification of lymph node metastasis, area of metastasis, number of metastatic foci, maximum diameter of metastasis, and tma . The ages of the 97 animals at the time of surgery ranged from 4 to 16 years (mean, 10.7 2.7 years). The dogs were predominantly purebred (68/93; 72.34%), and poodles (n = 31) were the most common breed . Mongrel dogs were observed less frequently (25/93; 26.60%), and the breed was not recorded for 4 dogs . The dogs were intact (52/76; 68.42%) and spayed (24/76; 31.58%); reproductive status was not available for 21 dogs . The inguinal and caudal abdominal glands were the predominant sites of the lesions (31/93; 33.33% each) in relation to the multicentric localization (15/93; 16.13%), cranial abdominal glands (12/93; 12.90%), and thoracic glands (4/93; 4.31%). In 4 cases, none of the animals had pulmonary changes on radiographic examination, changes in abdominal ultrasound findings, or both at the time of surgery . Initially, 178 lymph nodes (162 inguinal, 14 axillary and 2 location not confirmed) of 97 dogs were accessed . However, after ihc analysis, 161 lymph nodes of 90 dogs were considered for study . Among 161 lymph nodes, 49.7% (80) one axillary lymph node and multiple ipsilateral axillar lymph nodes were removed from 5.7% (5/88) and 3.4% (3/88) of dogs, respectively . One ipsilateral inguinal lymph node and multiple ipsilateral inguinal lymph nodes were identified in 39.8% (35/88) and 47.7% (42/88) of dogs, respectively . Both axillary and inguinal lymph nodes were excised from 3.4% (3/88) of dogs . In 2 dogs, dogs with lymph node metastasis were predominantly observed (57/90; 63.3%) relative to dogs without lymph node metastasis (33/90; 36.7%, table 1). The mean number of evaluated nodes per animal was 1.78 (sd, 1.01), with a maximum of 6 and a minimum of 1 lymph node . The mean number of metastatic lymph nodes per animal was 1.4 (sd, 0.75), with a maximum of 5 and a minimum of 1 metastatic lymph node . The inguinal lymphatic region was the predominant site of metastasis (45/55; 81.8%) relative to the axillary lymphatic region (8/55; 14.5%) and simultaneous occurrence (inguinal and axillary; 2/55; 3.6%). Distribution of histologic tumor types and lymph node features according to the adapted metastasis classification used in humans mtc, carcinoma in mixed tumor; sc, solid carcinoma; rtmt , rare type malignant mammary tumors (including micropapillary carcinoma, n = 14; lipidrich carcinoma, n = 1; pleomorphic lobular carcinoma, n = 1; malignant adenomyoepithelioma, n = 1; and malignant phyllodes tumor, n = 1); ptc *, (including papillary carcinoma, n = 10; and tubular carcinoma, n = 3); css, carcinosarcoma . Carcinomas in mixed tumors were the most common cancer, and the rarest tumors were lipidrich carcinoma, malignant adenomyoepithelioma, pleomorphic lobular carcinoma, malignant phyllodes tumor, and invasive micropapillary carcinoma, all of which were included in the group of rare types of malignant mammary tumors (table 1). The animals with carcinoma in mixed tumors, rare types of malignant tumors, solid carcinoma, papillary and tubular carcinoma and carcinosarcoma had mean respective survival times of 485.5 259.4 days (not reaching median survival), 227.1 235.2 days (median, 180 days), 228.2 253.4 days (median, 195 days), 401.7 196.8 days (not reaching median survival), and 163.3 115.7 days (median, 185 days). Animals with solid carcinoma, carcinosarcoma, and rare types of malignant mammary tumors had lower survival rates than animals with carcinoma in mixed tumors and papillary and tubular carcinoma (p <.0001; fig 2a). (a) kaplan meier survival curve for the animals according to the histologic types: mtc (carcinoma in mixed tumor; n = 32); ptc (papillary and tubular carcinoma; n = 11); sc (solid carcinoma; n = 16; median, 195 days); rtmt (rare type malignant mammary tumors; n = 18; median, 180 days); and css (carcinosarcoma; n = 7; median, 185 days). Animals with mtc and ptc not did reach median survival (p <(b) kaplan meier survival curve for the animals with metastasis (n = 53, median, 275 days) and without metastasis (n = 31, not reaching median survival) (p <.0001). (c) kaplan meier survival curve for the animals according to the number of lymph nodes involved: 0 (no metastatic lymph nodes; n = 31, not reaching median survival), 1 (1 metastatic lymph node; n = 37; median, 348 days), and 2 (2 metastatic lymph nodes; n = 16; median, 130 days) (p <.0001). (d) kaplan meier survival curve for the animals according to the classification of metastasis: a (absence of metastasis; n = 31), b (macrometastases; n = 11; median, 240 days), c (micrometastases; n = 14), d (isolated tumor cells; n = 8; median, 262 days), and e (no measurable metastasis; n = 20; median, 180 days) (p <.01) regarding the presence or absence of lymph node metastasis, the animals with metastasis had lower survival times (mean, 253.9 244.2 days and median, 275 days) compared with animals without metastasis (mean 530.6 286.8 days and not reaching median survival; p <.0001; fig 2b). When the number of lymph nodes involved was evaluated, the presence of 2 metastatic ipsilateral lymph nodes (equal or different lymphatic basins) was associated with lower survival (p <.0001; fig 2c). The mean survival times of animals with lymph nodes without metastasis, with 1 metastatic lymph node, and with 2 metastatic lymph nodes were 497.6 238.4 days (not reaching median survival), 275.3 223.2 days (median, 348 days), and 174.2 192.8 days (median, 130 days), respectively . Animals with isolated tumor cells (group d; mean, 200.1 108.0 days and not reaching median survival), nonmeasurable metastases (group e; mean, 191.8 186.9 days and median, 180 days), and macrometastases (group b; mean, 248.8 277.8 days and median, 240 days) had lower survival times than those without metastasis (group a; mean, 497.6 238.37 days and not reaching median survival) and those with micrometastases (group c; mean, 342.9 238.9 days and not reaching median survival). Lower survival with statistical significance was observed in animals from groups ba (p = .002), da (p = .001) and ea (p <.0001) and in animals from group e relative to group c (p <.001; fig 2d). Stratification of animals with measurable metastasis (i.e., macrometastases, micrometastases, and itc) distributed in groups according to the area of metastasis, number of metastatic foci, maximum diameter, and tma is presented in table 1 . The mean area of metastasis was 2.95 mm (range, 0.0001169.88 mm; sd, 17.16 mm; n = 33). When stratified by area, animals with areas <20.11 mm (mean, 207.1 93.4 days and not reaching median survival) had a greater survival than those with areas> 20.11 mm (mean, 103.0 107.1 days and median, 86.5 days; p = .0457; fig 3a). (a) kaplan meier survival curve for the animals according to the area of metastasis: <20.11 mm (n = 28; not reaching median survival) and> 20.11 mm (n = 5; median, 86.5 days) (p = .0457). (b) kaplan meier survival curve for the animals according to the number of metastatic foci: <28 foci (n = 30; median, 404 days) and> 28 foci (n = 3, not reaching median survival) (p = .629). (c) kaplan meier survival curve for the animals according to the maximum diameter of metastasis: <7.32 mm (n = 27; not reaching median survival) and> 7.32 mm (n = 6; median, 63 days) (p = .0068). (d) kaplan meier survival curve for the animals according to the total metastatic area (tma): <88.92 mm (n = 21; not reaching median survival) and> 88.92 mm (n = 3; median, 110 days) (p = .3329). The mean number of metastatic foci was 10 (range, 197 foci; sd, 18 foci; n = 33). There were no statistically significant differences in the survival of animals with <28 foci (mean, 237.1 174.5 days and median, 404 days) and> 28 foci (mean, 361.3 155.3 days and not reaching median survival; fig 3b). The mean maximum diameter of the metastases was 2.86 mm (range, 0.0616.92 mm; sd, 4.46 mm; n = 33). When stratified by maximum diameter, the survival of animals with diameters> 7.32 mm (mean, 95.8 97.4 days and not reaching median survival) was lower than animals with diameters <7.32 mm (mean, 212.6 90.6 days and median, 63 days; p = .0068; fig 3c). Finally, the mean tma obtained for animals with> 1 metastatic focus was 26.04 mm (range, 0.0196239.41 mm; sd, 62.88 mm; n = 24). When stratified by tma, no significant difference was found in survival of the animals with tmas <88.92 mm (mean, 303.3 210.1 days and not reaching median survival) and with tmas> 88.92 mm (mean, 317.0 399.9 days and median, 110 days; fig 3d). As in women, regional lymph node status has major impact on survival in dogs with mammary tumors.22, 23, 24, 25, 26, 27 in the present study, animals with metastasis had lower survival times than did animals without metastasis . These findings are similar to those reported in the literature.22, 23, 24, 25, 26 to our knowledge, this study is the first to describe the prognostic significance of identifying a higher number of lymph nodes positive for metastasis in dogs, as has been reported in humans.1, 39 another novel aspect is the use of a more accurate technique for measuring metastases, which yields a more detailed representation of the metastatic load of the lymph nodes in these animals . In this study, dogs with lymph node metastases with diameters> 7.32 mm had lower overall survival . Another interesting finding was that the presence of itc in the lymph nodes of dogs with malignant mammary tumors was associated with lower overall survival in comparison to dogs with no metastatic malignant mammary tumors . Histological type is one of the most important prognostic factors in dogs with mammary tumors and, our results are similar to those described in the literature.23, 24 we found that solid carcinomas are extremely aggressive histologically and have a worse prognosis compared with histologically welldifferentiated tumors, such as carcinomas in mixed tumors, papillary carcinoma, and tubular carcinoma . These tumors were associated with lower overall survival compared with other tumors, as described in previous studies.40, 41, 42, 43 in this study, metastatic involvement of> 1 lymph node showed a direct correlation with worse prognosis . This parameter may become an important prognostic factor in veterinary clinical practice and has not previously been described in dogs . In women, a higher number of affected axillary lymph nodes is associated with shorter survival times, which is a wellrecognized prognostic factor in breast cancer.1, 39 the presence of lymph node macrometastases is also a clinically relevant prognostic factor in dogs with mammary tumors.36 however, a novel finding of similar prognostic relevance was the identification of isolated cells in these animals . In this study, 33.33% of the animals diagnosed with itc had more aggressive tumors, such as carcinosarcoma and solid carcinoma . Such behavior may be similar to that observed in women with breast cancer, in whom the clinical relevance of identifying cell clusters <0.2 mm has shown conflicting results and may be directly related to the histologic type studied.13, 16, 18, 21 the differences in animal survival demonstrate that metastatic area measurements can be used to characterize regional lymph node metastases in female dogs with malignant mammary tumors . However, this characterization may not be practical in routine laboratory pathology because it would be necessary to measure all metastatic foci to obtain the mean metastatic area . The maximum diameter of metastases currently is used to classify metastases as macrometastases, micrometastases, or itc in humans.7 in the present study, the classification of metastasis used in humans was made, along with stratification to the maximum diameter (cutoff: mean plus sd = 7.32 mm). Lower survival was observed in animals with a maximum diameter of lymph node metastasis> 7.32 mm relative to animals with a maximum diameter of lymph node metastasis <7.32 mm . Thus, the behavior of macrometastases and staging in dogs can vary, and the current classification system based on humans (macrometastases: metastasis> 2 mm) may not be the most appropriate . Other authors have discussed the use of classification systems other than the classification systems used in humans that have been adapted to veterinary medicine.35 a subclassification system for macrometastases should be considered because the lymph node metastasis can reach considerable size in female dogs, similar to findings in the present study . The tma measurements in slns can predict nonsln metastasis in women with breast cancer.8 the present study found no differences in animal survival between a tma (cutoff: mean plus sd)> 88.92 mm and a tma <88.92 mm . Thus, to obtain prognostic information, we consider that the maximum diameter measurement could be one of the most suitable methods when analyzing single sections of lymph nodes in female dogs with malignant mammary tumors . In the clinical studies of humans, substantial advances are described in relation to the standardization of gross sectioning and the number of histologic sections to be analyzed in slns of women with breast cancer.44 in veterinary medicine, evaluating lymph node metastases, determining the prognostic value of metastatic size, and even establishing the number of histologic sections to be analyzed should be investigated in additional studies, especially when using sln mapping in mammary tumors of dogs . The findings in this study may help in future proposals for the standardization of these methods of analysis, showing the real prognostic relevance of the occurrence of lymph node metastasis in malignant mammary tumors of dogs . Corroborating previous findings,22, 23, 24, 25, 26 our study showed that the presence of lymph node metastasis is a negative prognostic factor . However, the analysis of these data with tumor size, histologic type, mitotic index, histologic grading, status of hormonal receptors, and cell proliferation markers may provide more consistent information in a multivariate analysis of the possible independent prognostic factors in dogs with malignant mammary tumors and may provide actual practical applications for the data presented in this study for clinical practice . However, the independent prognostic relevance of lymph node status in dogs with mammary tumors is still poorly established in multivariate analysis.23, 45, 46 the identification of 1 metastatic lymph nodes is considered a parameter for prognostic assessment in affected animals . Moreover, macrometastases and itc in regional lymph nodes are associated with a worse prognosis in dogs with malignant mammary tumors subjected only to surgical treatment . A more detailed classification system and staging that takes into account differences in the sizes of macrometastases identified in female dogs should be considered, and additional studies should be conducted to confirm these findings.
These lesions though may cause life threatening hemorrhage, watchful observation is still considered as a treatment option in these patients . A 71-year - old male presented with pulsatile swelling over the right side of face and over the forehead in october 2009 . Patient is a known diabetic and hypertensive for the past 9 years on regular medications . Patient was evaluated with computed tomography (ct) angiogram, which showed aneurysm of the external carotid artery with thrombus within and multiple aneurysm of the branches of external carotid arteries . Treatment option of excising the aneurysm with ligation of external carotid aneurismal branches was discussed with the patient . Since the swelling was asymptomatic for the past 34 years, patient was not willing for any procedure . He is assessed with clinical examination on each visit and ultrasound examination performed once in a year to assess the size of the aneurysm . The patient has been on follow up for the past 4 years without any new symptoms or increase in size of the aneurysm . Aneurysm of the external carotid artery and its branches have been described in cases due to trauma, iatrogenic injury, dental extraction, and also due to head and neck cancer because of tumor infiltration or due to radiation therapy . Geriatric patients with aneurysm are first consulted by a primary care physician and hence primary care physician at the community level has a major role in following these patients and referring these patients to specialized vascular centre . Increase in size of the aneurysm or impending rupture necessitates the need for referral to specialized center . The need for surgical intervention or endovascular stenting does not arise in our patient who has this aneurysm for 34 years and was on follow up for the past 4 years without any symptoms [figure 1]. Pulsatile swelling over the face, forehead, and scalp in this setting, the role of primary care physician is important to decide upon surgical or conservative management after having discussion with the patient . Multiple aneurysm of external carotid artery this case was discussed for the rarity of idiopathic multiple aneurysm [figure 3] of the external carotid artery [figure 4] and the need for individualized treatment protocol to be followed, as in this case, only watchful observation . In this world of evolving surgical techniques and newer treatment modalities, conservative treatment still has a role to play . Even in the era of advanced medical technology practices, treatment should be individualized and the choice rests with the patient . Primary care physicians have a major role in follow up of these patients and decide upon referring these patients to specialized vascular center thus lessen the burden of vascular centre in following these patients.
Tis education forms the common mind; just as the twig is bent, the tree s inclined, wrote alexander pope in 1734 . This is likely one of the earliest poetic descriptions of a current topic of great interest to psychiatrists and research neuroscientists, namely the impact of early psychosocial factors on subsequent neuronal development and ultimate phenotypic presentation . Michael meaney and colleagues at mcgill university have demonstrated, in an elegant series of experiments in rats, the effect of early maternal grooming of rat pups on stress resilience via hippocampal glucocorticoid receptor expression and activation of nerve growth factor inducible - a . Offspring interactions can dramatically influence the child s behavior as they age.1 the focus of this study is to examine retrospectively the potential relationship between maternal personality characteristics, as measured by the minnesota multiphasic personality inventory (mmpi), and drug dependence in their sons . The hypothesis is that certain personality traits in mothers may help predict an at - risk population of their sons for the development of drug dependence . This preliminary work is intended to explore a potential translational aspect of the work of meaney and others . Bucknall and robertson,2 have demonstrated the role of family in the etiology of drug abuse, with an even greater role in the maintenance of an individual s drug dependence . Andersson and eisemann,3 confirmed that a parental rearing behavior perceived both as rejecting and overprotective represents a link between dysfunctional parenting and the development of maladaptive psychosocial behaviors, like drug addiction . Drug addicts often come from families where there is frequently expressed ill will and hostility in the home . They have weaker family ties than do those who do not take drugs . In families in which there was contact with drugs, authority belonged to the mother to a greater degree than the father.4 handlarz et al5 studied drug addicts and their families and observed general characteristics common to all substance - dependent patients . Among these were vulnerability of personality and ego weakness, absent father, narcissistic mother, disaggregation of the family, and pathological communication among family members . Graeven and schaef 6 reported the relationship with the opposite sex parent had the strongest impact on both male and female heroin users . Kaufamn7 studied the family structure of drug addicts and their families who were in residential treatment at the time of the study . The most common familial pattern seen was that of a mother enmeshed with her addicted son . The father reported feeling excluded by the dyad and reacted with disengagement, brutality, or increased consumption of alcohol . This paper examines the personality of mothers and its relationship to severity of addiction of their sons . It is a follow up to our previous work on childhood parenting styles of the substance - dependent patient.8 patients were diagnosed according to diagnostic statistical manual (dsm iv).9 the control group consisted of 35 mothers of non - drug - dependent subjects . Mothers from both groups fulfilled the following inclusion criteria: age between 40 and 60.not suffering substance use disorders or any other psychiatric disorders.not suffering significant medical disorder . Age between 40 and 60 . Not suffering substance use disorders or any other psychiatric disorders . All study subjects were informed they were participating in a volunteer study examining the relationship between a mother s personality and drug dependence in their children . All study participants provided informed consent to a psychiatrist (sa) member of the study team . Both groups were administered the minnesota multiphasic personality inventory (mmpi),10 which is a self - reported inventory developed in 1937 by starke hanthaway and charnley mckinley . The items of each scale were selected for their ability to separate medical and psychiatric patients from normal control subjects . We applied the arabic version prepared by melika et al.10 substance - dependent patients were assessed with the addiction severity index (asi).11 the asi is a semi - structured interview designed to address 7 potential problem areas in substance use disorder patients: medical status, employment and support, drug use, alcohol use, legal status, family / social status, and psychiatric status . In 1 hour, a skilled interviewer can gather information on recent (past 30 days) and lifetime problems in all of the problem areas . The asi provided an overview of problems related to substance, rather than focusing on any single area . Personality profiles of mothers of both groups were compared, and those of mothers of dependent patients were correlated to the 7 dimensions of the asi . Differences between quantitative variables for the addicts group and the control group were assessed using student s t - test . The mmpi scales for mothers were classified into risk grades and differences between the study groups were assessed using pearson s chi - square test . The association between the different study scales for mother and their offspring was assessed using correlation analysis . The mean age of mothers of patients was 50, while that of the control was 44.8 years . They were age matched (p = 0.07) there were significant differences between both groups on depression, hysteria, and paranoia scales of the mmpi . Depression scores were higher than 70 (pathological) in 8% of study group compared to only 1% of control group . Depression scores were in the normal range (45 to 50) in 5% of study group compared to 22% of the control group (p = 0.003). Scores on the hysteria scale were higher than 70 in 4% of study group compared to none of the control group . Negative scores for hysteria were seen in 15% of the study group, compared to 40% of the control group (p = 0.02). Paranoia scale scores were positive in 4% of the study group, compared to 1% of controls (p = 0.03). No paranoia was detected in 10% of the study group, compared to 40% of controls . No significant differences were reported on any of the other mmpi scales (table 1). Significant positive correlations were observed among mothers of substance - dependent patients on scores of depression, hypochondrias, and psychopathic deviance (p = 0.003, and p = 0.01 respectively). A positive correlation was also detected between scores of hysteria, hypochondrias and psychopathic deviance (p = 0.001 and p = 0.004, respectively), and between paranoia and hypochondrias, depression, and psychopathic deviance (p = 0.005, p = 0.01, and p = 0.01, respectively). Pearson s correlation coefficient was calculated to measure the degree of association between mmpi scores and the asi . Results showed significant positive correlation between scores of hysteria and psychopathic deviance and asi legal status impairment (p = 0.023 and p = 0.019, respectively). There was a significant negative correlation between social introversion and asi scores of drug / alcohol use, and family history (p = 0.007 and p = 0.003, respectively). Significant positive correlations were observed between scores of social status impairment and those of psychiatric and legal status impairment (p= 0.005 and p = 0.04, respectively). The role of parenting in the development of drug abuse is a widely discussed topic . This is best demonstrated by 2,590,000 google hits displayed when the role of parenting on drug abuse is searched . Behavioral scientists appreciated the highly complex nature of parental interactions, particularly maternal, and have demonstrated the role of nurturing in early life to the development of pathophysiologic functioning in later years of the offspring . Schweitzer and lawton12 and brook et al1 have independently reported mothers assessed as cold, indifferent, controlling, and intrusive were a major risk factor for the development of adolescent drug use . Similarly, andersson and eisemann3 reported that parenting perceived as rejecting was linked to maladaptive psychosocial behaviors, including drug abuse . Our previous study confirmed these findings8 and demonstrated that substance - abusing patients had lower maternal protection scores than non - drug - abusing controls . This analysis of maternal mmpi scores in addicted offspring demonstrated a statistically significant correlation in the depression, hysteria and paranoia subscales . These results have significant health, mental health, and public health and health policy implications . The cost of drug abuse and addiction to the patient, their family, and society is enormous . Any primary interventions which might decrease pathologic drug use would be beneficial . These and other data strongly support the importance of early mother child assessment and intervention when clinically indicated . Although the role of family varies somewhat based on cultural norms, the effect of early maternal nurturing on neuronal development and subsequent phenotypic expression is not affected by socio - cultural variations . It is far more effective to work with mothers than to treat drug addiction in their grown children . Future research needs to focus on identifying cost - effective and clinically effective early intervention strategies through prospective, longitudinal trials . Clinical translational research drawing on results from pre - clinical reports will also be valuable.
Cardiovascular disease, the leading cause of death in western countries, is a preeminent health problem worldwide . Atherosclerosis, a chronic inflammatory - based disease (cibd), constitutes the single most important contributor for cardiovascular complications . Mainly, atherosclerosis results from an immune response to oxidized low - density lipoproteins (ldls). Induced by an atherogenic diet, monocytes are promoted to adhesion into the artherial endothelium and intimae (diapedesis). Once in the intimae, monocytes differentiate into macrophages and then modified lipoproteins (such as oxidized ldls) are accumulated as cytoplasmatic droplets . Some of these mediators are involved in proatherogenic processes, such as interleukin- (il-) 1, il-6, and tumor necrosis factor alpha (tnf-) that involve in the upregulation of the molecular adhesion on the endothelial cells . Others have demonstrated to have antiatherogenic properties such as il-10 that involve in the attenuation of the monocyte differentiation into macrophages . The use of medicinal plants, or extracts from them, has been traditionally practiced worldwide in the prevention and treatment of several chronic diseases such as cardiovascular diseases, intestinal inflammatory diseases, inflammatory bowel disease, arthritis, diabetes, allergies, multiple sclerosis, parkinson's and alzheimer's diseases, and others . Extracts from thyme have been used in traditional medicine for the treatment of several respiratory diseases like asthma and bronchitis and for the treatment of other pathologies thanks to several properties such as antiseptic, antispasmodic, antitussive antimicrobial, antifungal, antioxidative, and antiviral [7, 8]. Thyme oils have also been described as a strong bactericide against gram - positive and gram - negative bacteria and also as a bronchospasmolytic [9, 10]. For example, it has been reported that thyme oil reduces no production in j774a.1 murine macrophages . Major bioactive compounds of the extracts from thyme are carvacrol and thymol [12, 13]. Thymol exhibits multiple biological activities including anti - inflammatory, immunomodulating, antioxidant, antibacterial, antifungal, and free radical scavenging properties . Carvacrol also possesses antimicrobial, antifungal, and antioxidant activities [2022], as well as antimutagenic and anticarcinogenic effects . There have been demonstrated effects on the treatment of colitic mice with essential oils of thyme and oregano containing thymol and carvacrol as their principal bioactive compounds, decreasing levels of the proinflammatory cytokines il-1, il-6, and tnf-. However, mechanisms mediating these suppressive effects are unclear . Borneol, another compound present in thyme, has been also described as an anti - inflammatory since its dietary supplementation significantly decreases the concentration of the proinflammatory cytokines il-1 and il-6 in mice . Nowadays medicinal therapies for cibds involve treatment with nonsteroidal anti - inflammatory drugs, antibiotics, corticosteroids, and immunosuppressant, but the application of these drugs is limited due to their toxicity and side effects . Therefore, there is an increasing interest in finding alternative treatments with fewer side effects . Supercritical fluid extraction (sfe) is considered an attractive extraction method when compared to conventional techniques such as steam distillation or soxhlet extraction because it avoids solute contamination with solvent residues and the degradation of thermolabile compounds . In this sense, sfe with co2 is in increasing demand to produce high - quality essential oils from plant material with medicinal properties . During our on - going screening program, designed to identify natural compounds with anti - inflammatory potential, we have studied thyme oils from three different species (thymus vulgaris, thymus zygis, and thymus hyemalis). To determinate whether thyme oils could have immunomodulation properties and could mediate in inflammatory cytokines regulation, we study the effect of our extracts on ox - ldl - activated thp-1 macrophages, measuring the expression and release of several inflammatory mediators . Co2 sfe oil fractions composition from three different species of thyme (thymus vulgaris, thymus zygis, and thymus hiemalys) were determined by gc - ms (see table 1). The cytotoxicity effect on thp-1 macrophages of s1 and s2 co2 supercritical fluid extracts from thyme leaves was evaluated before the bioactivity study . Mtt assay was performed for periods of 24 of incubation (figure 2). After 24 hours of incubation neither extracts reduced significantly cell viability for concentrations from 5 to 25 g / ml . To activate the thp-1 macrophage, cu - oxidized ldls (ox - ldl) these ox - ldl - treated cells showed an increase in total protein secreted (data not shown). Treatment of activated cells with thyme fractions results in an overall reduction of proinflammatory cytokines release, tnf, il-1, and il-6 (figures 3 and 4), in a dose - dependent manner . To test anti - inflammatory effects of oil extracts, one group was treated with a small amount of diclofenac (5 g / ml). Extracts in general show a better anti - inflammatory effect than diclofenac at this small concentration, diminishing more the proinflammatory cytokines (tnf- and il-1) than diclofenac did and inducing anti - inflammatory release of il-10 that was not observed with diclofenac . After 24 hours of incubation, activated cells treated with any of the thyme fractions showed a very significant decrease in tnf- release when compared with nontreated cells . Both fractions of thymus zygis and thymus vulgaris had similar effects on tnf- secretion . For fraction concentrations of 15 g / ml and higher, the reduction of tnf- was such that these cytokine levels were much lower than the nonactivated basal levels . For thymus hyemalis, tnf- secretion was lower than the nonactivated controls for fraction concentrations of 25 g / ml . Despite the large reduction of tnf- secretion at 24 hours treatments, treatment with fractions of thymus zygis and thymus vulgaris for 48 hours showed a lesser reduction of tnf-. For these fractions, only concentrations of 25 g / ml induced a very significant reduction of tnf- levels, equal to nonactivated basal levels . At 48 hours, thymus hyemalis induced larger tnf- release inhibition, with all fractions concentration showing a decrease under the nonactivated basal levels . Regarding il-1 release at 24 hours, treatment of activated cells with any of the thyme fractions induced a decrease of this cytokine secretion in a dose - dependent manner (figure 3(b)). Both fractions of thymus zygis or thymus vulgaris induced similar reduction of il-1. 24-hour treatment with 15 g / ml of these fractions reduced il-1 secretion significantly to the nonactivated cells basal secretion . After 48-hour incubation with these thyme fractions, il-1 concentration in the medium was the same as the basal secretions of nonactivated cells . Regarding thymus hyemalis, at 24-hour treatments, both fractions of this species induced similar cell responses . There is a decrease in il-1 secretion with the increase of fraction concentration, although this il-1 decrease is only significant till 25 g / ml incubations . After 48 hours, incubations with these two fractions reduced il-1 secretion to nonactivated basal levels in the same manner as thymus zygis and thymus vulgaris did . Il-6 secretion was reduced significantly when activated cells were incubated with any of the fractions of either thymus zygis or thymus vulgaris at a concentration of 15 g / ml or higher . This reduction on il-6 secretion was dose dependent and was verified on both 24 and 48 hours of incubations . Incubations with 25 g / ml of these extracts reduced il-6 secretion to nonactivated cells basal levels . 48-hour treatment with thymus hyemalis, induced the same decrease as thymus zygis and thymus vulgaris, whereas at 24 hours, there was no significant reduction of il - secretion at the concentrations used . 24-hour treatment with any of the fractions induced an increase on il-10 secretion in a dose - dependent manner; higher fraction concentration induced higher il-10 secretion . Thymus zygis and thymus vulgaris increased significantly il-10 secretion of activated cells, with both fractions and at any of the concentrations used . As for thymus hyemalis, the increase in il-10 secretion was lower and only significant when the fractions were 15 g / ml or higher concentrations . 48-hour treatments with higher concentrations of the fractions induced an increase on il-10 secretion; the rest concentrations did not induce significant increases . In all experiments, thymus hyemalis had shown to be less effective than thymus vulgaris and thymus zygis, either in increasing or reducing cytokine release . A dose - dependent effect was observable in treatments with 24 and 48 hours of incubation . Changes on production were dose dependentent and according to the thymol content of each species (table 1). Relative quantification (rq) determinates the change in expression of a nucleic acid sequence relative to a control . Endogenous control represented in this figures was 18s rrna; similar results were showed using gapdh (data not shown). Similar to the observed cytokine release, gene expression of analyzed cytokines, but not il-10, increased in oxldl - activated cells compared with nonactivated cells in treatments during 24 hours . 48-hour treatments caused an increase only in tnf expression on oxldl - activated cells compared with nonactivated cells . Tnf gene expression decreases only in cells treated with thymus zygis s1 at 24 hours of incubation; other treatments did not change expression of this gene with respect to oxldl - activated cells . Similar to 24 hours of incubation, at 48 hours tnf- gene expression did not change with extract treatments; only thymus hyemalis s2 caused significant reduction of this cytokine gene expression . Expression of il-1 decreased in cells treated with all thyme extracts with respect to activated cells at 24 hours of incubation . At 48 hours of incubation, all treatments, except thymus hyemalis s2, caused reduction in gene expression . For thymus zygis s2, thymus vulgaris s1, and s2 and thymus hyemalis s1, expression of il-1 was lower than the nonactivated controls cells . Il-6 gene expression at 24-hour treatment with any of thyme extracts was reduced until level of nonactivated control cells which expression was decreased to half compared to activated cells . In contrast, at 48-hour treatment, expression was reduced using only thymus zygis s1 and s2 and thymus vulgaris s2 extracts with respect to activated cells . Il-10 expression gene increased twice with all thyme extracts at 24 hours of treatment compared with oxldl - activated cells . At 48 hours, gene expression in cells treated with thymus zygis s1 and s2 and thymus vulgaris s1 extracts increased significantly, but thymus vulgaris s2 and thymus hyemalis did not change expression of il-10 compared to oxldl - activated cells . Cytokines are considered to be key players in the inflammatory response involved in atherosclerosis and other chronic inflammatory - based diseases (cibds). Among these, interleukin (il)-1, il-6, il-10, and tumor necrosis factor- (tnf-) are expressed in atherosclerotic lesions by endothelium cells, macrophages, and smooth muscle cells [29, 30]. Some of them are involved in proatherogenic processes, such as upregulation of adhesion molecules on the endothelial cells, while others were proved to have an antiatherogenic role like attenuating the differentiation of monocytes in macrophages . The imbalanced expression of cytokines has been implicated in the progression of many diseases including cibds . Cytokines exhibit both beneficial and pathologic effects on their target cells and are produced by many cell types . Several natural compounds are known for their beneficial properties to some diseases or their derived complications and particularly concerning anti - inflammatory effects . In our experiments, were observed significant cellular responses elicited by the treatment of thp-1 cells with thyme fractions . The action of thyme fractions appears to involve the expression of the proinflammatory cytokine: tnf-, il-6, and il-1 and the anti - inflammatory cytokine il-10 . The most interesting observation made during these studies was that treatment of oxldl - activated thp-1 cells with thyme oils had different effects on proinflammatory and anti - inflammatory expression: tnf-, il-6, and il-1 expressions were inhibited while il-10 expression was enhanced . Proinflammatory cytokines levels decreased in a dose - dependent manner with any thyme fraction from thymus vulgaris, thymus zygis, or thymus hyemalis used (after 24 or 48 h of incubation). These results were in agreement with the ones regarding the expression of cytokines genes at 24 hours of incubation . Other authors have previously reported an increment in cytokine secretion in activated macrophages treated with ox - ldl [3, 33]. Similar results have been described for essential oils extracted from cinnamomum osmophloeum, a herb traditionally used in asia as food and as a medicine, which contains cinnamaldehyde . Murine macrophages were treated with essential oils from this plant; anti - inflammatory effects by decreasing tnf-, il-6, and il-1 secretions were reported . Main compounds present in supercritical thyme extract were thymol, 1,8 cineole, camphor, borneol, and carvacrol . Anti - inflammatory effect of thymol has been reported on human neutrophiles incubated with 10 or 20 g / ml of this compound . Mice edema has been reported to be reduced with a topical application of 100 g / cm of carvacrol . Moreover, antioxidant properties of thymol and carvacrol have been demonstrated in several studies, suggesting their use as nutraceutical ingredients in the development of novel functional foods . Derivatives of thymol and carvacrol have been described as antioxidant according to the dpph radical scavenging method [3537]. Essential oils of thyme and their components carvacrol and thymol inhibited 3-nitrotyrosine formation, biomarker of the oxidative stress, supporting the nutraceutical value of thyme and the potential of thymol and carvacrol in preventing the formation of toxic products by the action of reactive nitrogen species . Also, thymol and carvacrol prevent autoxidation of lipids . In the same way, the inhibitory activity of 1,8-cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes has been described: particularly, in monocytes, inhibition of tnf-, il-1, il-6, and il-8 was 99, 84, 76, and 65%, respectively, when monocytes were treated with 0.15 g / ml of 1,8-cineol . Borneol, one of the major compounds present in essential oils from sage, has been described as anti - inflammatory since its dietary supplementation significantly decreases the concentration of the proinflammatory cytokines il-1 and il-6 in mice . Results observed in this work suggest that supercritical thyme s1 and s2 fraction oils from thymus vulgaris, thymus zygis, and thymus hyemalis may act as effective inhibitors of oxldl - induced proinflammatory cytokines (tnf-, il-1, and il-6) secretion, and also as enhancers of the anti - inflammatory cytokine il-10 secretion, in macrophage thp-1 cells . In summary, co2 supercritical thyme extracts showed anti - inflammatory properties by (a) reducing the release of proinflammatory cytokines, and (b) increasing the anti - inflammatory secretion in activated macrophages . These results may suggest that essential oils from thyme extracts could be used as novel options for treatment of chronic diseases based on inflammatory processes . However, numerous and in - depth studies should be carried out for this purpose . Dried and cryogenic grinded leaves from three varieties of thymes (thymus hyemalis, thymus zygis, and thymus vulgaris) were subjected to supercritical fluid extraction with co2 . The supercritical extractions were carried out in a pilot - plant - scale supercritical fluid extractor (thar technology, pittsburgh, pa, usa, model sf2000) of 2 l capacity using pure supercritical co2 at a pressure of 300 bar and a temperature of 40c . Extracts from the three thyme species were fractionated using a two - cascade depressurized system and samples were collected in each of the two (separators 1 and 2) separators . Fractionation conditions were as follows: separator 1 was kept at constant pressure and temperature of 15 mpa and 40c, respectively, whereas separator 2 was maintained at a pressure of 2 mpa and a temperature of 40c . Composition of the supercritical thyme extracts was carried out by a gc-2010 (shimadzu, japan), equipped with a split / splitless injector, electronic pressure control, aoc-20i autoinjector, gcms - qp2010 plus mass spectrometer detector, and a gcms solution software . The column used was a zb-5 (zebron) capillary column, 30 m 0.32 mm i.d, and 0.25 m phase thickness . Helium, 99.996%, was used as a carrier gas at a flow of 1 ml / min . Oven temperature programming was 60c isothermal for 4 min, increased to 64c at 1c / min, and then increased to 106c at 2.5c / min . Oven temperature was then increased from 106c to 130c at 1c / min, then to 200c at 5c / min, and then to a final temperature of 250c / min at 8c / min which was kept constant for 10 min . Sample injections (1 l) were performed in split mode (1: 20). Injector temperature was of 250c and ms ion source and interface temperatures were 230 and 280c, respectively . The mass spectrometer was used in tic mode, and samples were scanned from 40 to 500 m / z units . Compounds thymol, carvacrol, borneol, and linalool were identified by comparison with standard mass spectra obtained in the same conditions and compared with the mass spectra from library wiley 229 . Rests of the compounds were identified by comparison with the mass spectra from wiley 229 library and by their linear retention index . The chromatographic method was to be based on the previously described by jordn et al . . Human thp-1 monocyte cell lines (american type culture collection, atcc) were maintained in suspension in rpmi 1640 culture medium (atcc) supplemented with 10% fbs (gibco), 100 u / ml penicillin (gibco), 100 mg / ml streptomycin (gibco), 0.05 mm -mercaptoethanol (sigma - aldrich), and 2 mm l - glutamine (gibco), at a density of 39 10 cells / ml at 37c in 5% air 95% co2 . Cells were pelleted via centrifugation and assessed for viability using the trypan - blue exclusion method . Viable cells were plated at a density of 5 10 cells / ml in 24 wells plates (100 l and 1 ml, resp .) And incubated with phorbol 12-myristate, 13-acetate (pma) 100 ng / ml (sigma - aldrich) for 48 h in fbs - free medium . Afterwards oil extracts toxicity was assessed using the mitochondrial - respiration - dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (mtt) reduction method . Thp-1 cells were plated in 96-well plates, differentiated and incubated with different concentrations of extract for 24 and 48 hours at 37c in 5% co2 . After treatment, the cells were washed with pbs and incubated with mtt 1 mg / ml in pbs for 2 hours at 37c in 5% co2 . Afterwards, formazan crystals produced from mtt by the mitochondrial hydrolase of the viable cells were solubilized in lysis buffer (10% sds in 50% dimetilformamida ph = 7). The absorbance of each well was then read at 540 nm using a microplate reader (sunrise remote, tecan). The optical density of formazan formed in control cells (without treatment with extract) was taken as 100% viability . Oil thyme extracts were dissolved in dimethyl sulfoxide (dmso; sigma - aldrich) to stock concentration of 10 mg / ml determined as the maximum doses not cytotoxic by the cell viability assays . After differentiation, the cells were washed with pbs and treated with or without cu oxidized ldls to activated or not activated them . Then, cells were incubated with the corresponding thyme extract diluted in fbs - free medium, for 24 or 48 hours at 37c in 5% co2 . Afterwards, the supernatant was frozen and rna isolated . Supernatants were centrifuged at 12,000 rpm to remove debris and then stored at 80c until cytokine analysis . Il-10, il-1, il-6, and tnf- were quantified using elisa kits from bd biosciences, according to the manufacturer's instructions . 100 l of 1: 10 diluted medium was added to anticytokine antibody - coated polystyrene wells and incubated for 2 h. after washing, the plates were incubated with biotin - labeled secondary antibody for 1 hour . The plates were washed and incubated for 30 min in the dark with substrate solution . Stop solution was added and the absorbance read at 450 nm with correction at 570 nm using a microplate reader (sunrise remote, tecan austria gmbh, grdig, austria). 5 10 cells were homogenized in 200 l of trizol reagent and, if necessary, stored at 80c . Following homogenization, samples were left to rest at room temperature for 5 minutes . After 40 l of chloroform was added, the tubes were vigorously shaken for 15 seconds and let rest at room temperature for 5 minutes . Tubes were then centrifuged at 12000 g, 4c for 15 minutes . The aqueous (upper and colorless) phase was transferred to a new tube . 100 l of isopropyl alcohol was added to the aqueous phase; the tube was then gently mixed and incubated at room temperature for 10 minutes . After incubation, samples were centrifuged at 12000 g, 4c for 10 minutes . The pellet was washed with 200 ml of 75% ethanol in depc - treated h2o and centrifuged at 7600, 4c for 5 min . Total rna was then dissolved in 15 l of depc h2o, incubated at 55c for 10 minutes, and stored at 80c for future use . Total rna isolated from thp-1 cells was quantification of il-1, il-6, il-10, tnf-, 18srna, and gapdh gene expression using real - time pcr . 10 ng/l total rna was used as template for cdna synthesis using the high - capacity archive kit from applied biosystems, according to the manufacturer's instructions . Real - time pcr was performed using taqman probes (applied biosystems) following the manufacturer's recommendations in an ab7900 ht fast real - time pcr system (applied biosystems). The taqman probes used were hs99999029_m1 for il-1, hs00174131_m1 for il-6, hs999999035_m1 for il-10, hs00174128_m1 for tnf-, hs99999901_s1 for 18s rrna, and hs99999905_m1 for gapdh . Gene expression quantification was determined using delta - delta ct method with correction for values of amplification efficiency and normalized to 18s rrna expression . For single variable comparisons, student's t - test was used . For multiple variable comparisons, data were analyzed by one - way analysis of variance (anova) followed by dunnett's multiple comparison test and bonferroni test when necessary using the graphpad prism statistical software (graphpad software inc.
The prevalence of obesity continues to rise worldwide with an alarming priority in developed and developing countries . Researchers, practitioners, and policy - makers call for updated valid evidences to monitor, prevent, and control of obesity . This information, mostly, provide through the publication and distribution of research results . Most of the times, quality of health services affected by the application of updated results of scientific productions . Aim to monitoring and assessment of scientific trends, scientometrics provides reliable, practical methods that measure, evaluate and analyze scientific products of specific fields . In these regards, using qualitative, quantitative and computational approaches, different indicators are increasingly employed to show the pattern of research performed by researchers, universities, institutes, and countries . One of its main indices is the number of published articles or science production in a specific field of sciences . In addition, citations of papers is another index that mostly is used as a proxy of application of papers . The collaboration in research conduct and papers publication and collaborative research centers consider as another citation indexes . Considering above, the aim of this paper is scientometrics analysis of obesity / overweight knowledge productions in the middle east region countries . The trends in published papers, citations, and collaborative researches in the fields of obesity / overweight, as the proxy of knowledge production, reviewed, during the past 24-year period . We specifically focused on iran and assessed its contribution in obesity / overweight researches by more details . Present study is scientometric analysis of obesity / overweight scientific papers among middle east countries from 1990 to 2013 . Reviewing the publication number, publication trends, citations, and collaborative institutions, iran has been compared with other countries of the region and also with global indices . For its most coverage in health and biomedicine discipline, we systematically searched scopus database . As it was the only sources for multidisciplinary citation reports, we select it as the valid source of citation reports of knowledge products . In addition, all of the collaborative papers extracted and analyzed for middle east countries . We also introduce top institutions, journals, and collaborative research centers in the field of obesity / overweight . Using methods of reviews and considering emtree, terms of obesity, overweight, and anthropometric indexes, search strategy has designed by researchers committee and validated by external scientific group . The name of middle east countries were identified according to free encyclopedia of wikipedia including; bahrain, cyprus, egypt, iraq, iran, israel, jordan, kuwait, lebanon, oman, palestine, qatar, saudi arabia, syria, turkey, united arab emirates, and yemen (in alphabetical order). Period of study was limited to 19902013, and there was no limitation for language . 2011 (stata statistical software: release 12 . College station, tx: stata corp lp . Given scopus data, globally, during 19902013, 415,126 papers have been published in the fields of obesity / overweight . From them, 136,595 were affiliated to usa, 33,927 to uk, and 20,019 to germany . During this period, 14,792 published papers were affiliated to middle east countries . Compare with other countries in the regions; after turkey (7092 papers: 47.94%) and israel (5214 paper: 35.25%), it has achieved to third position . The time trend of obesity / overweight publication by middle east countries, 19902013 pearson chi - square test confirmed a significant time trends of published papers between region countries (p = 0.000). Table 2 shows the p - trends of total number of papers and the total number of citations, in middle east's countries, during the 19902013 . P - trends of total number of papers and total number of citations of middle east's countries obesity / overweight related papers, 1990 - 2013 as it shows in table 2, based on the pearson chi - square test results, all of region countries have significant time trends in their obesity / overweight publications citations (p = 0.000). The comparative situations of citations time trends of obesity / overweight publications is presented in figure 2 . The time trend of citations of obesity / overweight publication by middle east countries, 19902013 in overall, in the field of obesity / overweight, iran had the most collaborative papers respectively with usa (180, 4.6%), uk (140, 3.6%), and canada (66, 1.7%). Turkey with 15 collaborative papers (0.4%) had the 16 rank of this list . Usa also had the most participation in publications of all other 16 middle east countries . The situation of contributions of middle east's countries in obesity / overweight related papers have been shown in table 3 . The diagonal diameter of table (green marked number) shows papers that there was no partnership between the countries in their publication . For example from bahrain publications, 197 cases have been published by contribution of only internal researchers and academic members of bahrain scientific centers . Red marked zeros refer to situations in which, there were no any joint contribution for common publications between two countries that have been indicated in the top of leading rows . Contributions of middle east's countries in obesity / overweight related papers, 1990 - 2013 in overall, in global level, most of obesity / overweight papers published in the field of medicine (76.7%), after that, the highest proportion of publications respectively, belonged to, biochemistry (23.3%), and agricultural and biological sciences (12%). In iran, publications approximately follow this distribution . Considering the results, 64% of iranian papers were completely related to medicine, 11% were published in the field of biochemistry, genetics and molecular biology, and 6% were aligned to the fields of agricultural and biological sciences . Figure 3 compares the distribution of publications subject area of obesity / overweight publications at national, regional and global levels . The distribution of subject area of obesity / overweight publication at national, regional and global levels considering the role of universities or other scientific institutes in publication of obesity / overweight papers; the first three effective institutes with the most publications are; va medical center, brigham and womans hospital, and inserm, respectively with 1.25%, 0.81%, and 0.78% of global participation in knowledge production . Harvard school of public health with insignificant difference is the fourth producing center of obesity publications (3,154 papers, 0.87%). In iran, after that, shahid beheshti university of medical sciences counterpart in 15% of knowledge productions . The third rank belonged to isfahan university of medical sciences (14% contribution) [figure 4]. Contribution of national universities / scientific institutes in publication of obesity / overweight papers, 19902013 regarding the sources of publications in global level, the three first journals are; international journal of obesity (5396; 1.30%), plos one (4280; 1.03%), and obesity surgery (4033; 0.99%). In iran, iranian journal of endocrinology and metabolism, journal of research in medical sciences, and archives of iranian medicine, respectively with 3.47%, 2.78%, and 2.19% of national publication contribution, were the top three sources of obesity - related publication . During this period, the most prevalent type of obesity - related publications was original articles that consist 90% of all knowledge products . After these review articles (4%) and letters (2%) set on next levels . Given scopus data, globally, during 19902013, 415,126 papers have been published in the fields of obesity / overweight . From them, 136,595 were affiliated to usa, 33,927 to uk, and 20,019 to germany . During this period, 14,792 published papers were affiliated to middle east countries . Compare with other countries in the regions; after turkey (7092 papers: 47.94%) and israel (5214 paper: 35.25%), it has achieved to third position . The time trend of obesity / overweight publication by middle east countries, 19902013 pearson chi - square test confirmed a significant time trends of published papers between region countries (p = 0.000). Table 2 shows the p - trends of total number of papers and the total number of citations, in middle east's countries, during the 19902013 . P - trends of total number of papers and total number of citations of middle east's countries obesity / overweight related papers, 1990 - 2013 as it shows in table 2, based on the pearson chi - square test results, all of region countries have significant time trends in their obesity / overweight publications citations (p = 0.000). The comparative situations of citations time trends of obesity / overweight publications is presented in figure 2 . The time trend of citations of obesity / overweight publication by middle east countries, 19902013 in overall, in the field of obesity / overweight, iran had the most collaborative papers respectively with usa (180, 4.6%), uk (140, 3.6%), and canada (66, 1.7%). Turkey with 15 collaborative papers (0.4%) had the 16 rank of this list . Usa also had the most participation in publications of all other 16 middle east countries . The situation of contributions of middle east's countries in obesity / overweight related papers have been shown in table 3 . The diagonal diameter of table (green marked number) shows papers that there was no partnership between the countries in their publication . For example from bahrain publications, 197 cases have been published by contribution of only internal researchers and academic members of bahrain scientific centers . Red marked zeros refer to situations in which, there were no any joint contribution for common publications between two countries that have been indicated in the top of leading rows . Contributions of middle east's countries in obesity / overweight related papers, 1990 - 2013 in overall, in global level, most of obesity / overweight papers published in the field of medicine (76.7%), after that, the highest proportion of publications respectively, belonged to, biochemistry (23.3%), and agricultural and biological sciences (12%). In iran, publications approximately follow this distribution . Considering the results, 64% of iranian papers were completely related to medicine, 11% were published in the field of biochemistry, genetics and molecular biology, and 6% were aligned to the fields of agricultural and biological sciences . Figure 3 compares the distribution of publications subject area of obesity / overweight publications at national, regional and global levels . The distribution of subject area of obesity / overweight publication at national, regional and global levels considering the role of universities or other scientific institutes in publication of obesity / overweight papers; the first three effective institutes with the most publications are; va medical center, brigham and womans hospital, and inserm, respectively with 1.25%, 0.81%, and 0.78% of global participation in knowledge production . Harvard school of public health with insignificant difference is the fourth producing center of obesity publications (3,154 papers, 0.87%). In iran, after that, shahid beheshti university of medical sciences counterpart in 15% of knowledge productions . The third rank belonged to isfahan university of medical sciences (14% contribution) [figure 4]. Contribution of national universities / scientific institutes in publication of obesity / overweight papers, 19902013 regarding the sources of publications in global level, the three first journals are; international journal of obesity (5396; 1.30%), plos one (4280; 1.03%), and obesity surgery (4033; 0.99%). In iran, iranian journal of endocrinology and metabolism, journal of research in medical sciences, and archives of iranian medicine, respectively with 3.47%, 2.78%, and 2.19% of national publication contribution, were the top three sources of obesity - related publication . During this period, the most prevalent type of obesity - related publications was original articles that consist 90% of all knowledge products . After these review articles (4%) and letters (2%) set on next levels . The results of the present study verify iran's position in obesity / overweight publications between other middle east countries. Scientific evidences have emphasized that overweight and obesity become one of the most important health priority, with increasing trends, which need special attention and quick response . Designing and implementation of these preventive or controlling programs require to accurate information and scientific evidences that have provided through scientific papers and reports ., number of citations have become increasingly important as instruments for analyzing scientific activities and their relationship with economic and social development . Based on the iran's 20-year national vision document, iran is pictured as the highest developed country in science / technology by 2025 . Considering that, iran follow one of the fastest growth rates in scientific production in the whole world . Compare with other countries in the regions, after turkey and israel, respectively with 7,092 and 5,214 papers, iran has achieved to third position with 3,886 papers . Citation as one of the representative indexes for paper's application and quality, in most of region countries, has ascending pattern . In a closer inspection, these recent rapid increasing trends in research outputs can be attributed to the attention to research by country policy - makers up to the highest level of leadership that has caused a national comprehensive commitment on research policy, facilities, and resources . These collaborations, potentially, provide more facilities for increasing the citations and applications of papers . Israel between the region countries had the most citation for obesity - related papers that is directly associated with its higher rate of collaborative papers . In this regard, after turkey, iran had the third ranks of high citation order . Between out of regions countries, usa had the most participation in the publication of all 17 middle east countries . Inside the region, saudi arabia and egypt with 145 collaborative papers had the first collaborative position . In the field of obesity / overweight, iran had the most collaborative papers respectively with usa (4.6%), uk (3.6%), and canada (1.7%). Between region countries, the increase in the number of related multidisciplinary faculties as well as research centers and consequently the increase of related specialists, students, research projects, and dissertations are inevitably positive factors which influence the rise in the number and citations of papers in this field . First, we focused on specific obesity / overweight domain to clarify the exact situation of knowledge production . Third, we assessed collaboration between middle east countries in related research fields; and fourth, we explain the situation of iran as one of the most effective knowledge productive country in the region in details . To the best of our knowledge, this is the first scientometrics analyses of obesity / overweight knowledge productions in the middle east region countries that provide practical information for better research planning in related multidisciplinary fields . Despite the ascending trends in research outputs, more efforts required for promotion of collaborative partnerships . These results also could be useful for better health policy and more planned studies in this field.
The sagittal alignment of the spine has important ramifications to the health - related quality of life.1 it varies with age,2 3 gender,4 spinal disease,5 6 and body position.7 8 9 10 many researchers have evaluated the sagittal alignment of the cervical spine . However, they used different neck positions,7 8 as well as different radiographic views . Most commonly, these are cervical spine lateral radiographs and whole - spine lateral radiographs.4 11 12 13 14 however, to the best of our knowledge, there is no literature comparing the sagittal cervical parameters between these two views . The purpose of this study is to determine if the radiographic parameters for sagittal cervical alignment are comparable between these two views . This retrospective study included adult patients from a single institution who had the radiographs taken to evaluate the neck pain . The criteria for inclusion were as follows: (1) age 20 years or older at the time of evaluation; (2) no contraindication for radiographic exposure (e.g., pregnancy, tumor); (3) agreement of patients to take both radiographs . Lateral radiographs were obtained with a 10 12-inch cassette at a 72-inch (182 cm) distance with the radiographic tube centered at the c4c5 disk space with no magnification (fig . Subjects were instructed to stand in a comfortable position and keep their eyes forward with their arms extended on their chests (fig . 1). Immediately after taking the cervical lateral radiographs, whole - spine lateral radiographs were taken with a 14 17-inch cassette at a 98.4-inch (250 cm) distance with the tube centered at the xiphoid process and with the subjects in a comfortable standing position, keeping their eyes forward and their arms crossed upon their chests without magnification (figs . 2, 3). The digital x - ray images were obtained and measured on the pacs system (view, infinitt, seoul, korea). Cervical spine lateral radiographs were obtained with a 10 12-inch cassette at a 72-inch distance with the radiographic tube centered at the c4c5 disk space with subjects in a comfortable standing position, keeping their eyes forward and their arms extended on their chests without magnification . Whole - spine lateral radiographs were taken with a 14 17-inch cassette at a 98.4-inch distance with the tube centered at the xiphoid process with subjects in a comfortable standing position, keeping their eyes forward and their arms crossed upon their chests without magnification . Cervical spinal morphology was measured using the standard techniques to obtain the following parameters for the two different radiographs: occipital c2 angle: cobb angle between the mcgregor line and the inferior end plate of axis (fig . 4) c2c7 angle: cobb angle between the inferior end plate of c2 and the inferior end plate of c7 (fig . 4) c7sternal angle: cobb angle between the inferior end plate of c7 and the anterior border of sternum (fig . 4) sternal slope: cobb angle between the anterior border of sternum and the horizontal line (fig . 4) t1 slope: cobb angle between the superior end plate of t1 body and the horizontal line (fig . 5) c2 central offset distance: the distance between the center of c2 body and the line tangential to the posterior cortex of c7 body (fig . 6) angle (occipital c2) is formed by mcgregor line and the inferior end plate of the axis; angle (c2c7), by the inferior end plate of c2 and inferior end plate of c7; angle (c7sternal), by the inferior end plate of c7 and the anterior border of sternum; angle (sternal slope), by the anterior border of sternum and the horizontal line . C2 central offset distance (a) is the distance between the center of c2 body and the line tangential to the posterior cortex of c7 body; t1 slope () is the angle between the superior end plate of t1 body and the horizontal line . The distance between c2 and c7 plumb lines (b to c) is the distance between c2 plumb line and c7 plumb line, the difference of b and c; c4 ap diameter (d) is anteroposterior diameter of c4 vertebral body . Additionally, we calculated the ratio of c2 central offset distance to c4 ap diameter and the ratio of plumb lines' distance to c4 ap diameter to reduce the radiographic magnification (figs . 5, 6). The statistical analysis was performed using spss version 13.0 (ibm corporation, armonk, ny, united states). The differences in continuous variables between the two groups were examined with a paired t test . The power analysis was performed by gpower version 3.1.5 (universitt kiel, germany). The power was 0.8 for paired t tests with the effect size of 0.5 and error probability of 0.05 . The statistical significance level was set at p <0.05 . The intraobserver and interobserver reliability were calculated using the reliability statistics by intraclass correlation for the cobb angle and the distance . The intraclass correlation values were graded using previously described semiquantitative criteria: excellent for values in the 0.9 to 1.0 range, good for 0.7 to 0.89, fair / moderate for 0.50 to 0.69, low for 0.25 to 0.49, and poor for 0.0 to 0.24 . Finally, we compared the statistical difference of the previously mentioned parameters with the errors of measurement for the two different radiographs . This retrospective study included adult patients from a single institution who had the radiographs taken to evaluate the neck pain . The criteria for inclusion were as follows: (1) age 20 years or older at the time of evaluation; (2) no contraindication for radiographic exposure (e.g., pregnancy, tumor); (3) agreement of patients to take both radiographs . Cervical spine lateral radiographs were obtained with a 10 12-inch cassette at a 72-inch (182 cm) distance with the radiographic tube centered at the c4c5 disk space with no magnification (fig . Subjects were instructed to stand in a comfortable position and keep their eyes forward with their arms extended on their chests (fig . Immediately after taking the cervical lateral radiographs, whole - spine lateral radiographs were taken with a 14 17-inch cassette at a 98.4-inch (250 cm) distance with the tube centered at the xiphoid process and with the subjects in a comfortable standing position, keeping their eyes forward and their arms crossed upon their chests without magnification (figs . 2, 3). The digital x - ray images were obtained and measured on the pacs system (view, infinitt, seoul, korea). Cervical spine lateral radiographs were obtained with a 10 12-inch cassette at a 72-inch distance with the radiographic tube centered at the c4c5 disk space with subjects in a comfortable standing position, keeping their eyes forward and their arms extended on their chests without magnification . Whole - spine lateral radiographs were taken with a 14 17-inch cassette at a 98.4-inch distance with the tube centered at the xiphoid process with subjects in a comfortable standing position, keeping their eyes forward and their arms crossed upon their chests without magnification . Cervical spinal morphology was measured using the standard techniques to obtain the following parameters for the two different radiographs: occipital c2 angle: cobb angle between the mcgregor line and the inferior end plate of axis (fig . 4) c2c7 angle: cobb angle between the inferior end plate of c2 and the inferior end plate of c7 (fig . 4) c7sternal angle: cobb angle between the inferior end plate of c7 and the anterior border of sternum (fig . 4) sternal slope: cobb angle between the anterior border of sternum and the horizontal line (fig . 4) t1 slope: cobb angle between the superior end plate of t1 body and the horizontal line (fig . 5) c2 central offset distance: the distance between the center of c2 body and the line tangential to the posterior cortex of c7 body (fig . 6) angle (occipital c2) is formed by mcgregor line and the inferior end plate of the axis; angle (c2c7), by the inferior end plate of c2 and inferior end plate of c7; angle (c7sternal), by the inferior end plate of c7 and the anterior border of sternum; angle (sternal slope), by the anterior border of sternum and the horizontal line . C2 central offset distance (a) is the distance between the center of c2 body and the line tangential to the posterior cortex of c7 body; t1 slope () is the angle between the superior end plate of t1 body and the horizontal line . The distance between c2 and c7 plumb lines (b to c) is the distance between c2 plumb line and c7 plumb line, the difference of b and c; c4 ap diameter (d) is anteroposterior diameter of c4 vertebral body . Additionally, we calculated the ratio of c2 central offset distance to c4 ap diameter and the ratio of plumb lines' distance to c4 ap diameter to reduce the radiographic magnification (figs . 5, 6). The statistical analysis was performed using spss version 13.0 (ibm corporation, armonk, ny, united states). The differences in continuous variables between the two groups were examined with a paired t test . The power analysis was performed by gpower version 3.1.5 (universitt kiel, germany). The power was 0.8 for paired t tests with the effect size of 0.5 and error probability of 0.05 . The statistical significance level was set at p <0.05 . The intraobserver and interobserver reliability were calculated using the reliability statistics by intraclass correlation for the cobb angle and the distance . The intraclass correlation values were graded using previously described semiquantitative criteria: excellent for values in the 0.9 to 1.0 range, good for 0.7 to 0.89, fair / moderate for 0.50 to 0.69, low for 0.25 to 0.49, and poor for 0.0 to 0.24 . Finally, we compared the statistical difference of the previously mentioned parameters with the errors of measurement for the two different radiographs . Fifty - nine adults made up the study group (31 men and 28 women). The measurements of the cobb angles in the cervical spine lateral radiographs had 0.979 intraobserver reliability and 0.966 interobserver reliability . The difference of the intraobserver measurements for the cobb angles in the cervical spine lateral radiographs was 0.58 2.74 degrees, and the difference of the interobserver measurements was 0.73 3.43 degrees . The measurements of the c2 offset distance had 0.978 intraobserver reliability and 0.926 for interobserver reliability . The difference of the intraobserver measurements for the cervical spine distance in the lateral radiographs was 0.26 3.05 mm, and the difference of the interobserver measurements was 0.90 5.48 mm . For the whole - spine lateral radiographs, the measurements of the cobb angles had 0.969 intraobserver reliability and 0.803 interobserver reliability . The difference of the intraobserver measurements for the cobb angles in the whole - spine lateral radiographs was 0.36 3.65 degrees, and the difference of the interobserver measurements was 4.08 7.78 degrees . The measurements of the c2 offset distance had 0.982 intraobserver reliability and 0.979 interobserver reliability . The difference of the intraobserver measurements for the distance in the whole - spine lateral radiographs was 4.67 9.00 mm, and the difference of the interobserver measurements was 6.09 9.48 mm . C2 angle, sternal slope, and c4 ap diameter were similar, but the c2c7 angle, c7sternal angle, t1 slope, c2 central offset distance, distance between c2 and c7 plumb lines, ratio of c2 central off distance to c4 ap diameter, and the ratio of plumb lines' distance to c4 ap diameter were statistically different (table 1). However, the error of measurement, especially interobserver measurements for the cobb angles in the whole - spine lateral radiographs (4.08 7.78 degrees) and distances (6.09 9.48 mm), were greater than the angular statistical differences (2.51 7.86 degrees of c2c7 angle) and linear statistical differences (4.38 10.04 mm of c2 central off distance) between the two types of radiographs (table 1). Several methods have been described to evaluate the sagittal alignment of the cervical spine.4 7 8 11 12 13 however, they have used different neck positions and different radiographic techniques,7 8 including cervical spine lateral radiographs and whole - spine lateral radiographs.4 11 12 13 14 however, to the best of our knowledge, there is no literature comparing the sagittal cervical parameters obtained from cervical spine lateral views versus whole - spine lateral views . In the current study, the occipital however, the c2c7 angle, c7sternal angle, t1 slope, c2 central offset distance, distance between c2 and c7 plumb lines, ratio of c2 central off distance to c4 ap diameter, and ratio of plumb lines' distance to c4 ap diameter were different . However, the angular and linear differences between the two types of radiographs were smaller than the error of measurement for the two types of radiographs . The lordotic curvature of the cervical spine is considered as normal3; however, the exact values and the recommended methods of measurement are not clearly described . Normal lordotic angles for c2c7 have been reported to range from 20 to 35 degrees,13 but these values are highly related to the method of measurement used and the positioning of the patients while taking a radiograph . Moreover, the head posture can influence the sagittal curve of the cervical spine.8 in addition, the arm or shoulder posture can influence sagittal spinal balance.9 10 according to vedantam et al,10 positioning the arms at 90 degrees rather than 30 degrees resulted in a negative shift of the sagittal vertical axis . According to marks et al,9 shoulder flexion of 30 degrees is the best position to use when a lateral radiograph is made to repeatedly measure the sagittal vertical axis . Also, cervical lordosis is increased when the thoracic kyphosis of the trunk is increased.4 14 in analyzing spinal sagittal alignment, it is most important to standardize the patients' posture while taking radiographs . We tried to keep the patients' standing posture as identical as possible to limit the effect of positioning . Spine radiographs can be distorted by parallax.15 to limit the distortion, one can move the x - ray source away from the patient to reduce the divergence of the beam (conventional teleradiography) or translate the multiple focuses and coupled receptors simultaneously to scan the entire spine (digitalized teleradiography).15 the reasons for the differences in measurements for the two views include the differences in the centering target of the radiation beam, the distance from x - ray source to the cassettes, and the posture of arms between the two radiographs . Cervical spine lateral radiographs were taken at a 72-inch distance from the radiograph tube and centered at the c4c5 disk space with the subject in a comfortable standing position with eyes forward and arms extended upon their chests . Whole - spine lateral radiographs were taken at a 98.4-inch distance from the tube and centered at the xiphoid process of the subject with eyes forward and arms crossed upon their chests . The 95% confidence limits for intra- and interobserver variability of the cobb method in the cervical lateral radiographs were 5 and 9 degrees with the traditional manual method.16 in many cases, a 5-degree variation between the measurements in the whole - spine lateral radiographs by the traditional manual cobb method is acceptable.17 in the current study, the mean difference of the intra- and interobserver measurements for the cobb angles in the cervical spine lateral radiographs with digital methods was 0.58 and 0.73 degrees . The mean difference of the intra- and interobserver measurements for the cobb angles in the whole - spine lateral radiographs was 0.36 and 4.08 degrees . The reproducibility of the cobb angle measurements with digitalized images appears better than those with traditional manual methods.18 the difference in the intraobserver measurements on the digitalized images of the distance in the cervical spine lateral radiographs was 0.26 3.05 mm and the difference in the interobserver measurements was 0.90 5.48 mm in the current study . The reproducibility of the distance measurements with digitalized images may be better than those with traditional manual methods, similar to cobb angle measurements in the current study . However, we did not compare the reproducibility of the distance measurements using digitalized images with those using traditional manual methods . As with any study, the present investigation has limitations . Arm posture was one reason for the difference between the cervical and whole - spine lateral radiographs . Ideally, we should have taken the two radiographs in the same arm posture to reduce the variables . However, we tried to compare the real - life situation in clinical practice and clinical research . To the best of our knowledge, this study is the first comparing sagittal cervical parameters between the cervical spine lateral view and whole - spine lateral view . In conclusion, most numerical values of parameters for the two radiographic views appear to be different . However, the angular and linear numerical differences between the two radiographic views were smaller than the inter- and intraobserver errors of measurement for the two radiographic views . Our results suggest that to determine the alignment of only the cervical spine, there is no advantage to using whole - spine lateral radiographs . However, this finding does not take into account the overall spinal alignment, for which whole - spine lateral radiograph are necessary.
Since the beginning of mankind, organisms producing natural products have provided a reservoir of therapeutic remedies and medicines for various diseases . A subset of these drugs has been classified as antitumor antibiotics based on their ability to block cell growth by interfering with dna, the genetic material in cells . Key general features of an antitumor antibiotic include interference with dna synthesis, membrane transport, and production of reactive oxygen species . One of the most notable examples of an antitumor antibiotic is mitomycin c, a microbial metabolite that is used currently for the treatment of breast and bladder cancer . Among other antitumor antibiotics, daunorubicin and its semisynthetic derivative doxorubicin represent chemotherapeutic leukemia agents in clinical settings . The search for new antitumor antibiotics led to the discovery of chlorothricin (1), a complex polyketide produced by various streptomyces strains . Its intriguing chemical structure and bioactivity defined a new family of microbial metabolites, commonly referred to as spirotetronate polyketides . This family is identified by the presence of a cyclohexene ring spiro - linked to a tetronic acid moiety (figure 1, fragment a) that is embedded in a macrocycle (figure 1, fragment b). In several cases, the structure also contains a trans - decalin ring (figure 1, fragment c) and is decorated by various deoxy oligosaccharides (figure 1, fragment d). In terms of biological profile, spirotetronate polyketides exhibit potent antibacterial and antitumor activities and a documented value as tools in the elucidation of new biological pathways . As such, they represent highly promising leads in drug discovery . To appreciate their untapped potential, in this review we group the known spirotetronates based on common structural elements and biosynthetic considerations . Recently sssmuth and co - workers proposed a classification of tetronates based on two main categories: the linear tetronates and the spirotetronates . On the basis of biosynthetic considerations, the latter subgroup can be divided into two classes: class i (generic structure 4), which contains the spirotetronate moiety within a varying size macrocycle, and class ii (generic structure 5), which additionally contains a decalin moiety (figure 2). Representative members of the class i spirotetronates in order of increasing macrocyclic length are abyssomicin c (6) (containing a c11 macrocycle), okilactomycin d (7) (containing a c13 macrocycle), and spirohexenolide a / b (8/9) (containing a c15 macrocycle). Representative members of the class ii spirotetronates include maklamicin (11) (containing a c11 macrocycle), tetronolide (12) (the aglycon of tetrocarcin a containing a c13 macrocycle), and chlorothricolide (2) (the aglycon of chlorothricin containing a c13 macrolactone). In this class is also included versipelostatin aglycone (13), which contains the largest c17 macrocyclic motif isolated to date . Quartromicins 10, unusual spirotetronate polyketides containing four spirotetronate subunits, lie outside these two classes due to their peculiar structure and unique biosynthesis . The above classification stems from a common biosynthetic pathway that accounts for construction of these compounds . In general, the biosynthesis of spirotetronates occurs through condensation of acetic acid units via the type i polyketide synthase pathway (scheme 1). As shown in the biosynthesis of abyssomicin c and okilactomycin, construction of the class i spirotetronates proceeds by elongating their carbon chain via incorporation of propanoyl and/or acetyl units (14/15) to the acyl carrier protein (acp). Incorporation of a glyceryl unit, via coa intermediate 17, forms tetronate 18 likely via a claisen condensation followed by lactonization . The precise mechanism for the elimination of the c-5 hydroxy group was recently elucidated by the sun and leadlay groups and shown to proceed via acetylation and subsequent elimination, thereby forming dienophile 19 . An intramolecular diels the resulting substrates subsequently undergo peripheral oxidations to produce the final structures of the natural products . The biosynthetic pathways of the class ii polyketide spirotetronates have been elucidated for chlorothricin, tetrocarcin a, kijanimicin, and versipelostatin . Following chain elongation, the diene and dienophile groups of 20 undergo an imda to construct the characteristic decalin moiety of 21 . Glyceryl coa (17) is then inserted to generate tetronate 22, which following a second imda gives rise to the aglycones of the class ii spirotetronates (figure 2). Oxidations and/or glycosylations at the periphery of the aglycone lead to various natural products of the class ii spirotetronates . For instance, chlorothricolide (2), the aglycone of chlorothricin, contains an acyl - oxy tetronic acid moiety . This functionality (i.e., oxygenation at the c-2 position) is proposed to result from a baeyer a similar biosynthetic scenario can be proposed for the construction of pa-46101 a and b (see structures 57/58). Another interesting example of post - translational modification is found in the structure of tetronolide (12), the aglycone of tetrocarcin a (see structure 47). This functionality was proposed to result from oxidation at c-32 to the corresponding aldehyde followed by double - bond migration to c-22c-23 and further allylic oxidation at c-21 . Several class ii spirotetronates are subject to glycosylation mostly with 2-deoxycarbohydrates such as d - tetronitrose (26, d - kijanose), amicetose (27), and digitoxose (28). These carbohydrates are proposed to arise from thymidine diphosphate (tdp)-6-deoxy-4-keto - d - glucose (24), which, in turn, is available from d - glucose-1-phosphate (23) (scheme 2). Biosynthesis of the uncommon tetronitrose is proposed to occur from 25 via aminotransferase and methylation, while the precise mechanism for the carbamate biosynthesis still remains elusive . The majority of spirotetronates have been subjected to biological assays that aim to define their bioactivity as antibiotic and/or anticancer leads as well as compounds that regulate metabolism . With this in mind, we have grouped these molecules in three major classes that describe the commonality of their bioactivities . Isolated from a marine verrucosispora, abyssomicin c (6) and its atropisomer (29) (figure 3) are the first known natural products to block para - aminobenzoic acid (paba, 41) biosynthesis.paba is a biosynthetic precursor of folic acid (vitamin b9), and as such, it is essential for dna synthesis / repair and cell survival (scheme 3). On the other hand, lack of folic acid importantly, blocking the paba pathway is detrimental to bacteria but inconsequential to humans since the latter cannot produce folic acid but only absorb it through their diet . Studies on the effect of the abyssomicin motif in paba biosynthesis have been performed with atrop - abyssomicin (29) and are highlighted in scheme 3 . Amino-4-deoxychorismate (adc) synthase, a heterodimeric protein, catalyzes the biosynthesis of amino-4-deoxychorismate (40), a synthetic precursor of paba . Compound 29 was found to covalently react with the cys-263 of the pabb subunit of adc synthase at the c-9 enone center . The transiently formed c-8 nucleophile then reacts with the spirotetronate subunit at the c-16 center to form compound 39, thus irreversibly binding to adc synthase . Several natural products of the abyssomicin family have been tested for their ability to inhibit paba biosynthesis (scheme 3). Among them, only abyssomicin c (6), atrop - abyssomicin c (29), and abyssomicin j (31) have shown promising bioactivities . Specifically, 6 and 29 potently inhibit proliferation of methicillin - resistant staphylococcus aureus at mic values of 5.2 and 3.5 g / ml, respectively . Similar cytotoxicities have been reported against various tuberculosis - related mycobacteria . On the other hand, abyssomicin d (32) and related analogues lacking the c-7c-9 enone motif are inactive, attesting to the biological significance of this functionality . This increased potency has been attributed to an increased conjugation between the c-7 carbonyl group and the c-8c-9 alkene that renders 29 a stronger michael acceptor than 6 . The bioactivity of abyssomicin j (31), a thioether dimer of the abyssomicin scaffold, can be explained by considering that oxidation of the sulfur accelerates a retro - michael addition, producing the c-7c-9 enone functionality in situ . In fact, it has been suggested that 31 is a prodrug of 6, and as such, it represents a more attractive drug candidate . Isolated from various micromonospora bacteria, kijanimicin (43) and lobophorin b (42) are structurally defined by a c13 macrocycle (referred to as kijanolide, 44) in which the c-9 and c-17 hydroxy groups have been glycosylated (figure 4). Most members of this group show potent activity against gram - positive bacteria as well as cytotoxicity against various cancer cell lines . In addition, kijanimicin was shown to exhibit robust anticancer and antimalarial activities in mouse models . Moreover, fenical et al . Reported promising anti - inflammatory activities of lobophorins in a mouse ear edema model . Interestingly, this is the first report on the untapped potential of spirotetronates as small - molecule leads against inflammation . A similar framework is in the structures of pyrrolosporin a (45, c-9-glycosylation) and mm46115 (46, c-17 glycosylation). The glycopyranose motif of pyrrolosporin is also found in the structures of decatromicin a / b and nai414-a / b, which also exhibit similar antibiotic activity against various strains of gram - positive bacteria . In addition to its potent antibiotic activities mm46115 was found to exhibit promising antiviral activities . Along these lines, the structurally unrelated quartromicins 9(14a,14c) were shown to display potent bioactivity against herpes simplex virus (hsv) and human immunodeficiency virus (hiv) at low m concentration . Recent studies indicate that kijanimicin binds to the tetr family of transcriptional regulators that control expression of various cytoplasmic proteins in prokaryotes . This binding leads to (a) c-9-deglycosylation of kijanimicin, which results in loss of activity, and (b) overexpression of the receptor, thus increasing antibiotic resistance . The structurally related saccharocarcins are subject to a similar mechanism of deactivation and antibiotic resistance . Reported the isolation of spirohexenolides a (8) and b (9) and their potent cytotoxicities against various cancer cell lines (figure 2). Subsequent immunoaffinity - fluorescent labeling studies indicated that 8 targets human macrophage migration inhibitory factor (hmif). This interaction reduces the phosphorylation levels of pi3k / akt, ultimately leading to a reduction of tumor cell growth (figure 6). Conjugation of spirohexenolide a with fluorescent tags showed localization in the lysosome of hct-116 cells, suggesting that spirohexenolides interfere with cellular endocytosis of hmif . Isolated from various micromonospora bacteria, tetrocarcin a (47, also known as antlermicin a and ac6d) represents the archetype of the tetronolide family of natural products that also includes ac6h (48) and arisostatins a (49) and b (50) (figure 7). The antibiotic potential of these spirotetronates against several gram - positive bacteria has been reported . Animal studies have shown that 47 is about 4 times more potent than the commonly used antibiotic diminazene . Although 47 has a narrow safety margin, it can be used in combination with diminazene, providing a beneficial synergistic effect . Various reports on the potential anticancer profile of tetrocarcin a and related family members have been published . Initial studies showed tumor reduction in a mouse sarcoma model upon administration of 10 mg / kg of 47 over a period of 6 days . Similar treatment in a mouse leukemia p388 model led to an increased life expectancy . Comparable studies in b16 mouse melanoma showed that the life expectancy more than doubled at a single dose of 27 mg / kg of 47 . Moreover, ac6h 48 exhibited cytotoxicity against p388 leukemia and b16 melanoma cells at 6.25 and 25 g / ml, respectively . Ac6h also showed a moderate increase in the life expectancy of a p388 leukemia mouse model albeit less active than tetrocarcin a. studies in u937 cells indicated that arisostatin a (49) is equipotent to tetrocarcin a, while arisostatin b (50) was 10-fold less active . Arisostatin a was also found to be active in various breast and lung cancer cell lines at low micromolar concentrations . Mode - of - action studies in hela cells showed that tetrocarcin a (47) potently inhibits bcl-2, an important antiapoptotic protein that is often overexpressed in cancer cells (figure 6). Although there is no evidence of direct binding to bcl-2, the phenotypical response induced by 47 is very promising and suggests that this compound represents an important and unexplored lead against cancer . Studies in lymphoma cells showed that 47 induces a stress response of the endoplasmic reticulum (er), resulting in upregulation of the heat shock protein hsp70, ultimately triggering cell apoptosis (figure 6). Studies in breast cancer cells have suggested an alternative mechanism of action of 47 that proceeds by inhibiting phosphorylation of the pi3k / akt signaling cascade . Although the main cellular target of tetrocarcin a is still under investigation, preclinical studies have demonstrated its potential as a drug against chemoresistant cancers . In fact, 47 was reported to be more effective than paclitaxel at inducing cell apoptosis in breast cancer cells . Arisostatin a (49) was found to induce cell apoptosis by generating reactive oxygen species (ros), altering mitochondrial transmembrane potential, and releasing cytochrome c (cyt c) in amc - hn-4 cells (figure 6), ultimately leading to activation of caspase-3 and induction of apoptosis . However, bcl-2 activation was not altered by arisostatin a, indicating a different mode of action from that of 47 . Screening the potential anticancer and antimicrobial activities of naturally occurring tetrocarcins has produced the main structure these studies have led to the following observations: (a) the number of carbohydrate units (digitoxose and amicetose) attached at the c-9 center of tetrocarcin a is proportional to its antimicrobial activity; (b) c-21 acetylation and c-9 glycosylation of tetrocarcin a did not significantly affect bcl-2 activation . The results suggest that the attachments of amicetose (27) and digitoxose (28) at the c-9 position of tetrocarcin a enhance its antibacterial profile but have no significant effect on its anticancer potential . Versipelostatin a (51) was isolated from a strain of streptomyces versipellis (figure 8). Biological studies showed that 51 is the first known molecule to inhibit gene expression of grp78 . Together with its isoform grp94, these heat shock proteins are induced by stress responses in the endoplasmic reticulum and are essential for cancer cell survival . In addition to its role in cancer, er stress is considered to play a major role in the pathogenesis of various cns diseases, such as alzheimer s and parkinson s disease . Recent studies have shown that versipelostatin a (51) inhibits heat shock proteins and unfolded protein response (upr) under glucose deprivation conditions . As such, it appears to operate via a different mechanism as compared to that of rapamycin, an fda - approved immunosuppressive drug that activates grp78 independently of glucose availability . Thus, versipelostatin may offer a significant advantage due to its selective effect in hypoglycemic cells . Although there is no information for direct binding of 51 to a protein target, its effect on the upr signaling pathway offers a novel tool to understand er - induced stress and pharmacologically regulate related illnesses . Sar studies on this family have been limited to the bioactivities of naturally occurring versipelostatins . The results show that versipelostatins a (51), e (52), and f (53) are the only biologically active compounds, inhibiting grp78 expression at low micromolar ic50 values . Interestingly, 53 was found to be 10 times more potent than 51 in grp78 expression with an ic50 of 0.3 m . The data attest to the significance of the glycosylation motif to the grp78 expression and bioactivity . In addition to these studies, takahashi et al . Demonstrated the importance of the l - oleandrose sugar for the bioactivity, and changes in the oxidation state of c-7 had no effect on biological activity . Okilactomycin (54) was isolated from streptomyces griseoflavus and is noted for its potent antitumor activity against ehrlich ascites carcinoma in vivo at 2.5 mg / kg with a t / c of 145.7% for mice survival . In addition, 54 exhibited in vitro activity against p388 and l1210 leukemia cells, with ic50 values of 89 and 216 nm, respectively . Recently, okilactomycin was shown to inhibit rrna protein synthesis at low m concentrations, suggesting potential applications as an antibacterial agent . Although other natural okilactomycins were found to be inactive, the related chrolactomycin (55) was reported to exhibit antibacterial and anticancer activity at a low m concentration . It has been reported that 55 inhibits telomerase activity, thus blocking the ability of cancer dna to replicate . The most recently isolated 6-hydroxy chrolactomycin was less active than 55 against gram - positive bacteria . The potent antibiotic properties of pa-46101a (57) and b (58) have been reported . Recent efforts by igarashi and co - workers led to the isolation of maklamicin (11) and nomimicin (59), which contain the smallest macrocyclic ring of the class ii spirotetronates . Both compounds display potent activity against many gram - positive bacteria, while 11 also exhibits moderate antitumor activity against hela and mcf7 breast cancer cells . Chlorothricin (1) was shown to inhibit the activity of pyruvate carboxylase, a key enzyme that converts pyruvate to oxaloacetate, thus allowing consumption of glucose through the krebs cycle (figure 11). Inhibition of pyruvate carboxylase leads to an increase of pyruvate concentration in liver, which through gluconeogenesis accounts for accumulation of glucose, ultimately leading to diabetes . Moreover, an inhibitory effect of 1 on malate dehydrogenase, an enzyme that oxidizes malate to oxaloacetate in the krebs cycle, has also been reported . It should be noted, however, that the direct cellular target of 1 is under debate and may involve interaction with components in the cell membrane that may account for the observed downstream effects . Although the potential anticancer properties of chlorothricin (1) have not been investigated, c-31 hydroxychlorothricin (figure 1) was shown to exhibit antitumor activity at 40 mg / kg against implanted ehrlich carcinoma cells in mice with an ld50 of 295 mg / kg . C-28 methyl ester of chlorothricolide (2), the aglycone of 1 (figure 1), also inhibits pyruvate carboxylase albeit at higher concentrations than 1, suggesting that glycosylation enhances biological activity . Efforts to discover new gastric atp - ase inhibitors led to the isolation of a88696f (61) and its dehydroxylated counterpart a88696c (60). Hydroxylation at c-3 was found to enhance the biological activities since 61 was the most active, with an ic50 at 0.5 m, while 60 was considered inactive . Isolated from a streptomyces species, tetronothiodin (62) was shown to inhibit brain - type cholecystokinin (cck)-b receptor in rat cerebral cortex with an ic50 value of 3.6 nm . It is worth noting that cck receptors are structurally similar to gastrin and are used throughout the central nervous system (cns) and gastric tract . Interestingly, 62 has 27 000 times higher affinity for cck - b over cck - a in rat models . Thus, in addition to its pharmaceutical promise, 62 could be used as a tool to study the cck - b / cck - a signaling pathway . Chlorothricin (1) was shown to inhibit the activity of pyruvate carboxylase, a key enzyme that converts pyruvate to oxaloacetate, thus allowing consumption of glucose through the krebs cycle (figure 11). Inhibition of pyruvate carboxylase leads to an increase of pyruvate concentration in liver, which through gluconeogenesis accounts for accumulation of glucose, ultimately leading to diabetes . Moreover, an inhibitory effect of 1 on malate dehydrogenase, an enzyme that oxidizes malate to oxaloacetate in the krebs cycle, has also been reported . It should be noted, however, that the direct cellular target of 1 is under debate and may involve interaction with components in the cell membrane that may account for the observed downstream effects . Although the potential anticancer properties of chlorothricin (1) have not been investigated, c-31 hydroxychlorothricin (figure 1) was shown to exhibit antitumor activity at 40 mg / kg against implanted ehrlich carcinoma cells in mice with an ld50 of 295 mg / kg . C-28 methyl ester of chlorothricolide (2), the aglycone of 1 (figure 1), also inhibits pyruvate carboxylase albeit at higher concentrations than 1, suggesting that glycosylation enhances biological activity . Efforts to discover new gastric atp - ase inhibitors led to the isolation of a88696f (61) and its dehydroxylated counterpart a88696c (60). Hydroxylation at c-3 was found to enhance the biological activities since 61 was the most active, with an ic50 at 0.5 m, while 60 was considered inactive . Isolated from a streptomyces species, tetronothiodin (62) was shown to inhibit brain - type cholecystokinin (cck)-b receptor in rat cerebral cortex with an ic50 value of 3.6 nm . It is worth noting that cck receptors are structurally similar to gastrin and are used throughout the central nervous system (cns) and gastric tract . Interestingly, 62 has 27 000 times higher affinity for cck - b over cck - a in rat models . Thus, in addition to its pharmaceutical promise, 62 could be used as a tool to study the cck - b / cck - a signaling pathway . In this part of the review, we highlight the key steps toward the synthesis of selected spirotetronates . When possible, we compare the various strategies in terms of overall efficiency . Abyssomicin c (6) and its atropisomer 29 contain a rigid oxabicyclo [2.2.2] octane substructure that encapsulates the spirotetronate moiety . To date, three chemical syntheses of 6 and 29 have been reported . Sorensen s group used a biomimetic imda to construct spirotetronate moiety 65 from diene 63 . C-11c-12 epoxidation of 65 followed by c-16 intramolecular enol epoxide opening produced a 1:1 mixture of abyssomicin c (6) and atrop - abyssomicin (29). The nicolaou group s synthesis of abyssomicin c is highlighted by an intermolecular diels alder cycloaddition that furnishes cyclohexene 67 with the desired stereochemistry (scheme 4). A ring - closing metathesis (rcm) was used to generate the macrocyclic skeleton of 6 from diene 68 . Interestingly, the authors showed that treatment of 29 with lithium selectride led to formation of abyssomicin d (32). Interestingly, this finding supports the notion that abyssomicin c (6) is a biosynthetic progenitor of 32 and further validates the proposed mechanism of abyssomicin c deactivation as presented in scheme 3 . More recently, the groups of bihelovic and saicic reported a synthesis of 29 . Interestingly, this strategy produces exclusively atrop - abyssomycin c. it is likely that the restricted rotation around the c-2 and c-3 centers, due to the sp hybridization, affects the formation of the two isomers . In support of this hypothesis, the nicolaou group has shown that 29 can be converted to 6 by protonating the c-16 oxygen under mild acidic conditions . Other studies toward the abyssomicin scaffold have been reported in addition to the mentioned total syntheses . Ferrier union / rearrangement of 72(97) that yielded the 2,6-cis - tetrahydropyranone ring 73 . Grubbs second - generation catalyst was used to construct the 13-membered macrocycle of 54 . Key to this approach was a prins - type fragment assembly between cyclohexene 75 and -keto - ester 74 that formed the 2,6-cis - tetrahydropyranone ring of 76 . Similarly to the smith approach, an intramolecular ring - closing metathesis using grubbs second - generation catalyst constructed the macrocycle . Additional synthetic studies toward okilactomycin have been reported by the yoshii and paquette groups . Key to this strategy was an imda cycloaddition that formed spirotetronate 80 from precursor 79 . The overall synthesis proceeds in 13 linear steps (17 total steps) and 17% yield . The burkart group reported a strategy toward spirohexenolides based on an intermolecular diels alder cycloaddition (scheme 7). A ring - closing julia kocienski coupling was applied for the synthesis of macrocycle 82 . Although the projected intramolecular hemiacetalation to 84 failed due to an oxidative rearrangement of 82 to 83, the overall strategy has successfully installed the major skeletal features of spirohexenolides . Structurally tetronothiodin is highlighted by an -acyl tetronic acid moiety and tetrahydrothiophene moiety . Page et al . Have reported a synthesis of the spirotetronate subunit isomer 87 using a diels alder reaction with propenal and the hydroxyl diene 85 to install the desired stereochemistry of 86 (scheme 8). Several strategies have been employed for the synthesis of tetronolide (12), the aglycone of tetrocarcin a (47), kijanolide (44), the aglycone of kijanimicin (42), and chlorothricolide (2), the aglycone of chlorothricin (1). Tetronolide has been synthesized by yoshii and boeckman, while an improved formal synthesis has also been reported by roush . In general, these strategies rely upon independently constructing the spirotetronate and decalin moieties and then connecting them to form the c13 macrocycle . A remarkable synthesis of chlorothricolide (2) was reported by the roush group . The yoshii and roush syntheses of the decalin moiety 92, common to both tetronolide and kijanolide, are summarized in scheme 9 . In yoshii s approach a horner wadsworth emmons (hwe) olefination between 88 and 89 was used to construct polyene 90, which underwent an imda reaction to produce decalin 92 . The roush group implemented a suzuki coupling between 93 and 94 to form polyene 91, which, following further functionalizations, gave rise to decalin 92 via an imda cycloaddition . A synthetic approach toward spirotetronate 100 has been reported by yoshii and subsequently optimized by roush . This approach is based on constructing triene 97 via a hwe olefination between 95 and 96 . An intermolecular diels alder of diene 97 and chiral dienophile 98, followed by oxidative functionalization and double - bond migration, yielded enal 99 . Coupling of lithiated spirotetronate 100 with aldehyde 92 followed by subsequent functionalizations yielded sulfone 101, which, under julia coupling conditions, gave rise to the 13-membered macrocycle of 12 . Boeckman s group synthesis of 12 is highlighted by a tandem ketene - trapping [4 + 2] cycloaddition of diene 103 and alcohol 102 to form spirotetronate subunit 104 . Overall, this approach significantly reduces the number of steps required for completion of the tetronolide synthesis . Various synthetic studies toward kijanolide (44) have been reported by the groups of marshall, yoshii, and roush . Application of a julia coupling to the synthesis of 28,29-bisnor-(+)-kijanolide has been reported by the yoshii group . A tandem intra / intermolecular diels alder reaction between polyene 105 and chiral dienophile 98 was implemented for the synthesis of chlorothricolide (2) (scheme 11). The reaction gave the desired cycloadduct in 40% yield together with partially reacted decalin 107 . Upon treatment with dienophile 98, 107 was converted to the desired product 106 in 58% yield . Construction of the spirotetronate unit followed by coupling with the allyl ester completed the synthesis of 2 . A late - stage imda reaction was used by yoshii s group for the synthesis of ()-24-o - methylchlorothricolide (scheme 12). Although the selectivity of the imda reaction was moderate, the overall strategy represents a noteworthy bioinspired approach toward these compounds . The groups of marshall, ireland, snider, schmidt, and meyers have also reported studies toward the synthesis of 2 . Numerous synthetic studies have been reported toward the total synthesis of versipelostatin a (51), but to date its total synthesis has not been completed . A synthesis of the versipelostatin (51) trisaccharide 114 is shown in scheme 13 . The resulting disaccharide 112 was deprotected and coupled with l - oleandrosyl imidate 113 to produce 114 (scheme 13). Further functionalization of glycosyl 114 and schmidt glycosylation with acetyl c-7c-9c-37 versipelostatin aglycone 13 (figure 2) yielded a versipelostatin derivative used for biological studies . On the basis of nmr and biological consideration, the oleandrose sugar was structurally reassigned from d to l. an alternate strategy used was adding each sugar individually to the versipelostatin aglycone, thus elongating the glycosyl chain . A stereocontrolled diels alder reaction has been implemented by the roush group for the synthesis of the quartromicin spirotetronate unit . In addition, this group reported a strategy of connecting subunits 115 and 116 together using lithium halogen exchange and cecl3 coupling . Group offered an alternative strategy of constructing the spirotetronate subunits using rcm, but to date no total syntheses of quartromicins have been completed . The discovery of penicillin revolutionized pharmaceutical research by demonstrating, for the first time, that microorganisms can produce secondary metabolites of value to medicine . Since then, cultured microorganisms have been recognized as prolific producers of secondary metabolites that are used either directly as drugs or have inspired the design of drugs . On the other hand, the intricate structures of these compounds represent exceptional tools to explore new biological pathways and unknown mechanisms of action . These qualities, although scattered, are observed in the family of spirotetronate polyketides and provide evidence for their significant but still untapped pharmacological value . More than 40 years after the discovery of chlorothricin, the spirotetronate family has grown to include over 70 macrocycles of various sizes that, in certain cases, are decorated with carbohydrate side chains . In addition to their potent antitumor and antibiotic activities, for example, versipelostatin was found to induce potent and selective cytotoxicity in glucose - deprived tumor cells . Moreover, abyssomicin c was found to be the first natural product to block paba biosynthesis, a pathway essential to bacteria but insignificant to humans . Impressive synthetic and chemical biology efforts were combined to decipher the mode - of - action of abyssomicins at the molecular level . This underscores the enormous significance of the spirotetronate polyketide family to biology in addition to their pharmacological potential . Several studies have documented the significance of the carbohydrate chains for the observed antibiotic activity of spirotetronates . However, with the exception of abyssomicins, there is no clear understanding of the biological significance of the spirotetronate aglycone core . At present, chemical strategies developed toward the synthesis of spirotetronates have uncovered the value of certain key reactions, such as diels alder cycloaddition, ring -closing metathesis, and julia olefination . Nonetheless, the vast majority of these strategies have not yielded sufficient amounts of compound for a methodical structure activity relationship study, thereby hampering rational drug design . It is evident that a methodical fragment - based approach to this structure, in combination with chemical biology studies, will be highly beneficial, as it could reveal the role of the spirotetronate motif, the effect of the macrocyclic size, and the role of the decalin system . In turn, this effort would allow a detailed evaluation and optimization of the spirotetronate pharmacophore . In addition to a dearly needed scalable synthesis, advances in microbial biosynthesis should offer a potential solution to large - scale production or semisynthesis of a lead candidate . A combination of these efforts should unveil the pharmacological value of spirotetronates and would have significant impact in current efforts toward personalized medicine.
The candida genome database (cgd, http://www.candidagenome.org/) is a freely available online resource, based on the saccharomyces genome database (sgd, www.yeastgenome.org; (1)), which collects, organizes, and makes available candida gene, protein and sequence information to the fungal research community . Cgd also provides web - based tools for the visualization and analysis of biological data . Candida albicans is the most thoroughly studied of the human fungal pathogens and also serves as a model organism for the study of other more experimentally challenging fungal pathogens (2). The frequency of fungal infections has risen dramatically over the past few decades, with the current annual incidence of invasive candida infections recently estimated to range between 72 and 228 infections per million (3,4). These infections have high mortality rates (> 35%) and, despite the availability of antifungal drugs, exceed those associated with bacterial infections among intensive care unit patients (5). As a result of medical progress, particularly with respect to chemotherapy, organ transplantation, intensive care and neonatal care, the population of immunocompromised patients susceptible to fungal infection, is expanding (68), and resistance to antifungal drugs is increasing (3,9,10). Furthermore, c. albicans is not the only disease - causing species in the genus; of great concern is an emerging clinical prevalence of non - albicans candida species (11,12). Among these, c. tropicalis is common, virulent, and increasingly resistant to antifungal therapy (13), c. parapsilosis is particularly problematic, causing severe infections in neonates (14), and c. glabrata exhibits a special ability to evade the immune system and survive after cellular engulfment, and can resist antifungal treatment (1517). In fungal taxonomy, c. albicans is placed in the order endomycetales, to which the family saccharomycetaceae also belongs (18,19), though saccharomyces cerevisiae and c. albicans are separated by 140850 million years of evolution (20). Despite sharing many similarities, including 3453 pairs of orthologous genes, the two fungi inhabit very different environmental niches, with s. cerevisiae existing as a saprophyte, and c. albicans living in close association with its mammalian hosts . Transcriptional rewiring has occurred in their regulatory networks, such that evolved divergence in the activity of orthologous transcription factors, or divergence in cis - regulatory elements, has created distinct regulatory programs in each class (e.g. Galactose metabolism (21), mating type (22,23) and ribosomal proteins (24)). Unlike s. cerevisiae, c. albicans exists primarily in a diploid state, with no conventional sexual reproduction that leads to meiosis . Instead, c. albicans undergoes a parasexual cycle that involves mating between two diploid cells of the opposite mating type and formation of a tetraploid cell that subsequently returns to diploidy by chromosome loss (25). Alternatively, chromosome loss may occur prior to mating yielding transient haploids that mate and return to a diploid state (26). Meiosis has not been observed in either case, and the homologous chromosomes remain mostly intact making the haplotypes much more stable than in other organisms . Thus, in order to understand and combat candida pathogenesis, it is important to study candida biology directly, and having access to the phased genome assembly opens up multiple new research opportunities . Since its inception in 2004, cgd has been tasked with maintenance of the primary genome sequence and annotation for the c. albicans reference strain sc5314 . Cgd was originally based upon assembly 19, a diploid, contig - level assembly built from the genomic sequence that had just been completed (27), and which included gene models, sequence corrections, and functional annotations provided by a group of volunteer researchers known as the annotation working group (28). Cgd then took over the task of curating all the candida - related information based on published experimental data, comparative genomics predictions and improved sequence information (29). In 2006, assembly 20 was incorporated into cgd (30); assembly 20 was the first chromosome level assembly for c. albicans, though it consisted of what are referred to as reftigs, wherein each of the chromosomes in the assembly consist of a mosaic of the two haplotypes (31). Assembly 20 was superseded by assembly 21, because a sequence from a different strain, wo-1, had been inadvertently incorporated into assembly 20 . A major update to assembly 21, based on additional sequence data and comparative analyses (32) was subsequently also incorporated into cgd . The most recent major update to the sequence is assembly 22 (33), which was generated using illumina - based sequencing of sc5314 as well as a collection of congenic strains homozygous for specific chromosomes, which allowed the construction of a phased diploid assembly of the genome . Incorporation of assembly 22 into cgd entailed careful reconciliation of sequence ambiguities, potential assembly errors and differences from previous assemblies . We have established a pipeline that takes advantage of all available data, including the illumina - based sequences from muzzey et al . (33), roche 454 sequences generated at cgd, as well as independently published, gene - specific sequence data derived from our curation of the candida literature . We have used this pipeline to correct the sequences for more than 300 features, eliminating many reading frame errors and resolving multiple sequence ambiguities (table 1). We have also re - analyzed the haplotype assignments for the entire chromosome 3, which led to an exchange of 845 kb between chromosome 3a and chromosome 3b . Improving the reference sequence is an ongoing project and we are currently focusing our efforts on intergenic regions, repetitive sequences and segmental duplications . Since 2010, cgd has used a versioning system to track genome sequence releases, and their associated annotations . The version designation appears in the name of each of the relevant sequence and feature annotation files that are available at cgd, so the exact source of the sequence data is always clear . Version designations appear in the following format: sxx - myy - rzz where xx, yy, and zz are zero - padded integers . Xx is incremented when there is any change to the underlying genomic (i.e., chromosome) sequence . Yy is incremented when there is any change to the coordinates of any feature annotated in the genome (e.g. Any change in location or boundary, or addition or removal of a feature from the annotation). Yy is reset to 01 when xx is incremented (when a sequence change is made). Zz is incremented in response to curatorial changes that affect information that appears in the gff file, specifically gene names, gene aliases, gene ids, gene descriptions, feature types (e.g. Gene or pseudogene), and orf classifications or qualifiers (e.g. Verified, uncharacterized, deleted, merged). Files are checked on a weekly basis, as well as any time that a gff file is regenerated manually, to determine if changes have occurred that warrant a change in the zz number . Zz is reset to 01 when either xx or yy is incremented (when a sequence change is made, or when the coordinates of any feature are updated). All versions are archived on the cgd download site . As a hypothetical example, say that we start with s05-m01-r01 as the current version . When the next weekly file check is performed, and the new file is noted to contain curatorial updates to gene names in the database, but no new changes to the structural annotation or to the sequence itself, the new version designation becomes s05-m01-r02 . Subsequently, the chromosomal coordinates of a gene are changed, based on curation of a paper that provides evidence for updating the gene model . Later, a change to correct a sequencing error is made, and the new version designation becomes s06-m01-r01 . Cgd recommends that authors note in their materials and methods sections of published papers what version of the genome they were working with when they performed an analysis, so that the analysis is reproducible . With assembly 22, it became possible to implement a new position based systematic nomenclature for chromosomal features . The previous systematic names, dating back to assembly 19, consisted of the orf19 prefix followed by a unique but somewhat arbitrary number . The new systematic name is based on the known chromosomal location and haplotype, and it consists of the chromosome (c1-c7 and cr for the eight nuclear chromosomes, cm for the mitochondrial chromosome), a unique number indicating the order of features along chromosomes, the strand (w for watson or c for crick) and the haplotype (a or b). For example, c4_03570w_a denotes a feature located on chromosome 4, watson strand and haplotype a. feature numbers start at the left end of the chromosome and increase by 10 to allow for adding new features in the intervening spaces as they are discovered . Since systematic identifiers from all the previous assemblies orf19 names, continue to be used by researchers, cgd includes all the previous identifiers as searchable aliases to allow seamless and unambiguous transition between various nomenclature systems . Locus summary pages (lsp) exist for every chromosomal feature in cgd and provide a primary way to access gene - specific information . In addition to locus - specific data, such as description, functional annotations (gene ontology terms, mutant phenotypes), or orthologous genes in other species, lsps now also provide access to allele - specific data: dna and protein sequences for both alleles, as well as a listing of allelic variations if they exist . Lsps also show graphical representations of the chromosomal context for each allele as thumbnails that lead to genome browser windows and allow farther exploration of a chosen haplotype . Assembly 22 allows powerful, allele- and haplotype - specific analyses of the overall genome structure, function and evolution . To facilitate genome - wide research, cgd provides access to all the data in downloadable files that can be used by researchers' own bioinformatics tools . All dna sequences for the entire chromosomes, coding and non - coding regions, as well as translation products, are available for download at http://www.candidagenome.org / download/. An important part of cgd's mission is to incorporate and annotate large - scale datasets from published experiments on candida species, and to make them easily available for exploration and analysis by our users . For several years we have provided genexplorer (34) to display and analyze microarray datasets . Recently, to provide access to the growing number of datasets from experiments that use high - throughput sequencing technologies, we have deployed the jbrowse genome browser (35). Jbrowse is a mature and widely used application that is fast, intuitive, and compatible with most web browsers (36). Jbrowse allows users to quickly view large - scale sequence data in a genomic context, at multiple zoom - levels of resolution, from base pairs in individual sequence reads to read - density summaries across large genomic regions . The display includes parallel tracks of annotated sequence features, allowing seamless navigation between jbrowse and the locus summary pages for each feature (figure 1). Quantitative tracks graphically display comparative information, such as relative expression level or sequence conservation . Jbrowse is highly flexible and customizable: users may easily load their own sequence datasets and analysis tracks, for display in the context of genomic features, or for comparison with datasets and tracks provided by cgd . Jbrowse display of the region around the c. albicans serum - inducible gene hwp1, showing aligned rna - seq reads from serum - treated cells (37). The red and blue bars in the top track of the main display window show genes annotated at cgd: red for genes encoded on the w strand (+), blue for genes on the c strand (). Clicking on a bar brings up an information window for that gene, and includes a link to its cgd locus summary page . The green bar graph below the gene track shows the density of aligned rna - seq reads along to the chromosome, plotted on a log scale . The bottom track shows all the aligned rna - seq reads along the chromosome: each short bar in the bottom track represents a unique read . In this example the sequence reads are strand - specific: pink bars indicate reads transcribed from the w strand, and light - blue bars indicate reads transcribed from the c strand . Clicking on a bar brings up information about the read, including the sequence and quality score for each base . Menus and controls at the top of the browser provide navigation, zoom and search functionalities, and allow users to load their own data . We provide the high - throughput dna sequence data (33) that was the basis of assembly 22, described above . Optional tracks highlight the sequence variation between the two assembly 22 haplotypes, as well as the variation between the common strains sc5314 and wo-1 (32). We also make available rna - seq datasets from a number of gene expression studies in c. albicans, including comparisons of different stress conditions (37), hyphae - inducing conditions (37), biofilm vs. planktonic growth (38), white - opaque switching (39), and allele - specific expression differences (40). In addition, we provide chromatin occupancy data (chip - seq) for the wor1p transcription factor during white - opaque switching (41). We also have rna - seq datasets for two other candida species: gene expression under ph and nitrosative stress in c. glabrata (42), and during biofilm vs. planktonic growth in c. parapsilosis (43). We will add new datasets to jbrowse as they become available, and in response to user requests . The reference sequence for an organism is not static while sequencing technologies continue to advance (both in accuracy and read lengths), there is always the possibility that updates will improve the reference sequence . For example, either pacbio or oxford nanopore sequencing (very long reads, though with high error rates), coupled with existing illumina data (short reads, but low error rates) may better resolve telomeric and other repeat sequences, which are hard to resolve even with the original sanger reads . Another question is whether the sequence of a single instance of a strain is a reasonable representation of the reference sequence indeed, given candida albicans propensity to undergo rearrangement and loss of heterozygosity under stress, different lab isolates of ostensibly the same strain might have different sequences . It is likely in the near future that many distinct strains of candida species will be sequenced, and cgd will endeavor to incorporate these into the database as well . They will provide insight into the genomic variation that exists in each of the candida species . Additional high throughput sequencing datasets may also allow refinement of the genome annotations, such as novel additional transcripts, or the addition of 5 and 3 utrs to each of the genes . They may also allow the annotation of functional elements within non - transcribed regions, such as transcription factor binding sites from chip - seq studies . Cgd will strive to incorporate such refinements as soon as they become available to maintain the reference sequence and annotation current . Since its inception in 2004, cgd has been tasked with maintenance of the primary genome sequence and annotation for the c. albicans reference strain sc5314 . Cgd was originally based upon assembly 19, a diploid, contig - level assembly built from the genomic sequence that had just been completed (27), and which included gene models, sequence corrections, and functional annotations provided by a group of volunteer researchers known as the annotation working group (28). Cgd then took over the task of curating all the candida - related information based on published experimental data, comparative genomics predictions and improved sequence information (29). In 2006, assembly 20 was incorporated into cgd (30); assembly 20 was the first chromosome level assembly for c. albicans, though it consisted of what are referred to as reftigs, wherein each of the chromosomes in the assembly consist of a mosaic of the two haplotypes (31). Assembly 20 was superseded by assembly 21, because a sequence from a different strain, wo-1, had been inadvertently incorporated into assembly 20 . A major update to assembly 21, based on additional sequence data and comparative analyses (32) the most recent major update to the sequence is assembly 22 (33), which was generated using illumina - based sequencing of sc5314 as well as a collection of congenic strains homozygous for specific chromosomes, which allowed the construction of a phased diploid assembly of the genome . Incorporation of assembly 22 into cgd entailed careful reconciliation of sequence ambiguities, potential assembly errors and differences from previous assemblies . We have established a pipeline that takes advantage of all available data, including the illumina - based sequences from muzzey et al . (33), roche 454 sequences generated at cgd, as well as independently published, gene - specific sequence data derived from our curation of the candida literature . We have used this pipeline to correct the sequences for more than 300 features, eliminating many reading frame errors and resolving multiple sequence ambiguities (table 1). We have also re - analyzed the haplotype assignments for the entire chromosome 3, which led to an exchange of 845 kb between chromosome 3a and chromosome 3b . Improving the reference sequence is an ongoing project and we are currently focusing our efforts on intergenic regions, repetitive sequences and segmental duplications . Since 2010, cgd has used a versioning system to track genome sequence releases, and their associated annotations . The version designation appears in the name of each of the relevant sequence and feature annotation files that are available at cgd, so the exact source of the sequence data is always clear . Version designations appear in the following format: sxx - myy - rzz where xx, yy, and zz are zero - padded integers . Xx is incremented when there is any change to the underlying genomic (i.e., chromosome) sequence . Yy is incremented when there is any change to the coordinates of any feature annotated in the genome (e.g. Any change in location or boundary, or addition or removal of a feature from the annotation). Yy is reset to 01 when xx is incremented (when a sequence change is made). Zz is incremented in response to curatorial changes that affect information that appears in the gff file, specifically gene names, gene aliases, gene ids, gene descriptions, feature types (e.g. Gene or pseudogene), and orf classifications or qualifiers (e.g. Verified, uncharacterized, deleted, merged). Files are checked on a weekly basis, as well as any time that a gff file is regenerated manually, to determine if changes have occurred that warrant a change in the zz number . Zz is reset to 01 when either xx or yy is incremented (when a sequence change is made, or when the coordinates of any feature are updated). All versions are archived on the cgd download site . As a hypothetical example, say that we start with s05-m01-r01 as the current version . When the next weekly file check is performed, and the new file is noted to contain curatorial updates to gene names in the database, but no new changes to the structural annotation or to the sequence itself, the new version designation becomes s05-m01-r02 . Subsequently, the chromosomal coordinates of a gene are changed, based on curation of a paper that provides evidence for updating the gene model . Later, a change to correct a sequencing error is made, and the new version designation becomes s06-m01-r01 . Cgd recommends that authors note in their materials and methods sections of published papers what version of the genome they were working with when they performed an analysis, so that the analysis is reproducible . With assembly 22, it became possible to implement a new position based systematic nomenclature for chromosomal features . The previous systematic names, dating back to assembly 19, consisted of the orf19 prefix followed by a unique but somewhat arbitrary number . The new systematic name is based on the known chromosomal location and haplotype, and it consists of the chromosome (c1-c7 and cr for the eight nuclear chromosomes, cm for the mitochondrial chromosome), a unique number indicating the order of features along chromosomes, the strand (w for watson or c for crick) and the haplotype (a or b). For example, c4_03570w_a denotes a feature located on chromosome 4, watson strand and haplotype a. feature numbers start at the left end of the chromosome and increase by 10 to allow for adding new features in the intervening spaces as they are discovered . Since systematic identifiers from all the previous assemblies orf19 names, continue to be used by researchers, cgd includes all the previous identifiers as searchable aliases to allow seamless and unambiguous transition between various nomenclature systems . Assembly 19/21 identifiers, locus summary pages (lsp) exist for every chromosomal feature in cgd and provide a primary way to access gene - specific information . In addition to locus - specific data, such as description, functional annotations (gene ontology terms, mutant phenotypes), or orthologous genes in other species, lsps now also provide access to allele - specific data: dna and protein sequences for both alleles, as well as a listing of allelic variations if they exist . Lsps also show graphical representations of the chromosomal context for each allele as thumbnails that lead to genome browser windows and allow farther exploration of a chosen haplotype . Assembly 22 allows powerful, allele- and haplotype - specific analyses of the overall genome structure, function and evolution . To facilitate genome - wide research, cgd provides access to all the data in downloadable files that can be used by researchers' own bioinformatics tools . All dna sequences for the entire chromosomes, coding and non - coding regions, as well as translation products, are available for download at http://www.candidagenome.org / download/. An important part of cgd's mission is to incorporate and annotate large - scale datasets from published experiments on candida species, and to make them easily available for exploration and analysis by our users . For several years recently, to provide access to the growing number of datasets from experiments that use high - throughput sequencing technologies, we have deployed the jbrowse genome browser (35). Jbrowse is a mature and widely used application that is fast, intuitive, and compatible with most web browsers (36). Jbrowse allows users to quickly view large - scale sequence data in a genomic context, at multiple zoom - levels of resolution, from base pairs in individual sequence reads to read - density summaries across large genomic regions . The display includes parallel tracks of annotated sequence features, allowing seamless navigation between jbrowse and the locus summary pages for each feature (figure 1). Quantitative tracks graphically display comparative information, such as relative expression level or sequence conservation . Jbrowse is highly flexible and customizable: users may easily load their own sequence datasets and analysis tracks, for display in the context of genomic features, or for comparison with datasets and tracks provided by cgd . Jbrowse display of the region around the c. albicans serum - inducible gene hwp1, showing aligned rna - seq reads from serum - treated cells (37). The red and blue bars in the top track of the main display window show genes annotated at cgd: red for genes encoded on the w strand (+), blue for genes on the clicking on a bar brings up an information window for that gene, and includes a link to its cgd locus summary page . The green bar graph below the gene track shows the density of aligned rna - seq reads along to the chromosome, plotted on a log scale . The bottom track shows all the aligned rna - seq reads along the chromosome: each short bar in the bottom track represents a unique read . In this example the sequence reads are strand - specific: pink bars indicate reads transcribed from the w strand, and light - blue bars indicate reads transcribed from the c strand . Clicking on a bar brings up information about the read, including the sequence and quality score for each base . Menus and controls at the top of the browser provide navigation, zoom and search functionalities, and allow users to load their own data . We provide the high - throughput dna sequence data (33) that was the basis of assembly 22, described above . Optional tracks highlight the sequence variation between the two assembly 22 haplotypes, as well as the variation between the common strains sc5314 and wo-1 (32). We also make available rna - seq datasets from a number of gene expression studies in c. albicans, including comparisons of different stress conditions (37), hyphae - inducing conditions (37), biofilm vs. planktonic growth (38), white - opaque switching (39), and allele - specific expression differences (40). In addition, we provide chromatin occupancy data (chip - seq) for the wor1p transcription factor during white - opaque switching (41). We also have rna - seq datasets for two other candida species: gene expression under ph and nitrosative stress in c. glabrata (42), and during biofilm vs. planktonic growth in c. parapsilosis (43). We will add new datasets to jbrowse as they become available, and in response to user requests . The reference sequence for an organism is not static while sequencing technologies continue to advance (both in accuracy and read lengths), there is always the possibility that updates will improve the reference sequence . For example, either pacbio or oxford nanopore sequencing (very long reads, though with high error rates), coupled with existing illumina data (short reads, but low error rates) may better resolve telomeric and other repeat sequences, which are hard to resolve even with the original sanger reads . Another question is whether the sequence of a single instance of a strain is a reasonable representation of the reference sequence indeed, given candida albicans propensity to undergo rearrangement and loss of heterozygosity under stress, different lab isolates of ostensibly the same strain might have different sequences . It is likely in the near future that many distinct strains of candida species will be sequenced, and cgd will endeavor to incorporate these into the database as well . They will provide insight into the genomic variation that exists in each of the candida species . Additional high throughput sequencing datasets may also allow refinement of the genome annotations, such as novel additional transcripts, or the addition of 5 and 3 utrs to each of the genes . They may also allow the annotation of functional elements within non - transcribed regions, such as transcription factor binding sites from chip - seq studies . Cgd will strive to incorporate such refinements as soon as they become available to maintain the reference sequence and annotation current . National institute of dental and craniofacial research at the us national institutes of health [r01 de015873]. Funding for open access charge: nih . Conflict of interest statement.
All study subjects were from the ongoing, prospective, population - based korean genome and epidemiology study (koges) cohort . The original study was designed to establish a representative adult cohort in an urban area, the city of ansan, and to identify the epidemiologic characteristics, frequency, and determinants of chronic diseases in koreans . Initially, 5,015 participants (2,521 men and 2,494 women 4069 years old) who participated in a comprehensive health examination and onsite interviews at korea university ansan hospital formed a longitudinal cohort from june 2001 to january 2003 ., subjects signed an informed consent form, which was approved by the human subjects review committee at the korea university ansan hospital . The fifth biennial examination was conducted from march 2009 to february 2011 and the sixth examination from march 2011 to february 2013 . Polysomnography (psg) was included randomly in the study protocol in september 2009 in about one - half of the koges participants . Although psg will be administered to the entire study population during the 4-year period, the present study includes only the subset of the sample with psg data acquired between september 2009 and november 2011 . After excluding the subjects who had missing data and extreme outliers of glucose concentrations, 1,344 subjects (706 men and 638 women) further details from the protocol and design of the koges are described elsewhere (11). All participants responded to an interviewer - administered questionnaire and underwent a comprehensive physical examination . Lifestyle characteristics were smoking status and alcohol consumption categorized as never, former, and current . Level of exercise was categorized as never, lightly (<3 times / week, 30 min / session), or regularly (3 times / week, 30 min / session) during the previous month . The presence of chronic illnesses, including diabetes, hypertension, dyslipidemia, and cardiovascular disease (cvd), was noted as were prescribed medications . Subjects with documented events or medical records of myocardial infarction, angina, heart failure, stroke, or peripheral artery disease were considered to have cvd . Diabetes was defined by american diabetes association criteria, using a 75-g oral glucose tolerance test (fasting plasma glucose [fpg] 7.0 mmol / l), and medical history (12). For subjects without diabetes, glucose tolerance status was assessed by american diabetes association criteria (12) as follows: ifg only (5.6 fpg mmol / l), and ifg + igt (5.6 fpg <7.0 mmol / l and 7.8 2hpg <11.1 hypertension was defined as systolic blood pressure 140 mmhg, diastolic blood pressure 90 mmhg, or medical history (13). Height and body weight were measured to the nearest 0.1 cm and 0.1 kg, respectively . Wc was measured at the midpoint between the lower rib margin and the iliac crest in the standing position . Obesity was defined as bmi 25 kg / m according to the asian - specific bmi cutoffs from the world health organization report (14). Plasma glucose, serum triglycerides, hdl cholesterol, and ldl cholesterol levels were measured with an autoanalyzer (advia 1650; siemens, tarrytown, ny). Insulin was measured with an immunoradiometric assay kit (ins - irma kit; biosource, nivelles, belgium) using a packard counter system . Insulin resistance was estimated with the homeostasis model of assessment for insulin resistance (homa - ir) and calculated as fasting glucose (mmol / l) fasting insulin (u / ml) / 22.5 . Homa -cell function (homa-) (%) was calculated as 20 fasting insulin (u / ml) / fasting glucose (mmol / l) 3.5 (15). For subjects who participated in the fifth biennial examination (n = 820), single - slice computed tomography (ct) scanning (brilliance 64; philips, cleveland, oh) the scans were conducted at 120 kv with a slice thickness of 5 mm at the level of the l4l5 vertebral interspace . The total area of intra - abdominal fat was delineated by manual tracing within the muscle wall, and the visceral fat area (vfa) was defined as an area with an attenuation range between 190 and 30 hounsfield units . Overnight sleep study was performed at home with a portable device (embletta x100; embla systems, san carlos, ca). Two trained sleep technologists visited each subject s home in the evening, applied sensors, and instructed the subject on how to start and stop the recording . Subjects were required to record the lights - off and -on times and to report them the next morning . Recording channels were one for electroencephalography (c4a1), one for electrooculography (right upper outer canthus to left lower outer canthus), one for chin electromyography, one for modified lead ii electrocardiography, one for airflow from nasal airflow pressure transducer, two for respiratory effort from chest and abdominal respiratory inductance plethysmography, one pulse oximeter, and one position sensor . Data were scored by two well - trained technicians who had 5 years of experience with psg monitoring and scoring according to standard guidelines (16,17). Internal consistency for scoring the apnea - hypopnea index (ahi) was high (cronbach = 0.996 and 1.00 for each rater), and interrater reliability was strong (cronbach = 0.998). Although we did not perform a validity study to compare psg recordings obtained in the home and laboratory settings, the sleep heart health study clearly demonstrated that the median respiratory disturbance index was similar in the unattended home and attended laboratory settings, with differences of a small magnitude in some sleep parameters (18). Obstructive apnea was defined when airflow dropped by 90% of the baseline with ongoing chest and abdominal movement, and hypopnea was defined as a reduction in airflow by 30% associated with at least a 4% oxygen desaturation . The duration threshold for the respiratory events was 10 s. ahi was calculated, and osa was defined as an ahi 5 (1,16,19). Daytime sleepiness was assessed with the epworth sleepiness scale (ess) (20), a well - validated and frequently used subjective eight - item, self - administered questionnaire . Subjects were asked to score the likelihood of falling asleep in eight different situations with different levels of stimulation . The higher the ess score, the greater propensity for sleepiness implied . In the current study, eds was defined as ess scores> 10 (20). Subject characteristics at baseline were compared among groups stratified by the presence or absence of osa and obesity by student t test for continuous variables and test for categorical variables . Nonnormally distributed variables, such as hba1c, homa - ir, homa-, ahi, and triglyceride level, are presented as the median and interquartile range for each group, and the differences were tested after logarithmic transformation . Variables associated with glucose metabolism were compared by ancova after adjusting for age, sex, and bmi among the groups stratified by osa and obesity . For ancova, we confirmed the homogeneity of the slope between the covariates and the dependent variables in subjects with or without osa . The interaction term for osa and obesity on homa- was calculated to determine whether the association between osa and homa- was modified by obesity status . To exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism, the same analyses were repeated after excluding subjects who were taking these medications . To evaluate the impact of osa on ifg only, igt only, ifg + igt, and diabetes according to the presence or absence of obesity, the following five models were fit for each outcome: model 1 was adjusted for age and sex; model 2 was adjusted for age, sex, alcohol consumption, smoking status, exercise, presence of hypertension or cvd, and medication for dyslipidemia; model 3 was the same as model 2 and adjusted for bmi; model 4 was the same as model 2 and adjusted for wc; and model 5 was the same as model 2 and adjusted for vfa in subjects who underwent abdominal ct scan . To exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism odds ratios (ors) for ifg only, igt only, ifg + igt, and diabetes were calculated according to the tertile of ahi to demonstrate the dose - response relationship . In addition, to examine the additive effect of eds and osa on glucose metabolism, ors for ifg only, igt only, ifg + igt, and diabetes were evaluated in subjects with osa according to the presence or absence of eds compared with those without osa . For logistic regression these models showed no evidence of lack of fit according to the hosmer - lemeshow statistic . All statistical analyses were performed with sas version 9.1 for windows software (sas institute inc ., cary, nc). All participants responded to an interviewer - administered questionnaire and underwent a comprehensive physical examination . Lifestyle characteristics were smoking status and alcohol consumption categorized as never, former, and current . Level of exercise was categorized as never, lightly (<3 times / week, 30 min / session), or regularly (3 times / week, 30 min / session) during the previous month . The presence of chronic illnesses, including diabetes, hypertension, dyslipidemia, and cardiovascular disease (cvd), was noted as were prescribed medications . Subjects with documented events or medical records of myocardial infarction, angina, heart failure, stroke, or peripheral artery disease were considered to have cvd . Diabetes was defined by american diabetes association criteria, using a 75-g oral glucose tolerance test (fasting plasma glucose [fpg] 7.0 mmol / l or 2-h plasma glucose [2hpg] 11.1 mmol / l), and medical history (12). For subjects without diabetes, glucose tolerance status was assessed by american diabetes association criteria (12) as follows: ifg only (5.6 fpg mmol / l), igt only (fpg <5.6 mmol / l and 7.8 2hpg <11.1 mmol / l), and ifg + igt (5.6 fpg <7.0 mmol / l and 7.8 2hpg <11.1 hypertension was defined as systolic blood pressure 140 mmhg, diastolic blood pressure 90 mmhg, or medical history (13). Height and body weight were measured to the nearest 0.1 cm and 0.1 kg, respectively . Wc was measured at the midpoint between the lower rib margin and the iliac crest in the standing position . Obesity was defined as bmi 25 kg / m according to the asian - specific bmi cutoffs from the world health organization report (14). Plasma glucose, serum triglycerides, hdl cholesterol, and ldl cholesterol levels were measured with an autoanalyzer (advia 1650; siemens, tarrytown, ny). Insulin was measured with an immunoradiometric assay kit (ins - irma kit; biosource, nivelles, belgium) using a packard counter system . Insulin resistance was estimated with the homeostasis model of assessment for insulin resistance (homa - ir) and calculated as fasting glucose (mmol / l) fasting insulin (u / ml) / 22.5 . Homa -cell function (homa-) (%) was calculated as 20 fasting insulin (u / ml) / fasting glucose (mmol / l) 3.5 (15). For subjects who participated in the fifth biennial examination (n = 820), single - slice computed tomography (ct) scanning (brilliance 64; philips, cleveland, oh) was used to quantify intra - abdominal adipose tissue . The scans were conducted at 120 kv with a slice thickness of 5 mm at the level of the l4l5 vertebral interspace . The total area of intra - abdominal fat was delineated by manual tracing within the muscle wall, and the visceral fat area (vfa) was defined as an area with an attenuation range between 190 and 30 hounsfield units . Overnight sleep study was performed at home with a portable device (embletta x100; embla systems, san carlos, ca). Two trained sleep technologists visited each subject s home in the evening, applied sensors, and instructed the subject on how to start and stop the recording . Subjects were required to record the lights - off and -on times and to report them the next morning . Recording channels were one for electroencephalography (c4a1), one for electrooculography (right upper outer canthus to left lower outer canthus), one for chin electromyography, one for modified lead ii electrocardiography, one for airflow from nasal airflow pressure transducer, two for respiratory effort from chest and abdominal respiratory inductance plethysmography, one pulse oximeter, and one position sensor . Data were scored by two well - trained technicians who had 5 years of experience with psg monitoring and scoring according to standard guidelines (16,17). Internal consistency for scoring the apnea - hypopnea index (ahi) was high (cronbach = 0.996 and 1.00 for each rater), and interrater reliability was strong (cronbach = 0.998). Although we did not perform a validity study to compare psg recordings obtained in the home and laboratory settings, the sleep heart health study clearly demonstrated that the median respiratory disturbance index was similar in the unattended home and attended laboratory settings, with differences of a small magnitude in some sleep parameters (18). Obstructive apnea was defined when airflow dropped by 90% of the baseline with ongoing chest and abdominal movement, and hypopnea was defined as a reduction in airflow by 30% associated with at least a 4% oxygen desaturation . The duration threshold for the respiratory events was 10 s. ahi was calculated, and osa was defined as an ahi 5 (1,16,19). Daytime sleepiness was assessed with the epworth sleepiness scale (ess) (20), a well - validated and frequently used subjective eight - item, self - administered questionnaire . Subjects were asked to score the likelihood of falling asleep in eight different situations with different levels of stimulation . The higher the ess score, the greater propensity for sleepiness implied . In the current study subject characteristics at baseline were compared among groups stratified by the presence or absence of osa and obesity by student t test for continuous variables and test for categorical variables . Nonnormally distributed variables, such as hba1c, homa - ir, homa-, ahi, and triglyceride level, are presented as the median and interquartile range for each group, and the differences were tested after logarithmic transformation . Variables associated with glucose metabolism were compared by ancova after adjusting for age, sex, and bmi among the groups stratified by osa and obesity . For ancova, we confirmed the homogeneity of the slope between the covariates and the dependent variables in subjects with or without osa . The interaction term for osa and obesity on homa- was calculated to determine whether the association between osa and homa- was modified by obesity status . To exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism, the same analyses were repeated after excluding subjects who were taking these medications . To evaluate the impact of osa on ifg only, igt only, ifg + igt, and diabetes according to the presence or absence of obesity, multivariate logistic regression analyses were conducted . In the analysis, the following five models were fit for each outcome: model 1 was adjusted for age and sex; model 2 was adjusted for age, sex, alcohol consumption, smoking status, exercise, presence of hypertension or cvd, and medication for dyslipidemia; model 3 was the same as model 2 and adjusted for bmi; model 4 was the same as model 2 and adjusted for wc; and model 5 was the same as model 2 and adjusted for vfa in subjects who underwent abdominal ct scan . To exclude the potential confounding effect of medication for diabetes or dyslipidemia on glucose metabolism odds ratios (ors) for ifg only, igt only, ifg + igt, and diabetes were calculated according to the tertile of ahi to demonstrate the dose - response relationship . In addition, to examine the additive effect of eds and osa on glucose metabolism, ors for ifg only, igt only, ifg + igt, and diabetes were evaluated in subjects with osa according to the presence or absence of eds compared with those without osa . For logistic regression, we calculated adjusted r and hosmer - lemeshow statistics to assess model adequacy . These models showed no evidence of lack of fit according to the hosmer - lemeshow statistic . All statistical analyses were performed with sas version 9.1 for windows software (sas institute inc ., cary, nc). Table 1 shows the characteristics of the participants stratified by the presence or absence of osa and obesity . About 36.6% of nonobese and 58.2% of obese subjects had osa . However, the severity of osa was mostly mild and moderate (27, 8.8, and 0.8% of nonobese and 38.4, 14.6, and 5.2% obese subjects had mild, moderate, and severe osa, respectively). Among 396 subjects with diabetes, 351 had a history of diabetes of whom 142 were treated with oral hypoglycemic agents (n = 140) or insulin (n = 4). Subjects with osa were older and had a higher 2hpg, hba1c, bmi, wc, vfa, and homa - ir than those without osa, regardless of obesity . In the nonobese group, subjects with osa had higher fpg and triglyceride levels and lower homa- and hdl cholesterol levels than those without osa . The proportion of ifg + igt and diabetes was higher according to osa status in nonobese subjects, whereas only the prevalence of diabetes was higher in obese subjects with osa . Characteristics of subjects according to the presence or absence of obesity and osa table 2 presents the age-, sex-, and bmi - adjusted values for glucose metabolism according to osa and obesity status . In the nonobese group, fpg, 2hpg, and hba1c levels were significantly higher, and homa - ir was modestly higher in those with osa than in those without osa . However, in the obese group, only insulin and homa - ir levels were associated with osa . Although not statistically significant, homa- was lower in nonobese subjects with osa than in those without osa but higher in obese subjects with osa than in those without osa . Therefore, a significant interaction between osa and obesity was observed for homa- (p = 0.025). After excluding subjects who were taking medication for diabetes or dyslipidemia, in the nonobese group, fpg and 2hpg were still higher in those with osa than in those without . On the contrary, in the obese group, although glucose concentrations were not different, insulin, homa - ir, homa- were higher in those with osa than in those without (supplementary table 1). Variables associated with glucose metabolism according to obesity and osa status after adjusting for age, sex, and bmi multivariate logistic regression analyses were conducted to investigate the effects of osa on glucose metabolism according to obesity status (table 3). In subjects without obesity, the presence of osa showed higher ors for ifg + igt and diabetes, even after adjusting for several confounding variables, including bmi or wc . Osa was significantly associated with diabetes even after adjusting for vfa in nonobese subjects . In an analysis to show the dose - response relationship, nonobese subjects with the highest ahi tertile had the highest ors for ifg + igt or diabetes (supplementary table 2). In contrast, in those with obesity, none of the abnormal glucose categories was associated with osa . These findings were consistent even after excluding subjects who were taking medication for diabetes or dyslipidemia (supplementary table 3). Association between osa and abnormal glucose metabolism according to obesity status supplementary table 4 shows the joint effect of osa and eds on glucose tolerance categories . In the nonobese group, compared with subjects without osa, nonsleepy subjects with osa had higher ors for diabetes, but sleepy subjects with osa had the highest ors for diabetes (hazard ratio 5.78 [95% ci 2.0516.3]). In this large community - based cohort study, we demonstrated the differential association between osa and impaired glucose metabolism according to the presence of obesity . Of note, osa was significantly associated with abnormal glucose metabolism in nonobese subjects, even after adjusting for several confounding variables, including generalized or visceral adiposity . To our knowledge, this study is the first to show that the impact of osa on glucose metabolism is more evident in the nonobese than in the obese population . We also clearly demonstrated that in the nonobese group, fpg, 2hpg, and hba1c were significantly higher and that homa - ir was modestly higher in subjects with osa than in those without . The only previous study to analyze the effect of osa according to obesity status was the sleep heart health study, where the association between osa and abnormal glucose metabolism was not different between the nonoverweight and overweight / obese groups (3). We can hypothesize that osa may have a lesser impact on glucose metabolism in obese than in nonobese individuals because of preexisting derangements in those who are obese . In accordance with this theory, the improvement of insulin sensitivity in patients with osa receiving cpap therapy was minimal in those with a bmi of> 30 kg / m, whereas it was more prominent in those with less obesity (5). (21) demonstrated that the acute intermittent hypoxia caused insulin resistance and increased secretion of leptin and tumor necrosis factor- in lean mice . However, obesity was associated with striking increases in leptin and tumor necrosis factor- levels, which overwhelmed the effects of hypoxia . On the contrary, drager et al . (22) showed that the effect of chronic intermittent hypoxia on glucose intolerance, inflammation, and oxidative stress was prominent in obese mice but not in lean mice . The causes of these contradictory findings are not clear; however, the metabolic and proinflammatory responses to acute and chronic intermittent hypoxia may be different . An alternative hypothesis may be a presence or absence of eds in subjects with osa . Eds is reportedly associated with hyperglycemia and insulin resistance in subjects with osa (23,24). Intermittent hypoxemia has been shown to result in disturbed sleep architecture or to damage neuronal structures that promote wakefulness in animal models (25). Indeed, higher degrees of hypoxemia predict both sleepiness and insulin resistance (1,26). According to these studies, we can expect that the effect of hypoxemia on glucose metabolism may be more evident in patients with osa and eds . In the current study, we demonstrated that the joint effect of osa and eds on the or for diabetes was highly significant only in nonobese subjects . Therefore, the differential impact of eds on glucose metabolism according to obesity status may be responsible for the discrepant effect of osa in subjects with or without obesity in the current study . The association between homa - ir and osa was similar in subjects with and without obesity, as seen in previous research (1,3,27). However, few studies have evaluated insulin secretion according to osa status . Recently, in healthy young men without diabetes and obesity (most of them caucasian), subjects with osa had increased insulin resistance and compensatory hyperinsulinemia (10). In the current study, however, homa- was lower in nonobese subjects with osa than in those without osa but was higher in obese subjects . This finding was more evident in obese subjects after excluding those taking medication for diabetes or dyslipidemia and suggests that inadequate insulin secretion against the increased insulin resistance may be responsible for the abnormal glucose metabolism in nonobese individuals, which is characteristic for diabetes in asians (28,29). On the contrary, increased insulin secretion in response to insulin resistance in obese individuals with osa may explain why glucose metabolism is less impaired by osa . However, the hyperinsulinemic - euglycemic clamp technique should be used to further explore the mechanisms of the impairment of glucose metabolism by osa . Therefore, the prevalence of metabolically obese normal weight (monw) individuals who have not only a normal bmi, but also a cluster of obesity - related risk factors for diabetes and cvd is reportedly higher in asians (30,31). Higher levels of inflammatory adipokines and atherogenic ldl profiles in monw individuals (32,33) may mediate the increase in cvd and mortality in this population (34). The finding that a worse cardiometabolic profile was more prominent in nonobese subjects with osa in the current study supports the notion that these individuals may have similar characteristics as those with monw . Although the relationship between osa and abnormal glucose metabolism has been demonstrated in several previous studies (1,2,35), a few have evaluated the association between osa and distinct prediabetic groups (ifg and/or igt), where sleep - disordered breathing was associated with occult diabetes, ifg only, ifg + igt (3), or glucose intolerance (1,35) in population and clinic - based studies . Subjects with ifg had a greater impairment of early phase insulin secretion and increased endogenous glucose output, whereas igt was associated with peripheral insulin resistance (36). In the current study, however, osa was associated with neither ifg only nor igt only . Because more severe metabolic abnormalities are present in individuals with ifg + igt, diabetes develops more rapidly and unfavorable cardiovascular risk factors and mortality are increased compared with individuals with ifg only or igt only (3638). From this point of view, the significant association between osa and ifg + igt in the current study suggests that subjects with osa are at a greater risk for developing diabetes and cvd and have a higher risk of mortality . Compared with previous studies, which had several methodological limitations in terms of the study population (39,40) or lacked rigorous control for numerous confounders, especially obesity, the current study used a large community - based sample and differentiated the effect of osa according to the presence of obesity in addition to adjustment for generalized or visceral obesity . Another strength of the current study was the evaluation of osa status during sleep at home, which provides a more realistic estimation of osa severity than hospital - based studies because of the maintenance of regular daily habits of sleep, physical activity, and diet in the general population . The major limitation of the current study was its cross - sectional design, which makes it difficult to determine whether there is a causal relationship between osa and impaired glucose metabolism . Secondly, because we did not have a sufficient number of subjects with severe osa (ahi 30), it is unclear whether the present findings would be consistent in those with severe osa . However, the positive association between the ahi tertile and glucose tolerance categories in the nonobese group supports a dose - response relationship . In addition, the small numerical, but significant differences of some metabolic variables between those with and without osa would be more evident if we had more subjects with severe osa . However, the analysis of the association between ahi tertile and glucose tolerance categories showed that subjects with the third ahi tertile had the highest or for ifg + igt and diabetes in the nonobese group, which supports a dose - response relationship (data not shown). Finally, because bmi and body fat distribution in this population are different from other ethnicities, the present findings may not be generalizable to non - asian populations . In summary, the current study provides original evidence that the presence of mild to moderate osa in nonobese individuals confers a higher risk for impaired glucose metabolism, even after adjusting for important risk factors . Osa can be responsible for the future risk for diabetes and cvd in nonobese individuals, whereas the prognostic implication of osa in obese individuals is unclear . Whether this finding is universal across different populations with diverse ethnicity and obesity status should be studied in the future.
Approximately 24% of non - hodgkin's lymphomas are considered to be of extranodal origin . Primary uterine non - hodgkin's lymphomas are extremely rare accounting for <1% of all extranodal non - hodgkin's lymphomas . However, the diagnosis of primary uterine lymphoma is often delayed due to its low incidence and nonspecific clinical presentation ., we present magnetic resonance imaging (mri) and fluorine-18-fluorodeoxyglucose (f - fdg) positron emission tomography (pet)/ct findings in a patient with primary uterine peripheral t - cell lymphoma . A 27-year - old female was admitted to our hospital because of intermittent fever with neutropenia for 7 months . Laboratory tests showed decreased white blood cell count (1.4 10/l; range 410 10/l). Serum cancer antigen 125 was elevated (193 u / ml; range <35 u / ml). Mri showed an ill - defined mass involved both the uterine corpus and cervix, resulting in diffuse enlargement of the uterus . This mass showed inhomogeneous hypointensity on unenhanced t1-weighted images, hyperintensity on diffusion - weighted imaging, relative hypointensity compared to the surrounding myometrium on t2-weighted images, and lower enhancement than the surrounding myometrium on enhanced t1-weighted images (figure 1)., f - fdg pet / ct was performed showing intense fdg uptake in the thickened wall of the uterine corpus and cervix with suvmax of 26.9 . There were multiple hypermetabolic lymph nodes in the pelvis and retroperitoneum (figure 2). Transverse unenhanced t1-weighted image (a) showed an inhomogeneous hypointense mass (arrows) in the uterus . Corresponding transverse diffusion - weighted imaging (b) showed peripheral hypointensity of the mass . Saggital t2-weighted mr image (c) showed the mass (arrows) involved both the endomerium and myometrium, resulting in diffuse enlargement of the uterus . This mass showed relative hypointensity compared to the surrounding myometrium . On transverse (d), coronal (e), and saggital (f) enhanced t1-weighted images, this mass showed lower enhancement (arrows) than the surrounding myometrium . Maximum intensity projection pet (a) showed intense fdg uptake of the enlarged uterus (arrow) and multiple hypermetabolic lymph nodes (arrowheads) in the pelvis and retroperitoneum . Transverse ct (b, c), corresponding pet (d, e), and fused (f, g) images showed intense fdg uptake in the thickened wall of the uterine corpus (arrows) and cervix (arrowhead) with suvmax of 26.9 . Ct = computed tomography, fdg = fluorodeoxyglucose, mri = magnetic resonance imaging, pet = positron emission tomography . Microphotograph revealed diffuse myometrial infiltration by small round tumor cells with multifocal coagulative necrosis (figure 3a). The tumor cells were positive for cd2, cd3 (figure 3b), cd43, and lat, and negative for cd19 and cd20 . The imaging and pathologic findings were consistent with primary peripheral t - cell lymphoma of the uterus (stage, ii e). After 3 circles of chemotherapy, a follow - up mri showed decreased thickness of the uterine wall (figure 4). Histopathology at higher magnification (a, hematoxylin - eosin, original magnification 400) revealed diffuse myometrial infiltration by small round tumor cells . The tumor cells were positive for cd3 staining (b, original magnification 400). A follow - up enhanced t1-weighted image showed decreased thickness of the uterine wall after 3 circles of chemotherapy . Primary uterine non - hodgkin's lymphomas are extremely rare accounting for <1% of all extranodal non - hodgkin's lymphomas . The mean age of patients with primary uterine non - hodgkin's lymphoma at presentation is 55 years and abnormal uterine bleeding is the most frequent complaint . The combination of chemotherapy and irradiation is the most effective treatment regimen for uterine lymphomas . On mri, diffuse enlargement of the uterus, relatively homogeneous signal intensity, and multinodular growth pattern are helpful for diagnosing uterine lymphoma . In this case, on t2-weighted mr images, the tumor showed relative hypointensity compared to the surrounding myometrium, which may be due to the hypercellularity and less necrosis of the tumor . On diffusion - weighted imaging, the tumor showed remarkable hyperintensity indicating marked restricted diffusion of the water molecules due to the dense microstructure . On enhanced t1-weighted mr images, the tumor showed lower enhancement than the surrounding myometrium, which may be due to the minimal tumor angiogenesis and destroying of the normal uterine vessels . Mri may be helpful for the differential diagnosis between uterine lymphoma and uterine leiomyosarcoma or abscess . Unlike uterine lymphoma, hemorrhage and necrosis are common in uterine leiomyosarcoma . Therefore, uterine leiomyosarcoma commonly manifests as a large infiltrating myometrial mass with heterogeneous hypointensity on t1-weighted mr images, and intermediate - to - high signal intensity with central hyperintensity on t2-weighted mr images . Uterine abscesses commonly show high - signal intensity on t2-weighted mr images, which is different from relative hypointensity of uterine lymphoma . Fdg pet / ct may be useful for detection, staging, and treatment evaluation of uterine lymphoma . High - grade uterine lymphomas usually show intense fdg uptake, whereas the low - grade ones show lower fdg uptake . Therefore, fdg pet / ct may be helpful for assessment of the tumor grade . This case indicates uterine lymphoma should be included in the differential diagnosis of abnormal uterine fdg accumulation along with physiological endometrial uptake, postpartum uterus, adenomyosis, endometrial hyperplasia, leiomyoma, cervical cancer, endometrial cancer, choriocarcinoma, uterine sarcoma, and uterine metastasis . In conclusion, primary uterine non - hodgkin's lymphoma should be taken into consideration when a uterine tumor shows large size, relative hypointensity on both t2-weighted images and enhanced t1-weighted images compared to the surrounding myometrium, and intense fdg uptake on pet / ct . Ethical approval was obtained from the ethics committee of changhai hospital, shanghai, china . Written informed consent was obtained from the patient for publication of this case report and any accompanying images.
An early cross - sectional study of mild hypertension reported that the predominant hemodynamic feature was a high cardiac output and this observation has been confirmed consistently since . Although not invariably so, the high cardiac output is accompanied by an increased heart rate and several studies have demonstrated that when oxygen consumption is measured, both cardiac output and this parameter are raised . In the mild early stages of essential hypertension the peripheral resistance is low although the crucial point is that it remains appropriately high for the corresponding cardiac output . A 20-year longitudinal study of the haemodynamics of essential hypertension, confirmed the finding of initially increased cardiac index heart rate oxygen consumption and blood pressure with normal peripheral resistance . However, over this period the high cardiac index and normal total peripheral resistance pattern changes to a low cardiac index high resistance pattern . Again, the inappropriately high level of vascular resistance for the increase in cardiac output is a hallmark of early hypertension . Such a haemodynamic profile could be ascribed to a high sympathetic nervous tone with the resulting increased drive to the heart, peripheral circulation and metabolic receptors, which would then promote enhanced oxygen consumption . Other studies demonstrated that autonomic blockade of the heart in mild hypertensive patients restored cardiac output to normal and there was a combination of increased sympathetic tone and decreased parasympathetic activity in such individuals [4, 5]. This combination of increased sympathetic discharge coupled to a reduced parasympathetic activity suggests that the abnormality in essential hypertension is one of integrated function in the medulla oblongata . Recent data have proposed that this might be a consequence of neurovascular compression on the left ventrolateral medulla . Therefore it should follow that the resulting increased sympathetic activity would be distributed to all innervated organs and vascular beds producing uniform vasoconstriction and the predicted cardiac indices . There is evidence for this in the heart, kidney and skeletal muscle but it has not been confirmed in studies of the hepatomesenteric circulation . In other words, it is difficult to provide evidence for a ubiquitous abnormality of sympathetic function in all vascular beds although the overall integrated haemodynamic profile can be ascribed to increased sympathetic discharge . Since the work of bright and johnson it has been recognized that the walls of medium - sized arteries are thickened in hypertension . However, it has to be conceded that their contribution to vascular resistance is small . The histopathological change appears to be hypertrophy, and indeed the heart and medium - sized blood vessels demonstrate this hypertrophic response to blood pressure . In addition, until the heart dilates it is of interest to note that the hypertrophic response takes place at the expense of the ventricular cavity: in other words there is inward encroachment on the chamber space . Therefore in any form of sustained hypertension there is an alteration in the architecture of the circulation that inevitably occurs in consequence . At the level of the resistance artery where the internal diameter of the blood vessels is around 250 m or less, there is evidence of a reduced lumen diameter and increased media thickness: lumen diameter ratio . This was originally reported in necropsy specimens and subsequently confirmed in segments of artery mounted as isometric ring preparations on wires and in vessels perfused in vitro . Detailed histological analyses more recently carried out have suggested that eutrophic inward remodelling occurs at this point in the circulation (fig . It is meant that there is a narrowing of the vascular lumen without having to invoke a growth response of the arterial wal1 . A small amount of hypertrophy may be observed in some pathological states where hypertrophy may supervene and is an adverse prognostic sign . A cartoon representation of small arteries in transfer section . The number of smooth muscle cells in both arteries is the same as is their size indicating that there is no hypertrophy but the cells have re - orientated themselves and as a result produced an increased number of smooth cell layers and decreased the external and internal diameters of the vessel . The most compelling issue is whether structural changes in these small blood vessels occur before blood pressure rises or in consequence of the hypertension? The corollary of what has been described above, namely the existence of excessive sympathetic nervous activity in the early stages of the development of hypertension being the trigger for the majority of this disorder then it is tempting to regard the small artery as changing shape in consequence . In a study of small artery structural characteristics in first degree offspring of hypertensive patients, in whom one would predict an increasing propensity to developing hypertension, there was no evidence of any alteration despite mean blood pressures being higher . Also, in another study in young genetically hypertension - prone spontaneously hypertensive rats (shr), where non - constricting ligatures were placed on the iliac artery and the animals allowed to mature until a point when the ligature compressed gradually on the vessel, the pressures in the hind limb were kept low and structural changes were not observed . However, most recently it has been reported that mice mutant for emilin 1, a cysteine - rich secreted glycoprotein expressed in the vascular tree, display hypertension, increased peripheral vascular resistance and reduced blood vessel size . Emilin 1 inhibits tfg- signalling by binding specifically to protgf- precursor and prevents its maturation by furin convertases in the extracellular space . Therefore in this model the other important issue to consider at this time is whether structural alterations amplify vasoconstrictor responses? Korner and angus developed a theory with respect to the role of an increased wall: lumen ratio in the resistance vasculature, which maintains the elevated peripheral resistance in hypertension . This is based on calculating resistance from pressure and flow recordings and consequently calculating an arbitrary radius from the fourth route of 1/r (poiseuille s law). The calculations indicate a constant narrowing in vessel radius from maximal dilatation to maximal constriction in hypertension, which acts to amplify changes in resistance . A number of studies has consistently reported structural changes predicted and described above in small arteries with an internal diameter between 200 and 300 m from patients with essential hypertension and in various animal models with the disease . However, direct supporting evidence needs the measurement of the lumen diameter of blood vessels demonstrated to contribute to the control of resistance in a vascular bed but under extremely rigorous conditions . In this context particular emphasis this has been carried out using studies of functional and structural characteristics of small mesenteric arteries cannulated and pressurized in vitro . Such arteries have spontaneous myogenic tone at physiological pressures, which is a major determinant of diameter in the resistance vasculature unlike the upstream mesenteric arteries frequently studied in hypertension research . Distal mesenteric arteries were pressurized to 63% of the mean aortic pressure of each rat . It is the pressure recorded at this level of the mesenteric / intestinal vasculature in both rat strains indicating a location within the resistance vasculature . In the absence of tone, however, with spontaneous myogenic tone, lumen diameter became identical in the two strains, and importantly remains identical as tone was increased throughout the complete noradrenaline concentration response curve . Such data do not support the hypothesis of an increased wall lumen ratio acting as an amplifier in hypertension . Studies in isolated segments of small artery or in isolated intact vascular beds can provide clear insights into basic mechanisms, which can only be considered as hypothesis generating regarding the relevance of a particular mechanism . Surprisingly few studies have actually been carried out to date in intact conscious animals and overall the results are not consistent with an amplifier hypothesis . In a recent study, blood pressure and total peripheral resistance responses to infusion of the agonist phenylephrine at two rates at the development phase of hypertension in shr aged 826 weeks was compared with aged matched wistar kyoto rats before and after ganglionic blockade . At 16 weeks of age more complete dose total peripheral resistance responses to phenylephrine were significantly less in shr versus wky rats at all ages in the study . The higher infusion rate increased mean arterial pressure by approximately 80 mmhg and nearly doubled the tpr . In contrast, blood pressure and tpr responses to methoxamine were enhanced in shr in low rates of infusion but did not differ at the higher rates . Assuming that methoxamine is the most specific agonist, these results are suggestive for functional rather than structural changes being contributory to the hyper - responsiveness to modest receptor stimulation . From a pathophysiological perspective, changes in tpr in response to low / modest stimulation appear to be a relevant parameter of tpr reactivity . More marked stimulation leading to 5- or 6-fold increases in tpr may lead to increased responsiveness but these are unlikely to be relevant for the maintenance or development of most hypertension . In other words, in vivo studies do not actually support the hypothesis that structural changes amplify vasoconstrictor responses throughout the circulation . To understand how hypertension produces non - hypertrophic changes in small arteries, one must look at the physiological role of the resistance vasculature . At normal pressures, these vessels exhibit a level of contraction (myogenic tone), which is independent of neurohormonal influences and in functioning in this way the response enables arteries to constrict or dilate in response to changes in upstream pressure . This process known as the myogenic response is only observed in smaller resistance arteries, which mediate autoregulation of blood flow and stabilize capillary pressure . This ensures that target organs downstream are supplied with oxygenated blood at a constant flow and pressure . Hypertrophy is observed in vessels which do not possess myogenic tone whereas in smaller resistance arteries, initial increase in pressure will bring about an increased myogenic constriction . If an individual has untreated hypertension then there will be prolonged myogenic constriction as the resistance vasculature endeavours to protect the target organs downstream from pressure - induced damage brought about by an increase in blood flow . The structural difference between large conduit and medium - sized arteries and downstream resistance vessels is apparent in many models of hypertension: for example, in a hypertensive model brought on by chronic nitric oxide synthase inhibition . In addition, the magnitude and duration of an increase in intraluminal pressure plays a role in determining the remodelling response . Eutrophic inward remodelling is a process of structural adaptation observed in most forms of hypertension including the onset of hypertension and milder hypertensive states . However, a few animal models of hypertension, such as a model developing hypertension independent of the renin angiotensin system (bph-2 mice), demonstrate hypertrophy as the predominating structural change . Inward eutrophic remodelling is a relatively fast functional adaptation observed after prolonged vasoconstriction and is thought to be an energetically favoured mechanism to preserve a lumen diameter for long periods . The process is also the preferred physiological mechanism by which wall stress can be normalized while maintaining vasomotor tone . Studies of the well - characterized tgr (mren2) 27 rat which develops fulminant hypertension from 4 weeks of age have demonstrated that eutrophic inward remodelling occurs from 4 weeks and is dependent on the integrin av3, a multifunctional extracellular matrix receptor . The extracellular matrix of resistance arteries is subject to tensile force exerted by blood pressure, which is transferred through integrins across the cell membrane and linked by molecular complexes to the cytoskeleton . Using peptides and specific antibodies, it has been shown that integrins v3 and 51 indirectly regulate the myogenic response by influencing the control of calcium flow through ion channels; 51 is responsible for the initial calcium influx required to establish vascular tone and v3 mediates force maintenance by calcium sensitization of contractile components . These integrins can form complexes, which regulate cytoskeletal dynamics and maintain a vascular myogenic force at a given pressure (fig . Cytoskeletal proteins such as heat - shock protein 27 activated by rhoa kinases have been shown to regulate myogenic tone . It is now clear that rhoa signalling plays a central role in both calcium sensitization and regulation of actin dynamics in small artery remodelling . These figures show a percentage control diameter of isolated arterioles exposed to step increments and intraluminal pressure before and after abluminal treatment with a control antibody anti - rat major histocompatibility complex . Anti-5-integrin function blocking antibody or an anti-1-integrin function - blocking antibody, or anti-3-integrin function - blocking antibody . In both cases one can see the amelioration of the myogenic response in consequence implicating these two integrins in the control of myogenic tone (data taken from). In contrast to molecular signalling mechanisms behind the vascular myogenic response relatively few data are available on the role of integrins and the underlying biochemical pathways of the next stage of vascular adaptation for hypertension . Remodelling involves a migratory process following prolonged vasoconstriction whereby existing vascular smooth cells reposition themselves in the vascular wall and thereby produce a narrow lumen . A characteristic of migrating cells in vitro is the presence of lamealae podial and filopodial protrusions containing focal adhesion kinase which provide a substrate for other cytosolic proteins such as src and interact with actin - associated cytoplasmic components . Recently it has been shown that the migration of vascular smooth muscle cells of arteries in vivo is more subtle and limited to elongation of tape in smooth muscle cells and an increase in cellular overlap . It is thought that cytoskeletal re - arrangements in subsequent force generation play a central role in these changes . Integrin v3 is necessary for the pressure - induced inward remodelling process but the rest of the biochemical sensing mechanism is still uncertain . If the physiological response to a raised blood pressure in small arteries is eutrophic inward remodelling then the integrity of the circulation appears to be preserved until this breaks down . As indicated above in the tgr (mren27) rat there is evidence of the development of vascular wall hypertrophy in small arteries from week 6 onwards . This rat model of hypertension develops a severe form of the disorder and indeed dams are unable to breed if they do not receive antihypertensive medication . Therefore against this background it would appear that the breakdown of autoregulation (at the myogenic tone) is associated with the vascular wall developing a growth response (hypertrophy) in an attempt to offset the increased wall stress . Recent work on the small blood vessels of patients with type 2 (maturity onset) diabetes mellitus has demonstrated that there is vascular wall hypertrophy . These patients were selected as already having evidence of downstream target organ damage because they demonstrated microproteinuria and their myogenic tone was disordered . In other words, the onset of hypertrophy is a consequence of disruption of normal myogenic tone and the delivery of blood at a higher perfusion pressure causing cellular damage . In the kidney, this would inevitably lead to a loss of filtration capability and protein leak . In terms of cardiovascular risk, recent data from italy have demonstrated that there is an increased risk of development of cardiovascular events in patients whose small arteries demonstrate hypertrophy rather than eutrophic inward remodelling (figs 3 and 4). It has been demonstrated that the myogenic response of the middle cerebral artery from pre - stroke spontaneously hypertensive stroke - prone rat (shrsp) are impaired compared with the shr . This observation would explain the deranged cerebral autoregulation that has been observed in the shrsp before stroke occurs . Also this is associated with a re - distribution of collagen throughout the blood vessel wall . Other studies have demonstrated that the myogenic component of renal autoregulation is impaired in the fawn hooded rat (the tubuloglomerular feedback component of renin autoregulation is unchanged) compared with controls thereby causing glomerular hypertension and hyperfiltration, which explains why the kidneys are susceptible to the deleterious effects of moderate hypertension . Furthermore, the brown norway rat is normotensive but has a greater than normal life expectancy . However, when hypertension is induced, these animals have a high incidence of cerebral haemorrhage and mortality compared with the long evans rat . Also the brown norway rat is very sensitive to hypertension - induced renal injury and recently the myogenic component of renal autoregulation has been found to be abnormal in normotensive brown norway rats . Therefore it seems reasonable to speculate that the cerebral vessels from the brown norway animals exhibit weaker myogenic responses compared with cerebral vessels from wistar rats, which would explain the susceptibility of the brown norway rat to hypertension - induced cerebral haemorrhage . Inhibition of the renin angiotensin system markedly delays the development of cerebral haemorrhage and mortality in salt - loaded shrsp [25, 26]. In the fawn hooded rat early angiotensin - converting enzyme (ace) inhibition prevents renal damage and this protection is associated with a normalization of glomerular pressure . The protective effect of ace inhibition in the kidney has been presumed to be a consequence of an inhibition of angiotensin ii induced efferent arteriolar myogenic tone . Of course it could be argued that the effects of blood pressure lowering would be important on stroke development and, in consequence, ace inhibition is working by its antihypertensive effects . However, dexamethasone or thyroxine increased blood pressure in the shrsp to a greater extent than salt loading but stroke does not occur . Also, the anti - stroke effect of captopril on salt - loaded shrsp which occurs without an antihypertensive effect is unchanged when blood pressure is increased with dexamethasone . Therefore it seems that the renin angiotensin system inhibition improves myogenic responses and survival in salt - loaded shrsp largely independent of changes in blood pressure although this remains to be confirmed . Individuals with a medial lumen ration greater than the medium of 9.8% show a larger number of cardiovascular events compared with those with smaller wall thickening v (data from). A further analysis of the data from reference 17 indicating that those individuals with marked vascular hypertrophy rather than eutrophic inward remodelling have an increased number of cardiovascular events (data taken from). There is little evidence to suggest that hypertension is associated with abnormalities of contractile function . Both in vitro and in vivo studies have suggested that contraction is normal although there is controversy about whether the structural alterations in the vascular wall lead to exaggerated constriction and vascular amplification (see above). This has been the subject of intense debate over a number of years although some work in intact animals really seems to suggest that vascular amplification seen in isolated vascular beds is not something which is observed when the whole of the circulation is integrated and examined . The problem with interpreting studies which have been published is that many other risk factors are often abnormal and accompany hypertension . For example, there is often associated dyslipidaemia and there is clear evidence that oxidized low - density lipoprotein (ldl) can reduce the bioavailability of nitric oxide and as a result of this there is evidence of abnormal endothelium dependent dilator function which has been reported in patients with high blood pressure and dyslipidaemia, patients with dyslipidaemia and coronary artery disease or the subcutaneous vasculature of patients with hypercholesterolaemia . In addition endothelial function is recognized to decline as individuals age and therefore it is obviously complex to dissect out whether endothelial function is abnormal as a result of hypertension per se or as a result of other demographic abnormalities and the cohorts being examined . The overall impression that one is left with is that it is the level of oxidized ldl that is important in the bloodstream of individuals with hypertension and that blood pressure per se is not responsible for endothelial dysfunction . With regard to improvement in endothelial function the use of statins has been demonstrated to restore endothelial integrity to near normal as soon as the cholesterol levels are improved . There is also evidence that the use of ace inhibitors or angiotensin receptor blockers can also ameliorate abnormal endothelial function . This is because hypercholesterolaemia is associated with an increased expression of a type 1 angiotensin receptor (atl) and that the binding of angiotensin ii to the atl receptor is associated with an increase in oxidative stress and a reduced bioavailability of nitric oxide . Animal experiments have demonstrated that the use of angiotensin receptor blockers independent of their antihypertensive effect can be associated with an improvement in endothelial function and a reduction in plaque load throughout the vasculature . Of course, longitudinal experiments in human beings are awaited but it is clear that endothelial function can be improved with the use of angiotensin receptor antagonists and ace inhibitors and recent studies have demonstrated in human beings that the combination of an angiotensin receptor blocker and a statin is an extremely powerful one for improving endothelial function . The molecular basis of the physiological remodelling response to hypertension is slowly being understood . The replacement of eutrophic inward remodelling by a pathological change such as hypertrophy appears to herald the development of an increased risk of circulatory disease . Future research should look at why this occurs and whether effective antihypertensive medication can reverse this and improve outcome for individual patients . The obvious attraction is that the identification of the, at risk patient will mean that scarce healthcare resource can be targeted at such individuals with maximum benefit in terms of circulatory protection . The challenge ahead is to identify those that are going to develop hypertrophic remodelling against a background of sustained high blood pressure and more indirect measurements of structural changes in blood vessels are urgently required . The authors state that there is no conflict of interest with the view state in this paper.
Annexin a2 (a2) belongs to the annexin family of ca - regulated phospholipid binding proteins, which are expressed in plants, animals, and protists throughout the phylogenetic tree . A2 is a 36-kilodalton protein produced by endothelial cells, monocytes, macrophages, trophoblast cells, and some tumor cells and exists both free in the cytoplasm and in association with intracellular and plasma membrane surfaces [2, 3]. The human anxa2 gene consists of 13 exons distributed over 40 kb of genomic dna on chromosome 15 (15q21). Among mammalian species for which a2 has been sequenced, identity is approximately 98% at the amino acid level . When a2 is membrane associated, the tightly packed, alpha - helical 33-kda core domain forms a disk whose convex face is associated with membrane phospholipid and whose concave face is oriented away from the membrane . Membrane binding is mediated by at least two potential ca - binding annexin repeats, features common to all annexin family proteins . While the core domains of the annexin proteins are relatively well conserved, the hydrophilic amino - terminal tail or interaction domains are highly variable and essentially unique to each family member . Protein s100a10, also known as p11, is a well - described binding partner of a2 [7, 8]. As a member of the s100 family of proteins, p11 contains ca - binding helix - loop - helix motifs and confers increased phospholipid binding affinity on a2 . Typical s100 proteins undergo a conformational change upon ca - binding that places helix iii (hiii) in a perpendicular orientation relative to helix iv(hiv), thus forming a cleft that can accept associated target proteins . This calcium activation rule, however, does not apply to p11, which has permanently assumed a calcium - on state, due to replacement of the bidentate e by s, and the monodentate d with c . The published crystal structure of p11 in complex with the n - terminal 13 amino acids of a2 suggests that the basic unit of p11 structure is a noncovalently linked homodimer, each component of which can bind the a2 tail peptide to form a heterotetramer . Upon binding, the a2 tail peptide assumes an -helical conformation that presents key hydrophobic residues (v, i, l, and l) within a cleft formed by loop l2 and helix hiv of one monomer and helix hi of the other . The c - terminal region of p11, particularly its hydrophobic residues within the c - terminal extension (yfvvhm), such as y and f, contributes critical contact points for binding to a2 . The primary fibrinolytic protease, plasmin, is formed upon cleavage of plasminogen at a single peptide bond at position r - v by either of two serine proteases, tissue plasminogen activator (tpa), produced by vascular endothelial cells, or urokinase (upa) [1113]. Tpa - dependent plasminogen activation is dramatically accelerated in the presence of fibrin, and to a lesser extent by cell surface fibrinolytic receptors . Upar is expressed by monocyte / macrophages, tumor cells, and activated endothelial cells [14, 15], while the (a2p11)2 complex is expressed on both resting and activated endothelial cells [16, 17]. In addition, an interesting array of plasminogen - binding receptors, including -enolase, tata - box protein interacting protein (tip49), histone h2b, m2 integrin, amphoterin, and plg - rkt, have been identified on many cell types . On cell surfaces, the (a2p11)2 complex serves as an assembly site for plasminogen and tpa [16, 17, 25, 26]. Although it is clear that heterotetramer - mediated colocalization of activator and substrate accelerates plasmin generation, there are, interestingly, two main theories as to the exact site of interaction of plasminogen and tpa with components of the heterotetramer complex . While one group suggests p11 as the key ligand interaction site and annexin a2 as the molecule that anchors it to the plasma membrane, another proposes annexin a2, in complex with p11, as the ligand binding site . A third group has suggested that, in the context of the cell surface and its proteolytic milieu, both annexin a2 and p11 may have exposed lysine residues that are accessible to the lysine binding kringle domains of both tpa and plasminogen . Translocation of a2 to the outer leaflet of the plasma membrane of the endothelial cell is a key regulatory step governing vascular fibrinolysis [16, 17]. Although cell surface appearance of a2 has been linked to plasma membrane fusion of multivesicular bodies in nih 3t3 fibroblasts, and as a consequence of membrane disruption upon exocytosis of secretory granules in chromaffin cells, it is not clear whether similar mechanisms apply to the endothelial cell . Endothelial cell translocation, which can occur within minutes, is initiated by several factors including heat stress, thrombin stimulation, and hypoxia [3032] and is known to require the presence of adequate p11 as well as src kinase phosphorylation at y. a2 was originally identified as a src kinase substrate, and translocation is driven by activation of pp60src . Translocation of a2 to the cell surface is dependent upon the abundance of p11 . In the endothelial cell, p11 is stabilized by a2, which, upon binding, masks a critical degron, or polyubiquitination site on p11 . In the absence of sufficient a2, p11 is polyubiquitinated and targeted to the proteasome for degradation . In anxa2 mice, which demonstrate low to nondetectable p11 expression, treatment with bortezomib, a proteasome inhibitor, restored p11 expression, verifying its regulation via a proteasome - linked pathway in vivo . In nonendothelial cells, p11 may be stabilized by one or more of its other partner proteins, which include a number of transmembrane channels and membrane receptors, such as the tetrodotoxin - resistant sodium channel nav1.8, the two predomain k channel task-1, the acid - sensing ion channel asic1a, the transient receptor potential channels trp5 and trp6, and the 5ht-1b serotonin receptor . Protein kinase c-(pkc-) mediated phosphorylation of s or s residues on a2 appears to represent an additional regulatory pathway . Serine phosphorylation within the tail domain of a2 dissociates the heterotetramer complex, preventing further translocation to the cell surface by allowing polyubiquitination of p11 and its degradation in the proteasome [40, 41]. This event appears to be initiated by plasmin, which, once generated, signals activation of conventional pkc and thus limits its own generation . This mechanism appears to require cleavage of a2 by plasmin as well as activation of toll - like receptor 4 . In this paper, we summarize evidence from both animal models and human studies on the in vivo functions of the annexin a2/s100a10 system . The concept of the annexinopathy was first proposed in 1999, and expanded in several subsequent reviews [4347]. Here, we focus exclusively on the growing body of evidence that annexin a2 and its partner protein p11 contribute to human health and disease . The anxa2 mouse has been highly informative in investigating the role of the annexin a2 system in vascular homeostasis in vivo . Although a2-deficient mice display normal development, fertility, and lifespan, fibrin accumulation is evident in both intravascular and extravascular locations within the lungs, spleen, small intestine, liver, and kidney (figure 1). Microvascular endothelial cells isolated from anxa2 mice, moreover, lack the ability to support tpa - dependent plasmin generation in vitro, and arterial injury in vivo leads to an increased rate and severity of vascular occlusion in the anxa2 mouse . Recently, fibrinolysis was also assessed in p11-null mice, which also displayed increased vascular fibrin, reduced clearance of thrombi, and impaired neovascularization of matrigel thrombi . Interestingly, mice with diet - induced hyperhomocysteinemia share this phenotypic feature with the anxa2 mouse (figure 1). Homocysteine (hc) is a thiol - containing amino acid that is generated during the conversion of methionine to cysteine . Elevated levels of circulating hc have been associated with both thrombotic and atherosclerotic vascular disease, although therapies that lower plasma hc have not been shown to reduce the risk of recurrent cardiovascular disease . Pretreatment, but not cotreatment, of endothelial cells with hc blocks their ability to bind tpa and inhibits endothelial cell - related, tpa - dependent plasminogen activation . Incubation of purified a2 with hc, moreover, interferes with its ability to bind tpa . When wild type mice were subjected to diet - induced hyperhomocysteinemia, fibrin accumulated in multiple tissues (figure 1(d)), and extracted a2 failed to support tpa binding or tpa - dependent plasmin generation, revealing that hc - induced blockade of the cell surface a2 pathway can occur in vivo . The potential clinical utility of recombinant annexin a2 protein (ra2) in ischemic stroke has emerged from thrombosis models in rats . Animals were treated with low - dose tpa with or without ra2 at 2 or 4 hours following the initiation of focal embolic stroke . Those receiving both agents had a significantly lower infarct size and greater cerebral blood flow compared to animals treated with low - dose tpa alone . In similar experiments, in which animals underwent middle cerebral artery embolization with autologous clot, pretreatment with ra2 not only improved blood flow but also reduced infarct size compared to saline - treated controls . These findings are significant in view of reported neurotoxicity and cerebral hemorrhage associated with the use of tpa in the treatment of thrombotic stroke in humans [5759]. Thus, ra2 or related agents may constitute a useful adjunct to tpa alone for the restoration of cerebral blow flow . A2 or its analogs might also prove efficacious in the treatment of peripheral arterial occlusion . When carotid artery thrombosis was induced by adventitial application of fecl3, administration of recombinant full length, but not truncated, a2 was associated with improved cerebral blood flow and reduced thrombus size in comparison with untreated control animals . This treatment had no effect on bleeding time, prothrombin time, or activated partial thromboplastin time, indicating that global clotting parameters remained intact . Thus, a2 or its analogs may constitute a useful adjuvant to conventional thrombolytic treatment by reducing the effective dose of tpa, thereby limiting its potential toxicity . The fibrinolytic system appears to modulate the development of plaque - like vascular lesions in mouse models of atherosclerosis in a complex fashion . Mice deficient in both plasminogen and apolipoprotein e (apoe), for example, display an enhanced tendency toward atherosclerosis compared to those lacking apoe alone, suggesting that plasminogen protects against lesion formation . On the other hand, when macrophages overexpressed upa in apoe mice, plaque development was accelerated through a plasminogen - dependent pathway . When apoe deficiency is combined with global deficiency of either upa or tpa, however, the predilection for early fatty streaks and advanced plaque development was similar to that seen in mice with isolated apoe deficiency . These data suggest that the fibrinolytic system acts at multiple levels in the regulation atherogenesis . In order to determine whether blocking plasmin(ogen) binding to a2 on the surface of macrophages is an effective strategy to reduce the development of atherosclerosis, apoe mice were crossed with anxa2 mice to generate double nulls (figure 2). Following weaning, apoeanxa2 mice were placed on a western chow diet (30% fat) and sacrificed at 12 or 24 wks . Aortas were removed and evaluated for lesion development by en face oil red o staining and morphometry of histologic sections taken through the aortic root at the base of the heart . There was no difference in en face lesion area or lesion size in apoeanxa2 mice as compared to apoeanxa2 mice . Therefore, we conclude that the redundant nature of plasmin(ogen)-binding sites on macrophages renders targeting a single binding site ineffective in modulating lesion development in this model system . Although embryonic vasculogenesis appears to be normal, anxa2 mice display diminished neovascularization in several in vivo assays, including matrigel implant, corneal pocket, and oxygen - induced retinopathy (oir) models . Mice with diet - induced hyperhomocysteinemia also display impaired corneal neoangiogenesis, which can be corrected upon intravenous injection of recombinant annexin a2 . Microvascular endothelial cells from anxa2 mice, as well as hc - treated human endothelial cells, moreover, migrate less efficiently in growth factor - enriched matrigel . Together, these data suggest that absence of a2, or its modification by hc, leads to impairment of angiogenesis - related endothelial cell function . Interestingly, annexin a2 is upregulated in oir (figure 3). In this model, newborn mouse pups are transitioned to room air after 5 days in a 75% oxygen environment, whereupon relative hypoxia initiates a robust vascular proliferative response in the retina . The return to 21% oxygen also triggers a2 synthesis out of proportion to the increase in vascular endothelial cell abundance . A2 expression is also increased in the endothelial cell under true hypoxia through the direct action of hypoxia - inducible factor-1 (hif-1) with the a2 promoter . Electrophoretic mobility shift experiments, chromatin immunoprecipitation studies, and luciferase promoter reporter assays all indicate binding of hif-1 and hif-1 to a hypoxia - responsive element within the promoter region of the human a2 gene, leading to its activation . Although oir - associated retinal neovascularization is impaired in the anxa2 mouse, it can be reestablished upon treatment of anxa2 mice with a subretinal injection of an a2-encoding adenovirus, which restores a2 expression . In addition, neovascularization of the hyperoxia - treated anxa2 retina can be repaired upon treatment with the defibrinating agent ancrod, which depletes fibrinogen, thereby preventing fibrin formation . Together, these findings provide a link between fibrin accumulation and diminished neoangiogenesis and imply that new therapeutic avenues for proliferative retinal vascular disorders, such as retinopathy of prematurity or diabetic retinopathy, could involve blockade of a2 in addition to inhibition of angiogenic growth factors . In the central nervous system, glioblastomas, malignant tumors derived from glial cells, are usually highly aggressive and refractory to treatment due to the early development of widespread infiltrative loci . Glioma - generated proteases, such as plasminogen activators and matrix metalloproteinases, contribute substantially to glioma cell invasion [67, 68]. High concentrations of annexin a2, similarly, are associated with the pseudopodia of invasive glioma cells, and knockdown of a2 reduces their migratory capacity in vitro . In both mouse and rat in vivo models, stable knockdown of a2 expression in glioblastoma cells retarded overall tumor progression upon implantation of the cells into rodent brains; cellular invasion, proliferation, apoptosis, and angiogenesis interestingly, when a2 expression was stably reduced by transfection of rnai directed against a2 in rat gl261 glioma cells, tumor growth and invasiveness were reduced, even though p11 expression persisted; this result indicated that the contribution of a2 to tumor invasiveness was p11 independent (figure 4). These data suggest that a2-directed treatment could offer a new therapeutic modality for human glioblastoma . In a xenograft model in which highly invasive and metastatic breast cancer cells were implanted into nude mice, both tumor growth and vascular density the tumor cells employed in this experiment expressed abundant a2, strongly supported tpa binding and tpa - dependent plasmin generation and exhibited plasmin- and a2-dependent cellular matrix invasion [72, 73]. These studies suggest that a2 may contribute to aggressive breast cancer cellular invasion and tumor angiogenesis through production of localized protease activity . In a third in vivo model, growth of lewis lung and t241 sarcoma tumors implanted into p11-deficient mice impaired tumor growth was correlated with diminished macrophage density within the tumors, and clodronate - mediated depletion of macrophages in wild type mice led to a similar reduction in tumor size . This study recapitulates the finding that thioglycollate - induced macrophage invasion into the peritoneum, and macrophage invasion of subcutaneous matrigel plugs, were also impaired in the p11 knockout mouse . A related study reveals that soluble (a2p11)2 tetramer activates human and murine monocyte - derived macrophages, that this activation requires toll - like receptor 4 (tlr-4), and that the tetramer modulates cytokine production in the macrophage . Thus, tumor infiltration by macrophages may in part be due to (a2p11)2 tetramer signaling . Nevertheless, aseptic osteolysis, due to generation of wear debris particles (wdp), is an emerging problem that leads to failure of 1030% of all joint replacements . Alkane polymers, 812 carbon atoms in length and derived from the breakdown of wdp, bind directly to toll - like receptors 1 and 2 and activate the downstream signaling pathway . In addition, endocytosed wdp can induce endosomal membrane damage and disruption in phagocytic cells, and this process is associated with dramatic recruitment of cytoplasmic annexin a2 to the endosomal membrane (figure 5). In the absence of a2, endosomal disruption leads to leakage of lysosomal cathepsins and h ions into the cytosol with subsequent activation of the nlrp inflammasome and an accelerated inflammatory response . A large body of work has focused on the role of p11 in neuropsychiatric function . P11 binds to both the serotonin 1b and serotonin 4 receptors, suggesting a role for p11 in regulation of mood . P11-deleted mice show depression - like behavior, characterized by increased immobility in the tail suspension test, increased thigmotaxis, and decreased responsiveness to a sucrose reward . In wild type mice, adenovirus - mediated deletion of p11 specifically within the nucleus accumbens (na) resulted in depressive behavior, indistinguishable from that seen in mice with global p11 deficiency; exogenous administration of p11 within the na of p11-deleted mice restored normal behavior . These data correlate with findings in human depression, in which p11 protein levels were reduced in the na . Furthermore, reduced p11 mrna levels in peripheral blood mononuclear cells may serve as a potential biomarker for patients at high risk of suicide . These studies raise the possibility that some forms of human depression may be reversible by augmentation of p11 expression . Indeed, commonly used anti - inflammatory drugs that attenuate the antidepressive effects of serotonin reuptake inhibitors may do so by inhibiting the effects of interferon, a known inducer of p11 [82, 83]. The tetrodotoxin - resistant sodium channel (nav1.8/sns), whose expression is restricted to sensory neurons, is the major pain perception receptor and is expressed in 85% of neurons emerging from the dorsal route ganglia . P11 binds to the amino terminus of the nav1.8 protein and promotes its translocation to the plasma membrane to produce functional channels . Deletion of p11 specifically in primary nociceptor sensory neurons was achieved using nav1.8 promoter - directed cre recombinase and led to a loss of tetrodotoxin - resistant sodium current density, and severe compromise of noxious coding in sensory neurons from the dorsal root horn . Thus, directed p11 targeting may prove useful in the treatment of refractory pain disorders . At diagnosis, acute promyelocytic leukemia (apl) is commonly associated with life - threatening hemorrhage . In apl, clonal expansion of immature promyelocytes harboring a balanced chromosomal translocation (t(15; 17)(q2224; q1221)) gives rise to the transcriptionally active promyelocytic leukemia - retinoic acid receptor (pml - rar) fusion protein . Apl frequently responds to differentiation therapy with all - trans retinoic acid (atra), which triggers degradation of pml - rar . While disseminated intravascular coagulation promotes coagulopathy in apl, consumption of the plasmin inhibitor alpha2-antiplasmin and development of a hyperfibrinolytic state due to excessive plasmin generation high - level expression of annexin a2 occurs specifically in apl blast cells (figure 6). A2 was detected in blast cells recovered from 6 of 6 apl patients, all of whom had evidence of hyperfibrinolysis, as evidenced by elevated circulating fibrin degradation products and d - dimer and depletion of plasma fibrinogen . Nb4 cells, which carry the t(15; 17) translocation and express the pml - rar fusion protein, displayed steady state a2 mrna levels that were approximately 10-fold higher than those found on leukemia cells that lacked the fusion protein . Treatment of nb4 cells with the retinoic acid receptor ligand, all - trans retinoic acid (atra), attenuated a2 expression in a time frame associated with clinical resolution of bleeding . Intracranial bleeding, an unusually frequent problem in apl, may be due to the relatively high level expression of the a2 system on cerebral microvascular endothelial cells compared to those of other vascular beds . Elevated expression of p11 in nb4 cells was also recently demonstrated and shown to respond to treatment with atra . In a second clinical study, a cohort of 26 patients were studied prospectively and found to have enhanced fibrinolysis at diagnosis, despite normal tpa levels and increased pai-1 . Apl cells harvested from these subjects expressed 3-fold higher levels of a2, and their rate of tpa - dependent plasmin generation was similarly elevated over that seen in the presence of m1, m2, m4, or acute lymphoblastic leukemia cells . Both elevated a2 expression in blast cells and hyperfibrinolytic hemorrhage corrected in 23 patients upon treatment with differentiation therapy, consisting of all - trans retinoic acid (atra) or atra plus arsenic trioxide . This study confirms the role of the a2 system in fibrinolytic bleeding in patients with apl . Expression levels of annexin a2 have been examined in a variety of human malignancies . In some, such as renal cell [9092], gastric, prostate, pancreatic, breast carcinoma, and osteogenic sarcoma, increased expression levels appear to correlate with higher histologic grade and/or development of distant metastases . In human glioblastoma, a2 expression correlates with histologic grade and cns dissemination [49, 9799]. In human breast cancer, a2 appears to be associated with the surface of invasive, malignant cells, but not normal ductal or acinar epithelial cells, and expression correlated with neoangiogenic activity . Proteomic profiling of colorectal cancer, moreover, revealed differentially increased expression of a2 in tumors that had progressed to lymph node metastases versus localized tumors . Primary multiple myeloma cells harvested from a cohort of patients displayed 10-fold higher cell surface a2 expression than that observed on normal plasma cells; silencing of a2 in related cell lines suppressed expression of proangiogenic genes . A2 and a related a2-binding receptor has been reported to promote myeloma cell adhesion and growth in the bone marrow . On the other hand, a2 expression in oral squamous cell carcinoma or sinonasal adenocarcinoma these studies suggest that expression levels of a2 may have prognostic value in malignancy, but would need to be validated for each specific tumor . High levels of both p11 and a2 were found in 100% of anaplastic thyroid carcinomas, and correlated with their aggressive behavior . In a comprehensive study of s100 gene expression in over 300 primary breast cancers, both p11 (s100a10) and s100a11 were selectively upregulated in basal versus nonbasal breast cancer subtypes, but did not predict overall survival . Among 62 cases of human esophageal squamous cell carcinoma, 11 of 12 s100 genes, including p11, were downregulated, based on reverse transcription - polymerase chain reaction assays . P11 transcripts have also been reported to be increased in both renal cell and gastric carcinomas [92, 108, 109]. Further studies may define p11 expression as a viable biomarker or prognostic indicator in selected tumors . It is characterized by thrombosis and recurrent fetal loss in association with circulating antiphospholipid antibodies . The latter are distinct, often coexisting antibodies directed against either 2-glycoprotein i or other intravascular proteins, which may be found in complex with anionic membrane phospholipids . A2 has been identified as a prominent target of autoantibodies arising specifically in patients with aps with severe thrombosis and/or pregnancy morbidity [112, 113]. In vitro, patient - derived antiannexin a2 antibodies blocked endothelial surface tpa - dependent plasmin generation, and also activated cultured endothelial cells, inciting them to express elevated levels of the prothrombotic agent, tissue factor . Other groups have noted that a2 can serve as a binding site for (2)-glycoprotein i in aps and can initiate a2-dependent endothelial cell activation . Together, these data implicate a2 in the pathogenesis of aps - associated thrombosis through several possible mechanisms . Cerebral venous thrombosis is a rare disorder of unknown etiology that mainly affects children and young adults . Among a cohort of 40 consecutive patients studied 2 to 6 months following the index thrombotic event, 12.5% were found to have high titer anti - a2 antibodies compared to 2.1% in healthy subjects . Thus, anti - a2 may define a new subset of individuals with immune - mediated thrombosis, play a role in the pathogenesis of this disorder, and/or offer novel therapeutic targets . Both a2 and p11 are expressed on the brush border of the placental syncytiotrophoblast . In 60 patients with preeclampsia, a2 mrna and protein levels in placenta high titer anti - a2 antibodies, moreover, were detected more frequently in sera from subjects in the pre - eclamptic group and associated with increased placental vascular thrombosis . Impaired local fibrinolytic function due to blunted a2 expression may contribute to the pathogenesis of pre - eclampsia and maternal and perinatal infant morbidity . Sickle hemoglobinopathy arises from a point mutation within the 6th codon of the human -globin chain . Polymerization of abnormal hemoglobin under deoxygenating conditions induces erythrocyte shape change and non deformability, which leads to vascular occlusion, impaired vasodilatation, distal ischemia, and endothelial cell activation with adhesion of leukocytes . In children a recent analysis of 108 single nucleotide polymorphisms in 39 candidate genes revealed that variations in the annexin a2 (anxa2) gene, among several others, were associated with increased risk of stroke . A second independent study linked anxa2 polymorphisms to increased risk for stroke in sickle cell disease, while additional anxa2 snps have been associated with avascular necrosis of bone (osteonecrosis) in sickle hemoglobinopathy . These data suggest that annexin a2 may represent a significant modifier gene that shapes the clinical expression and natural history of sickle cell disease.
Pregnancy in patients with end - stage renal disease (esrd) is rare, and kidney disease before pregnancy is associated with poor fetal outcome . However, the outcome of pregnancy in dialysis patients has been much improved, and the overall rate of successful fetal outcome is reported to be 76% (1). This is mainly due to a multidisciplinary approach that incorporates intensive dialysis and aggressive management of anemia and hypertension . Autosomal dominant polycystic kidney disease (adpkd) is the most common inherited renal disease, and approximately half of the patients progress to esrd by age of 60 (2). With regard to pregnancy, fertility is not affected by adpkd in patients with normal renal function (3, 4). However, as kidney cysts grow, hypertension and deterioration of kidney function develop, which adversely affect pregnancy . In addition, massively enlarged kidneys may occupy the abdominal and pelvic cavities, preventing the normal growth of placenta and fetus . We present a case of successful pregnancy in an adpkd patient on hemodialysis (hd). She started hd 3 times a week after 3 yr of her initial diagnosis . At the time of presentation, she was normotensive and had been prescribed aspirin (100 mg daily), multivitamins, calcium - based phosphate binders, and erythropoietin (1,000 units every hd session). Her dry weight was 53.3 kg and interdialytic weight gain was 1.5 - 2.5 kg . She was confirmed to be 8 weeks pregnant by pelvic ultrasound and beta - hcg test . The physical examination revealed a blood pressure (bp) of 137/85 mm hg and a regular heart rate of 107 beats per minute . Laboratory findings at presentation were as follows: hemoglobin (hb) of 10.4 g / dl; hematocrit (hct) 31.5%; blood urea nitrogen (bun) 56.9 mg / dl; serum creatinine (cr) 9.0 mg / dl; total protein 5.9 g / dl; serum albumin 3.7 g / dl; serum calcium 9.3 mg / dl; serum phosphorus 3.3 mg / dl; potassium 5.5 mm / l; and normal liver function . The computed tomography scan taken 2 yr ago showed bilaterally enlarged kidneys filled with numerous renal cysts along with only a few liver cysts (fig . She was managed by a multidisciplinary team approach to optimize the patient and fetal outcomes . Firstly, the risk of pregnancy in a dialysis patient and the need for intensive dialysis were discussed with her family . Secondly, our team evaluated the risk of stunted intrauterine fetal growth by her massively enlarged kidneys . We considered a unilateral nephrectomy at the second trimester to secure the intraabdominal space, but the risk was determined to outweigh the benefits in this case . Finally, genetic counseling about the fetus's risk of inheriting adpkd and prenatal genetic diagnosis was provided but declined by the patient . At 10 weeks gestation, hd prescription was changed to 4-hr treatments 4 - 5 times a week, and a predialysis bun less than 50 mg / dl was targeted . Throughout pregnancy, predialysis bun her bp was well controlled without any antihypertensive medication: systolic bp remained at 110 - 140 mmhg and diastolic was consistently at 60 - 80 mmhg . Erythropoietin doses were adjusted to target a maternal hb between 10 - 11 g / dl . The median dose was 18,000 iu / week (range, 5,000 - 26,000 iu / week) during gestational weeks 12 - 36 . In addition, 100 mg intravenous iron sucrose was administered every session during gestational weeks 19 - 22 . The follow - up data of dry weight, predialysis bun and hb level are shown in fig . Routine fetal karyotyping was performed at 16 gestational weeks, but mutation screening was not performed . At 34.5 weeks of gestation, the membrane ruptured during hd, and vaginal delivery was performed without any complications . She delivered a healthy female weighing 2,100 g. one week after delivery, the patient was hospitalized for 5 days because of postpartum cardiomyopathy . Previous studies suggested pregnancy in women with adpkd and normal kidney function can result in a favorable outcome . (3) studied fertility and pregnancy complications between patients with polycystic kidneys (n=76) and a control group (no polycystic kidney) (n=61) of women at risk of adpkd . They found no significant distinction between the 2 groups with regard to fertility, spontaneous abortion, stillbirth, or urinary tract infection (3). Another study showed that overall fetal complication rates were not significantly different between women with and without adpkd (4). On the contrary, affected women with preexisting renal failure and hypertension developed various complications and were associated with poor fetal outcome . Other risk factors for fetal complications include maternal age> 30 yr and development of preeclampsia (4). Landesman and sherr (5) suggested a classification of pregnant women with adpkd based on severity of renal disease . The patient in this case would be classified into group c, which refers to patients in renal failure secondary to advanced pkd . (6) first described a successful pregnancy in a patient with group c disease, and other cases have also been reported (6 - 9). Prophylactic hemodialysis was needed in 1 case (8), and in another case series, we found a pkd patient who underwent nocturnal hd, which is seldom performed in korea (10). With this background, we sought to determine the best way to secure fetal survival and a good maternal outcome . First, the management of ckd - related complications and the hd schedule were reviewed . Pregnant women with a favorable outcome were found to have lower predialysis bun levels compared to those with adverse fetal outcomes (11). It is recommended to increase the hemodialysis frequency (usually 4 - 6 sessions / week) to maintain a predialysis bun below 50 mg / dl (12). This provides a less uremic environment for the fetus, allows better control of volume status and bp, and permits the mother a more liberal diet . It also reduces the risk of intradialytic hypotension, which may be associated with fetal distress and premature labor (12). Increasing the dialysis dose prolongs gestation, resulting in higher birth weights and a better chance of fetal survival (13). Hypertension is the most frequently reported maternal complication in hd patients, occurring in 42%-80% of women (14). Both the rate of fetal survival and birth weights were lower in hypertensive pregnant patients compared to normotensive patients . Maternal dry weight and interdialytic weight gain should be regularly evaluated and adjusted according to changes in fetal growth (12). A lower third trimester hematocrit was associated with risk of an adverse fetal outcome and low birth weight (11). Erythropoietin dose needs to be increased by approximately 50% in order to maintain a target hemoglobin level of 10 - 11 g / dl (13). Another important area of concern for adpkd patients is the mass effect of huge kidneys and/or a massive polycystic liver . This can cause chronic pain and compression of adjacent organs, resulting in indigestion, gastroesophageal reflux, malnutrition, and ascites (compression of ivc or portal vein). Although adpkd does not affect fertility, pregnancy in patients with large kidneys at an advanced stage of renal failure has not been reported . The patient in this case had a measured kidney volume of about 6 l which is 30-fold larger than normal . Fortunately, the patient's liver size was normal with only a few small cysts, and she had no symptoms related to a mass effect . Moreover, abdominal muscles during pregnancy can adapt to increased space requirements by increasing the intra - abdominal volume under the influence of various hormones, such as relaxin . This case illustrates that even a very large kidney volume has no significant adverse effect on pregnancy . Lastly, the risk of congenital anomalies and inheritance of mutant pkd genes should be evaluated . Prenatal genetic diagnosis can be offered by obtaining fetal dna through chorionic villus sampling or amniocentesis . However, demand for prenatal diagnosis for elective abortion seems to be low, as in our case, and only 4% of women with adpkd would terminate a pregnancy if they knew the inheritance status (15). Although pregnancy remains risky in adpkd patients with esrd undergoing long - term hemodialysis, outcomes can be improved by optimizing management through a multidisciplinary team of nephrologists, obstetricians, and neonatal care specialists . Intensified dialysis, proper anemia management, and improved preconception counseling is needed in all women on dialysis because most of the pregnancies reported were unplanned.
A 78-year - old woman presented with weakness, fatigue and hemoglobin of 8.3 g / dl, to another clinic in september, 2009, and she was given erythrocyte suspension replacement . She had regression in her existing complaints with blood replacement therapy, but after one month, same complaints reappeared with a low hemoglobin value and she was referred for further investigation to the outpatient clinic of hematology of our university faculty of medicine . Her hematologic parameters ar that time were as follows: white blood cell count of 6390/mm, neutrophils 3785/mm, hemoglobin 8.1 g / dl, mean corpuscular volume (mcv) 95 fl, and platelets 211.000/mm . With the provisional diagnosis of myelodysplastic syndrome due to an isolated anemia unrelated to nutritional status, bone marrow aspiration biopsy was performed . Her bone marrow aspiration showed normal cellularity; and biopsy revealed increase in the number of megakaryocytes, signs of dismegakaryopoiesis, minimal dysplasia in erythroid series, and normal morphology in myeloid series . Upon conventional cytogenetical studies she had been lost - to - follow up for approximately a year and during that time she had received a total of 4 units of erythrocyte suspension, and erythropoietin treatment was started . However, erythropoietin treatment discontinued because she did not respond to the maximum dose of 10.000 iu three times a week . She did not attend to her follow - up visits regularly and in the mean time she had transfusions monthly at another clinic . Since her transfusion dependency increased, she was considered for lenalidomide treatment in april, 2013 . After 10 days of treatment, her liver enzymes increased more than 3 times of normal values and she had thrombocytopenia (platelet count: 38.000/mm) then her treatment was postponed for two weeks she restarted lenalidomide treatment at daily doses of 5 mg after liver enzymes and platelet counts returned to normal . During further follow - up, increase in liver enzymes and severe thrombocytopenia were not detected; her hemoglobin value increased and transfusion dependency was not observed (figure 1). In myelodysplastic syndrome, interstitial deletion in the long arm of chromosome 5 is a common cytogenetical abnormality seen in 16 - 28% of the patients . It is characterized by hypoproliferative anemia and normal megakaryocytes with hypolobated nuclei or increased dysplastic megakaryocytes in bone marrow [7, 8]. Most of the patients are transfusion - dependent because endogen erythropoietin production is usually increased . One of the major problems during the progression of the disease in transfusion - dependent mds patients is the iron overload secondary to transfusion and the dependency for chelation therapy to treat iron overload . The immunomodulatory drug lenalidomide that has additional benefits in the regulation of erythropoiesis, inhibition of angiogenesis, and reorganization of erythroid production, targets the mds clone directly [9, 10]. This syndrome is rarely seen; therefore, there is not enough data about isolated 5q - syndrome as well as lenalidomide therapy in our country . Isolated 5q - syndrome presents with normal or increased thrombocyte counts in peripheral blood and anemia with frequent transfusion replacement dependency . In clinical studies lenalidomide provided long term blood transfusion independency and erythroid response in patients with low- risk del(5q) and without del(5q). In a phase ii study of low- risk del(5q) patients; after 24 weeks, evaluation of erythrocyte response showed that 67% of the patients with 5q deletion who were treated with lenalidomide became transfusion independent . In low- risk 5q deletion mds patients, daily doses of 10 mg for 21 days are used, and repeated with 28 day - intervals that can be modified if needed . In order to evaluate the response, adverse events can be easily overcome with dose modifications . In clinical studies, thrombocytopenia and leukopenia are frequently observed but these adverse reactions usually appear during the first 8 weeks of treatment . Therefore, weekly clinical follow - up at the beginning of the therapy is recommended . Less often adverse events of fatigue, rash and diarrhea can be noted . In conclusion, in isolated 5q - syndrome which is a mds subgroup and characterized by transfusion dependency; lenalidomide can decrease transfusion dependency with a tolerable adverse event profile that can be overcome with dose modifications . Assessment of all prevalent cases in our country will be helpful to reveal the efficacy and adverse event profile of lenalidomide.
Most clinical complications associated with hepatitis b are manifested in conditions, particularly cirrhosis and hepatocellular carcinoma (hcc) as consequences of chronic infection . Hepatocellular carcinoma (hcc) is the sixth most common in the world and the third leading cause of cancer - related death (1). An effective vaccine has been available for more than two decades, which has decreased the prevalence of hbv infection worldwide dramatically . The primary goal of hepatitis b prevention is reduction of chronic hbv infection and hbv - related chronic liver disease . A secondary goal is prevention of acute hepatitis b. in 1991, the world health organization recommended that all countries include hepatitis vaccine in their routine infant immunization program, especially in areas where hepatitis b is endemic (2). The global reported decline in hbsag prevalence, especially from hbv endemic area (such as south east asia, alaska, etc .) Has been come as something of a surprise . Naively speaking, one would expect that the ultimate decline is reaching; however, this is not the case; universally, about 5 - 20% of vaccine failure among recipients has been reported manifested by different levels of hypo- or nonresponsiveness to hbv vaccination (4, 5). Historically, the potency of immune response after immunization against hepatitis b has been assessed by measuring antibody to hbsag . The persistence of anti - hbs above the protection level (> 10 iu / ml) in vaccine recipient is the main goal . This persistence declines by age, particularly, during the first years after vaccination (6). On the other hand, a growing body of literature has shown that hbv vaccine - induced immunologic memory lasting persists more than 15 years after immunization (7, 8). In 1989, iranian ministry of health launched an immunization program in four provinces as a pilot plan . Afterwards, many researches conducted on the level of response to hbv vaccine among iranian children and ample data on the coverage rate of vaccine together with evaluation of vaccine responsiveness published in iran so far . In this study, a systematic review and meta - analysis was performed to provide the statistic power on investigations performed nationally and to provide a summary of the results . We performed a comprehensive search on pubmed, isi, scopus, iran medex and scientific information database (sid), on published describing anti - hbs levels in children below 15 years (after six months to 15 years old) who received three doses of hbv vaccine following epi . The primary outcome was frequency of persons with protective levels of anti - hbs (> 10 iu / ml) following hbv vaccination . The secondary outcome was frequency of nonresponders to hbv vaccine (<10 iu / ml). The main inclusion criterion was all studies that included subjects under 15 years old who received 3 doses of hbv vaccine (regardless of type and brand of vaccines). We excluded all studies in which individuals received vaccine episodes out of vaccination schedule (less or more administrations) and in those with any medical interventions (such as using adjuvant, booster doses etc . ). Moreover, we did not include surveys that investigated responsiveness to vaccine in those with medical conditions (children born to hbsag positive mothers, thalassemia, dialysis, received hb vaccine plus immunoglobulin, predisposing factors for immunodeficiency such as hiv, etc . ). Data was extracted from selected studies including the name of author, year of publication, the mean age of participants and the levels of anti - hbs in different case - studied . Subjects were divided into two categories: responders who showed anti - hbs levels> 10 iu / ml and nonresponders (anti - hbs <10 iu / ml). We merged the subjects who harbored levels between two extremes; 10 and 100 iu / ml, as responders (because some studies assigned these levels as hypo - responsiveness). The medical subject headings (mesh) including entry terms of pubmed and emtree of scopus with affiliation to iran for searching in english databases (hbv vaccine, anti - hbs, expanded program on vaccination, prevalence, responders and nonresponders) were used for conducting a more efficient search . Persian keywords equivalent to their english terms were used for searching in national search engines . The references of selected citations and non - published national surveys were hand - searched . The authors assessed the risk of bias in the included studies using a risk - of - bias tool . Studies that had an adequate handling of incomplete outcome data, were free of selective reporting, included an adequate intervention description, had appropriate criteria for participant recruitment and included an adequate outcome explanation were considered low - bias risk trials . The studies with one or more unclear or inadequate quality component were considered high - bias risk trials . The authors were not blinded to the names of studies authors, journals and results . Any disagreements were resolved through discussion among the authors until consensus was reached (kappa coefficient: 78%). Data are expressed as percentages for categorical variables and means and sds (standard deviation) for continuous variables . Statistical heterogeneity of reported prevalence was explored by chi - square (or chi2)-based q - test and was regarded to be statistically significant at the 10% significance level (p <0.10). To gain better insight into the prevalence of dyslipidemia and its heterogeneity throughout iran, we analyzed our findings using random - effects model with a 95% confidence interval (ci). The analyses were conducted with stata software, version 11.0, produced by statacorp, the usa . The primary outcome was frequency of persons with protective levels of anti - hbs (> 10 iu / ml) following hbv vaccination . The secondary outcome was frequency of nonresponders to hbv vaccine (<10 iu / ml). The main inclusion criterion was all studies that included subjects under 15 years old who received 3 doses of hbv vaccine (regardless of type and brand of vaccines). We excluded all studies in which individuals received vaccine episodes out of vaccination schedule (less or more administrations) and in those with any medical interventions (such as using adjuvant, booster doses etc . ). Moreover, we did not include surveys that investigated responsiveness to vaccine in those with medical conditions (children born to hbsag positive mothers, thalassemia, dialysis, received hb vaccine plus immunoglobulin, predisposing factors for immunodeficiency such as hiv, etc . ). Data was extracted from selected studies including the name of author, year of publication, the mean age of participants and the levels of anti - hbs in different case - studied . Subjects were divided into two categories: responders who showed anti - hbs levels> 10 iu / ml and nonresponders (anti - hbs <10 iu / ml). We merged the subjects who harbored levels between two extremes; 10 and 100 iu / ml, as responders (because some studies assigned these levels as hypo - responsiveness). The medical subject headings (mesh) including entry terms of pubmed and emtree of scopus with affiliation to iran for searching in english databases (hbv vaccine, anti - hbs, expanded program on vaccination, prevalence, responders and nonresponders) were used for conducting a more efficient search . Persian keywords equivalent to their english terms were used for searching in national search engines . The references of selected citations and non - published national surveys were hand - searched . The authors assessed the risk of bias in the included studies using a risk - of - bias tool . Studies that had an adequate handling of incomplete outcome data, were free of selective reporting, included an adequate intervention description, had appropriate criteria for participant recruitment and included an adequate outcome explanation were considered low - bias risk trials . The studies with one or more unclear or inadequate quality component were considered high - bias risk trials . The authors were not blinded to the names of studies authors, journals and results . Any disagreements were resolved through discussion among the authors until consensus was reached (kappa coefficient: 78%). Data are expressed as percentages for categorical variables and means and sds (standard deviation) for continuous variables . Statistical heterogeneity of reported prevalence was explored by chi - square (or chi2)-based q - test and was regarded to be statistically significant at the 10% significance level (p <0.10). To gain better insight into the prevalence of dyslipidemia and its heterogeneity throughout iran, we analyzed our findings using random - effects model with a 95% confidence interval (ci). The analyses were conducted with stata software, version 11.0, produced by statacorp, the usa . Twenty - eight eligible articles were found in the literature review, all were potentially related to the iranian epi . Table 1 shows summary of iranian studies on immunization program . In total, these investigations were performed on 11639 children who received full three doses of vaccine . The age of subjects was between 6 months and 15 years old with the mean age of 5.21 3.64 years . Overall, 9311 (80%) responded to vaccine (anti - hbs> 10 iu / ml). On the other hand, 2328 (20%) were nonresponders (anti - hbs <10 iu / ml). The mean titers of antibody for responders and non - responders were 287.05 332.80 iu / ml and 4.08 1.70 iu / ml, respectively which was statistically significant (p = 0.024). Among responders, the mean anti - hbs titers showed differences between a cut - off of three years old; 500.95 484.19 and 164.82 139.44 iu / ml for those who were under 3 years old versus children more than 3 years old . Meta - regression analysis showed that with increase in age, the number of responders to vaccine decreased significantly (p = 0.001) (table 2). Put another way, the number of nonresponders to vaccine increased as age of vaccines increased (p <0.001). The other finding was that between years 1995 and 2014, there was no significant difference in the mean anti - hbs titers between studies (p = 0.428). However, results from 24 publications indicated that of total 10562 children, 5070 (56.3%) of males and 3937 (43.7%) of females had antibody levels of> 10 iu / ml . In contrast, 1346 (59.0%) and 938 (41.0%) of males and females had antibody levels of <10 iu / ml, respectively . Final result for meta - analysis and model performed for total estimated prevalence is shown in table 3 . However, there was no strong difference between responders versus nonresponders to vaccine (p = 0.119). Forest plot of prevalence of hbv vaccine responsiveness in iranian general, male and female population is shown in figures 1 - 3, respectively . Despite some studies did not specify the type of vaccine used, using different vaccines (heberbiovac, cuba; engerix - b, gsk; recombivax, cuba; euvax b) with the same protocol for vaccine doses and administrations, did not show any significant differences regarding response rate to vaccine (p <0.001). Furthermore, prevalence of anti - hbc was between 0 and 7.5% in different reports (table 1). In those studies which compared the rate of responsiveness to hbv vaccine between urban and rural area, no significant differences were found between responders and nonresponders (results not shown). No significant complications and side effects were reported after vaccine administration (results not shown). Response to vaccine according to ethnic groups (turkish, arab, kurdish, turkmen, etc .) And geographic area was evaluated; however, no strong correlations were found (p 0.826 and 0.896, respectively). Data are presented as% for 10 t: total; m: male; f: female abbreviations: ni: non identified . Hbv expanded program on immunization was started in iran in 1993 . Accordingly, more than 98% of infants there are ample data on the vaccine coverage rate, response to the vaccine, amnestic response to booster doses etc . Among iranian healthy children . Moreover, most of those articles published in persian language, makes the interpretation of data more incomprehensible . In this review, the impacts of different variables including age, gender, type of vaccine etc . On the rate of responsiveness of 11639 children - studied, 9311 (80%) and 2328 (20%) were responders and nonresponders to vaccine, respectively . There have been numerous published data on the rate of response to hbv vaccine internationally; the reported rate of hbv vaccine responsiveness ranged between 41.6 and 98.3% (table 4). The reverse was also realistic for the extreme of no responsiveness to vaccine . In iran, the rate of responders to vaccine was between 47.6 and 98.3%; however, in most studies (24 out of 28 papers, 85.7%), responders were above 69% of population in the surveys . Regarding the impact of age on the rate of response to vaccine, we did not find a significant difference between subjects, despite finding cut - off levels for anti - hbs between 500.95 and 164.82 iu / ml for those who were under three years old versus children more than three years old, respectively . There are considerable variations between studies for association between the age of participants and response to vaccine . The massive gaps between the extreme of these results substantially correlated to the age of vaccines . For example, table 4 shows that more increase in the age of vaccines, the more decline in the rate of responders . The other important issue is the timing between the last dose of vaccine administration and measurement of anti - hbs . Globally, in various reports, the age of children ranged between 5 and 15 years old; which makes substantial variance between the levels of anti - hbs in different age groups . On the other hand, this review and other studies showed that with increase in age, the number of nonresponders to vaccine increased significantly . Versatile ethnic groups, geographic variations, different age groups between subjects and difference in the endemicity of hbv in those areas might be the reasons for such discrepancies . What is the impact of low level of anti - hbs on acquisition of hbv infection? Taken together, with exception of some rare reports on healthcare workers who were nonresponders to vaccine and subsequently infected with hbv (47 - 49), there is no data to support that after robust rising in anti - hbs following hbv vaccination, subsequent decrease in anti - hbs levels endanger the subject to hbv infection (50). Previous results showed that memory t cells maintain the response to the virus after encountering . This issue has been highlighted by the fact that after booster dose(s) of hbv vaccine in hypo / nonresponders, there has been amnestic response manifested by increase in the levels of anti - hbs (45, 51). Interestingly, between years 1995 and 2014, there was no significant difference in the mean anti - hbs titers between iranian studies (p = 0.428). This finding together with this issue that the type of vaccine used did not show any significant differences between the rate of response to vaccine (p> 0.05), indicate the reasonable potency and antigenicity of vaccine used in iranian epi, which were already assessed by our lab in vivo and in vitro studies (52, 53). Despite some studies showed some differences in response to hbv vaccine between rural and urban areas (54 - 57) furthermore, in our own yet unpublished study on 1200 samples from two categories of rural and urban districts, we did not find any considerable changes (jazayeri s unpublished manuscript). Overall, iranian surveys did not find any significant correlation between gender of participants and the rate of response to vaccine (p = 0.119), which was similar to other findings (table 4). Iran is considered as an intermediate area for hbsag prevalence (2.54%) (58). On the other hand, the rate of past hbv infection, manifested by anti - hbc, ranged between 0 and 7.7% in this review . Both relative high prevalence for hbsag (4.6%) and anti - hbc (7.7%) previous reports showed rates of 6.3% hbsag and 8% anti - hbc in general population in these areas, respectively (59, 60). However, our unpublished data on 821 samples from vaccinated children showed no positive cases for both two markers (jazayeri and alavian, unpublished). Altogether, 0 to 1.29% prevalence of hbsag together with 0 to 2.5% anti - hbc from different provinces underscored the usefulness of hbv vaccination for substantial reduction in hbv endemicity in iran . Second, the vaccine was effective in stimulating the immune response of vaccines reasonably, despite being different in generation, manufacturers and types . Third, there was no substantial difference between iranian and other international investigations in the rate of nonresponsiveness to hbv vaccine.
Improperly - placed implants are at risk of dislocation, increased component wear and loosening, and the need for revision surgery . Avoiding glenoid component malposition in terms of version and inclination is therefore an important technical goal, which may be very demanding due to a variety of issues related to patient anatomy and surgical technique . Altered anatomy in revision cases, joint contractures and complex patterns of glenoid bone loss with unreliable landmarks are commonly encountered in this patient population . In such cases, directing the glenoid baseplate along an appropriate axis with sufficient bone stock for fixation may be a challenging intra - operative task . Surgical planning for these patients can be improved using three - dimensional (3d) reconstructions of ct scans, but recreating that same plan at surgery can be difficult . Computer navigation has been employed for this purpose, with promising results . However, computer - assisted glenoid implantation techniques use an intra - operative setup using a tracking system, with some disadvantages: instrumentation is cumbersome to use in the shoulder; pins used for array fixation may cause iatrogenic lesions such as fracture or neurovascular injury; registration of anatomical landmarks may be inaccurate; and the need for resetting may result in an increase in operative time of more than 20% patient - specific instrumentation has the potential to provide a similar level of accuracy as computer - assisted navigation, but without such problems or additional surgical steps . This type of pre - operative planning has grown in popularity across a wide range of orthopaedic subspecialties including total hip and knee arthroplasty, pelvic and acetabular procedures and spinal deformities, with varying degrees of success . It is the most recent development in this field, and its effectiveness in both the version and inclination planes of glenoid component placement has been demonstrated for both total anatomical and reverse shoulder arthroplasties . Patients undergo a pre - operative thin - cut ct scan of the entire scapula and adjacent humerus following a predefined protocol . Original two - dimensional (2d) images are uploaded to a 3d image processing software system and reformatted into accurate 3d models of the scapula, in order to avoid errors in measuring actual version or inclination the plane of image acquisition (gantry angle) can deviate from a perpendicular angle to the plane of the scapula . This plane of the scapula is defined by three landmark anatomical points: the inferior scapular angle point, the glenoid centre point and the trigonum spinae point (intersection of scapular spine and medial border). A neutral inclination axis, or glenoid centre line, is defined between the last two (fig . 1). The version is therefore measured with respect to the scapular plane, and inclination with respect to the neutral axis, as the angle between a line perpendicular to it and a line from the superior to inferior rim of the glenoid . Three anatomical points (red dots) used as landmarks for the definition of the scapular plane and the neutral inclination axis (red line): the inferior scapular angle point, the glenoid centre point and the trigonum spinae point . The pre - operative planning is performed by the surgeon using adequate software, in a process that may vary according to each provider (fig . Virtual surgical planning requires the definition of the axis for the glenoid baseplate component, by creating patient - specific surgical guides . The guide is designed to fit onto the surface and border of the glenoid in each specific case, requiring minimal additional exposure, and is manufactured following a 3d stereolithography model . This guide, constructed from a sterilisable material (usually polyamide resin) has one or two drill cylinders positioned to orientate the drilling of the central glenoid guide pin . In addition to this glenoid guide, a patient - specific glenoid vault replica is also created to aid the surgeon in having the best perception of the placement of the guide on the glenoid during surgery (fig . A) radiograph and b) mri of a shoulder with extensive glenoid destruction for a reverse prosthesis; c) coronal, and d) transverse view of the pre - operative planning . A) sterilised bone model and glenoid guide, with b) one inferior - tilted pin hole for a reverse procedure and a second superior one for an anatomical procedure . The technique for exposure and the use of instruments for bone preparation over a guidewire is identical whether or not the patient - specific instrumentation is used . The only additional requirements are care not to resect bony prominences such as osteophytes that were contemplated in the development of the guide and are necessary for the proper seating of the guide, and a slightly more extensive exposure of the anterosuperior aspect of the glenoid and base of the coracoid . This fact, however, may make the use of the deltopectoral approach more favourable as it may facilitate glenoid exposure, which is in fact the only restriction the surgeon may find in using a superior transdeltoid approach . The guide is then fitted to a stable position on the native glenoid and the central kirschner wire (k - wire) is drilled into the glenoid until the far cortex is perceived (fig . This k - wire then guides further cannulated glenoid reaming and subsequent steps for both total and reverse shoulder arthroplasties according to standard guidelines of the manufacturers (fig . A) glenoid central pin placement for an anatomical prosthesis in a right shoulder; b) good exposure of the anterosuperior glenoid rim is required for the proper seating of the guide . A) reverse prosthesis in a left shoulder and guided central pin drilling; b) native glenoid and patient - specific bone models are compared to check accurate placement of the guide on a very deformed scapula; c) central screw drilling completed over a guide pin that keeps its stability due to the integrity of the far cortex with no glenoid vault perforation, made possible with an accurate drilling direction . In addition to single - use patient - specific instrumentation (psi), it should be noted that reusable and adjustable psis are also available from some manufacturers . In the longer term, this technology may prove to be of lower cost than single - use psis and should result in a reduction of the time from planning to use of the instrument . In addition to single - use patient - specific instrumentation (psi), it should be noted that reusable and adjustable psis are also available from some manufacturers . In the longer term, this technology may prove to be of lower cost than single - use psis and should result in a reduction of the time from planning to use of the instrument . No long - term clinical studies comparing patient - specific to standard instrumentations have been published to date, and the few reports on this subject that have been published are mostly laboratory studies on cadaver specimens, evaluating the accuracy of the baseplate positioning . The purpose of those studies was to examine the effectiveness of patient - specific planning and a patient - specific drill guide for glenoid component placement in shoulder arthroplasty . The conclusions drawn are similar in all . In 2011, suero et al evaluated the safety and accuracy of a custom glenoid jig created using pre - operative ct imaging, with 3d modeling for glenoid component implantation . Comparison between the pre - operative plan and the result on post - operative ct scans of seven patients showed that a ct - based custom alignment can reliably guide the placement of the glenoid component during conventional and reverse shoulder arthroplasty, without technical difficulties or complications . In their level i randomised prospective clinical trial, hendel et al concluded that there was a significant improvement in the accuracy of glenoid component placement with the use of patient - specific guides in patients with bone deformity and glenoid retroversion in excess of 16, and no difference in those with glenoid retroversion below 7. levy at al later concluded that the use of patient - specific guides in 14 reverse arthroplasties was highly accurate in reproducing a virtual 3d pre - operative plan, delivering the accuracy observed using computerised navigation without any additional surgical steps or technical difficulties . Likewise, walch et al demonstrated in an in vitro study that using 3d planning software and custom guides is a reliable and precise option in total shoulder arthroplasty . The most recent papers from 2015 further support those conclusions . In 36 prostheses, heylen et al found that patient - specific guidance reduced variability in glenoid component inclination, as well as the risk of extreme inclination errors for total and reverse shoulder arthroplasty when compared with standard pre - operative planning and instrumentation . In a multi - surgeon study in 70 arthritic cadaver specimens, throckmorton et al found that these patient - specific targeting guides were significantly more accurate (p = 0.01) for the combined vectors of version and inclination, and also had fewer instances of significant component malposition than traditional instrumentation . 3d surgical planning and patient - specific guidance reduce variability in glenoid component inclination, which should result in fewer malpositioned glenoids in both total and reverse shoulder arthroplasty . In severe glenoid deformations, namely when bone grafting is required, psi will aid in estimating the best alignment, in terms of version and inclination, as well as the best fixation on stable bone stock, avoiding glenoid vault perforation (figs 6 and 7). Good baseplate alignment on the same patient as shown in figs 2 and 5, with severe glenoid deformation requiring bone grafting . Digitised image from pre - operative plan of an anatomical glenoid component, displaying peg penetration from the vault posteriorly . (from zimmer - biomet software; with permission). However, there are a few important points that need consideration when using this technology . First, manufacturing the psi guide is based on an accurate 3d model of the patient s scapula made from ct - scan cuts, which may be conditioned by cartilage loss, severe bone deformity or calcified labrum . Second, seating the guide properly on the native glenoid demands good exposure of the anterosuperior glenoid rim, with removal of any soft tissues that may impair it . Third, reaming to the adequate depth and orientation is still dependent on the surgeon s ability, as bending or pushing the guide pin with the reamer can mislead further reaming . Likewise, overtightening screws on soft bone or graft may asymmetrically overcompress the glenoid component against bone and change its final position, despite adequate reaming over a well - positioned central pin . Hendel reported four in 15 cases with over 10 of deviation in version or inclination from the pre - operative plan, despite the use of psi, all performed by surgeons with little experience with this technology . By way of addressing these limitations, some manufacturers have developed multiple psi guides to help correct off - axis reaming, control the depth of reaming and assist in the orientation of the fixation screws . Current evidence suggests that the use of psi can assist in the avoidance of significant glenoid baseplate orientation errors, especially by less experienced surgeons, resulting in fewer malpositioned glenoid components . Custom - made guides will play an important role in the placement of the central pin on glenoids with severe deformation, in cases where the best anatomical landmarks may have vanished and the use of bone grafting is anticipated . However, the surgeon s expertise and intuition remains invaluable, both in the pre - operative planning and in the final component implantation, especially concerning the reaming depth . However, psi seems to offer a distinct advantage to lower - volume surgeons, by reducing the risk of guide - pin malposition, a step that is very much dependent on the surgeon s expertise . Furthermore, pre - operative planning time is longer and may be dependent on external technical support, limiting its usage to elective surgeries and making it unsuitable for acute cases such as fractures . Psi offers a greater chance of accurate alignment of the glenoid component, which seems to be an advantage . However, scapulae are not all the same, so when we try to align all glenoids in the same way, we may actually be changing the native alignment of some shoulders . Predicting the physiological glenoid version for a particular pathologic shoulder can be difficult, unless comparison with a normal contralateral shoulder (if present) is done . Scalise et al concluded that a 3d vault model, as described by codsi et al, could be used as a template to predict normal or baseline glenoid version for a particular patient . Youderian and iannotti have concluded from surgical simulation in a 3d virtual environment that additional use of a patient - specific glenoid vault can predict pre - morbid version, inclination and amount of volumetric bone loss, which can help to generate the best possible scenario of bony structural support for the glenoid implant and can minimise the amount of bone loss, by reaming . These findings are invaluable for further development of systems which may help surgeons in estimating the right degree to which pathological version should be corrected . Psi will help in the placement of the glenoid component on the best bone stock with the desired alignment . Clear advantages in total knee arthroplasty are the fact that it is very intuitive and effective in placing the components in the desired position, it is attractive to patients because they like a personalised patient - care approach and it increases efficiency in the operating room (or) due to the reduced number of instruments required, which leads to reductions in or setup time, turnover time and overall surgical time . While the former may also be observed in psi for shoulder prosthesis, the latter will not necessarily be met . The added cost of psi manufacturing will need to be judged against the potential benefit in clinical outcomes . The cost benefit analysis and long - term clinical studies remain lacking; these are necessary components in the evaluation of the potential benefits of this alternative . Ng has received financial support outside of the current work in the form of consultancy and lecture fees from zimmer biomet.
Despite expanded indications for conservative surgery of urothelial tumors (ut - formerly transitional cell carcinoma) of the upper urinary tract (uut), radical nephroureterectomy (nue) with complete removal of the distal ureter including the bladder cuff is the standard surgical technique used for most patients with ut of the uut . Choosing the best procedure for this group of patients in everyday clinical practice is frequently a challenging task . While laparoscopic nephrectomy as a part of nue was first described in 1991 and it is to - date broadly accepted, the approach to the distal ureter and the timing of the ureterectomy are still disputed . Several techniques have been developed to remove the distal intramural part of the ureter during laparoscopic nue and it is very difficult to choose the best procedure for a given patient in everyday clinical practice . There is a risk of residual tumor at the stapling site and titan clips may constitute a nidus for the formation of cystolithiasis [5, 6]. Two groups [79] have described a technique for the division of the ureterovesical junction with a thermo sealing system (ligasure atlas). We considered this modification of clnue as an excellent method and we have previously performed this on 14 patients . However, we found a significant risk of incomplete resection of the intramural part of the ureter . Exclusive laparoscopic sharp excision of the bladder cuff with intracorporeal suturing [1115] appears too difficult for us . Thus, we have decided to start clnue with excision of the ureterovesical junction with collin's knife followed by clnue . The main problem with this procedure is the risk of occlusion of the ureter to prevent spillage of urine containing tumor cells during laparoscopic pluck nephroureterectomy . Several methods have been described to date: (1) cauterization of the ureteric ostium only, (2) endoloop [16, 17], (3) hem - o - lok clip [1820], or (4) fibrin sealant . We chose to close the ureter with a lockable hem - o - lok clip, which was introduced through a 5 mm intravesical port in the suprapubic area rather than endoscopically [18, 19, 20]. We have labeled this technique as clnue - wilc (with intravesical lockable clip). In this study the study is prospective, but due to the absence of a standard technique for nue, the study was not randomized and comparative . From 1/2010 to 1/2012, 38 patients with suspected ut of uut were indicated for surgical treatment . Four underwent conservative surgery (one ho: yag ablation, one ureteroscopic resection, one nephroscopic resection with resectoscope, and one open resection of the ureter) and 34 nue . Thirteen underwent some type of open surgery (advanced cases with open nephrectomy or laparoscopic nephrectomy with open ureterectomy for tumor of the distal ureter) and 21 nue by clnue- ivlc (main inclusion criteria: not suitable for conservative treatment, no tumor of distal ureter, no advanced tumor by ct, no contraindications to laparoscopy, or no concomitant bladder tumor). The ureterovesical junction is excised transurethrally with collin's knife (the paravesical adipose tissue must be clearly visible). From the suprapubic region, the stump of the ureter is grasped with biopsy forceps and on the end of ureter, a hem - o - lok clip size ml is applied (the applicator is introducible through the 5 mm port). In broader ureters, the patient is rotated to the flank position and a standard laparoscopic nephrectomy via a transperitoneal approach is performed [10, 22]. The transperitoneal approach is more familiar to us than the retroperitoneoscopic one . One additional 5 mm port in the suprapubic region is introduced; the skin incision from the previous endoscopic phase is used . The ureter is dissected along and under the iliac vessels with a harmonic scalpel or ligasure advance or blunt tip 35 mm . This phase is delicate due to the relatively narrow operation space and the close relation of iliac vessels and the bowel . The ureter is completely separated and the hem - o - lok clip must be clearly visible to constitute proof of completion of the ureterectomy . A few points to emphasize include: four complications (clavien ii 2x, iiib and v) wound infection at the site of extraction (staphylococcus aureus), urine leakage from the pelvic drain for 6 days (bladder catheter was removed on the 8 postoperative day following cystoradiography that did not reveal leakage), one open prostatectomy on the 2 postoperative day because of an enlarged prostate (bph with hematuria), and one patient died of heart failure on the day of operation . In four cases (19.0%) application of the clip failed and clnue was concluded with a non - occluded ureter and the risk of dissemination of tumor cells in urine paravesically . In the first case the patient had a ureteral stent and the ureter was incrassate, which is why we did not want to apply 10 mm port to facilitate the introduction of the hem - o - lok clip size l. in another case, the stented ureter was also incrassate and we introduced a 10 mm port to the urinary bladder and applied a size l clip without difficulty . There was one incidence with the inability to grasp the ureter by endoscopic forceps . In two cases the laparoscopic nephrectomy was converted to the open surgery (flank incision and lumbolaparotomy) in one case this was due to extensive adhesions in the abdominal cavity (a history of open cholecystectomy with evacuation of subphrenic abscess), and in the other two cases it was due to advanced tumor growth with perirenal and periureteral adhesions . In three cases, laparoscopic nephrectomy was followed by open distal ureterectomy with the standard pluck method, because the laparoscopic approach was not feasible due to poor access of the laparoscopic instruments to the small pelvis . Four patients with non - ut histology were judged to be ut by preoperative imaging . Follow - up (mean 10, range: 0 - 22 months), including results of the control endoscopy, are known in all patients . One patient with uc of the renal pelvis pt2n0m0g2 had uc in the contralateral distal ureter and died after 11 months due to extensive metastatic disease, mainly to bone . Abbreviations: ae adrenalectomy for adenoma, bcg history of intravesical instillation of bcg, bt urinary bladder tumor, eskd end - stage kidney disease, l left, m male, mo right, turb transurethral resection of bladder tumor, up - um ureter proximal - middle, uc notes: all cases were n0m0; time of whole procedure (endoscopy, rotation of patient, laparoscopy); non - uc histology: tumors were described by radiologists on ct / mri as a suspicious uc tumor . It should be noted that the aim of this work is not to comprehensively discuss the whole complex problem of nue . Our experience prompted us to review this topic in two recent publications [10, 22] and now we will focus the discussion on complete laparoscopic nue with emphasis on the method of removing the distal part of the ureter including the bladder cuff . In our view, due to the disadvantages mentioned in the introduction, the method involving a stapler should be abandoned . The other options of distal ureterectomy as a part of clnue are as follows: (1) a thermosealing technique [7, 8, 9], (2) the sharp excision of the bladder cuff with intracorporeal suturing (a purely laparoscopic technique) [11, 12, 15] including modification with a bulldog clamp, (3) robotic, or (4) purse string technique . The steps of the endoscopic phase in lithotomy position . The ureterovesical junction is excised transurethrally with a collin's knife to the paravesical adipose tissue . The stump of the ureter is grasped with biopsy forceps and the end of the ureter is clipped with a hem - o - lok clip size ml (an applicator is introduced through the 5 mm port inserted as an epicystostomy). As pointed out previously, the thermosealing system technique has the risk of leaving the intramural part of the ureter intact . The laparoscopic ne with ensuing sharp excision of the ureterovesical junction and closing of the defect with suture is an ideal but challenging method . We consider the technique with any variant intracorporeal suturing technically more challenging and time consuming [10, 22]. The variant with the da vinci robotic system [24, 25] decreases the technical difficulty of intracorporeal suturing . The disadvantages of the da vinci system include: high cost, lack of tactile sensation, long set - up time, and unavailability of the robotic system in many hospitals . An exotic technique is the pneumovesicum approach in which three 5 mm ports are introduced to the bladder and insufflated with co2 pneumovesicum (10 - 12 mm hg). The distal ureter, bladder cuff, and intramural ureter are then completely dissected free using electrocautery . As soon as the distal ureter is dissected an endo - loop knot is used to ligate the ureter . We do not have experience with this technique and we feel this technique to be complicated . The ureter is excised through a laparoscopic single port introduced to the urinary bladder and the defect is closed with intravesical suture . Six ports, on the left side, but five usually suffice . The same skin incision as for the epicystostomy is used for the suprapubic port . Due to the factors mentioned above we prefer a variant of excision of the ureterovesical junction but with another method for sealing of the ureter . We have long - term experience with excision of ureterovesical junctions using collin's knife . Previously we used it as a pluck technique combined with open and thereafter laparoscopic or retroperitoneoscopic nephroureterectomy, and, later on, we also used it as a part of antegrade mini - invasive nue . 's idea [18, 19] of endoscopically closing the excised ureter with lockable clip to be excellent, although the introduction of the hem - o - lok clip via endoscope appears to be difficult . Pathak et al . Performed their technique in 25 cases with a mean total operative time of 164 (range: 105 - 235) minutes . No pelvic complications in were reported and there were no perivesical tumor recurrences with mean follow - up of 26 (range: 11 - 44) months . We have decided to apply the hem - o - lok clip size ml via an intravesical 5 mm port introduced through the suprapubic area . Regarding our first nine cases, we had found the exact same method described in the literature . Suprapubic transvesical single - port technique for control of lower end of ureter during laparoscopic nephroureterectomy . The dissected nephroureterectomy specimen during the operation . The tumor can be seen in the upper calyx with hem - o - lok size ml at the end of the ureter (see detail). Reportedly, occlusion of the ureter may be performed instead of clip with electro - coagulation only (it is probably less reliable) or with fibrin sealant injection . Clnue- ivlc is a relatively simple, reproducible, and minimally invasive method with minimal risks of tumor spillage and seeding . The main disadvantage seems to be the risk of an unclosed defect of the urinary bladder, but based on our own experience, as reported in this paper and the available literature, we have not found any significant complications emerging from this . Another disadvantage is failure in applying the hem - o - lok clip, in which the technique is concluded without closing the ureter, and is generally thought to carry a higher risk of extravesical tumor recurrence, but as described recently, this technique has comparable oncological results to the open distal ureterectomy [3, 6]. Failures in clip application were experienced only in early cases and, with increasing experience, this problem was avoided . Importantly, if needed, the endoscopic phase can be transformed to open nue or it can be combined primarily with open surgery . Some may consider conversion to open distal ureterectomy / nephrectomy as a failure of the method . The method whereby closing of the ureter is performed allows the procedure (nue) to be completed safely in complicated cases (obesity, advanced cases, previous intraabdominal surgery etc . ).
Segmental copy - number variations (cnvs), involving the gain or loss of several hundreds of bases to several hundred kilobases (kb) of the genome, can be an important source of genetic variation among human populations of different ethnic groups as well as among individuals . Molecular genetics analyses and cytogenetic analyses have provided significant information about these variations in the human genome, specifically as they relate to disease, such as cancer, and to congenital malformation (see [1, 2], and review in). Following the development of methodologies and the introduction of new research platforms [49], information regarding the nature and pattern of cnvs from representative populations have accumulated . Examinations of a relatively large number of individuals from various specific ethnic groups have recently been conducted using different array platforms, such as bac - arrays [1013], oligo - arrays [1416], and others . The results are not always consistent and it is likely that different human populations bear different cnvs . The numbers of japanese individuals examined to date are not so large compared to the studies for other ethnicities . Polymorphic cnvs have received considerable attention since they might play an important role in the etiology of common diseases . Therefore, more data regarding cnvs should be accumulated from japanese populations . In this report, we focus on cnvs which were observed at a high frequency (5.0% of the individuals) in the population residing in hiroshima and nagasaki, japan by acgh with bac - clones as targets . In the study, the population studies were conducted at two stages: stage (1): 80 unrelated japanese individuals were examined using bac - acgh with an array having 2,241 bac clones, and stage (2): 133 unrelated japanese individuals were examined using bac - acgh that contained 2,622 bac - clones . The majority of the clones used in stage (1) of this study were selected from the set of cytogenetically mapped p1-artificial chromosome (pac) clones and bacterial artificial chromosome (bac) clones reported by the bac resource consortium and obtained from either the children's hospital oakland research institute (oakland, ca, usa) or from invitrogen inc ., co. (carlsbad, ca, usa). In stage (2), in addition to bac clones used in stage (1), an additional 381 bac clones were used, a majority of which were collaboratively obtained from dr . N. matsumoto of yokohama city university . The 2,241 clones of chromosomal fragments from chromosome 1 to chromosome 22 were used in stage (1) and 2,622 clones were used in stage (2), respectively . That is, the additional 381 bac clones were examined for only 133 unrelated individuals in stage (2). Those clones are distributed every 1.2 mb across all of the human autosomes in stage (1), and 1.1 mb in stage (2), respectively . In addition to autosomal clones, four kinds of x - chromosomal clones were used as internal references . With respect to examination for stage (1), three sets of arrays were constructed and imprinted: slide no.1, consisted of 698 clones on chromosomes 1 to 4; slide no.2 consisted of 718 clones on chromosomes 5 to 10, plus two bac clones on chromosome 3, and six clones on chromosome 4; and slide no.3 consisted of 817 clones on chromosomes 11 to 22 . For stage (2), all of the clones were printed onto one glass slid . The genomic dna samples used in this study were principally the same as those used in a previous study . The dna samples used for reference purposes were extracted from mononuclear cells of two physically and clinically normal volunteers (a 57-year - old japanese male and a 54-year - old japanese female). The dna used for testing and analyses of this population was extracted from lymphoblastoid cell lines obtained from the offspring of atomic - bomb survivors . High molecular weight genomic dna was isolated using conventional methods as described in detail elsewhere . Lymphoblastoid cell lines were derived from a cryopreserved archive of approximately 1000 families consisting of father, mother, and offspring from hiroshima and nagasaki for whom permanent cell lines have been established by epstein - barr (eb) virus transformation of peripheral b - lymphocytes . The composition of the families has been reported elsewhere . Three hundred five offspring were initially screened . Since the offspring include some siblings, we selected one representative offspring to construct unrelated individuals to avoid double counting of polymorphic cnvs from families containing two or more siblings . We selected the offspring who first visited our institution for donating blood rather than other siblings . The 213 offspring selected as unrelated individuals included 124 offspring from hiroshima and 89 from nagasaki . Cloned dnas for microarray targets were isolated from bacterial cultures using nucleobond bac 100 (nippon genetics, tokyo). With respect to stage (1), dna was digested by noti followed by phenol - chloroform - isoamyl alcohol (25:24:1) extraction and ethanol - precipitation . On the other hand, in stage (2), cloned dna was digested with msei followed by phenol - chloroform - isoamyl alcohol (25:24:1) extraction and ethanol precipitation . The fragmented dnas were amplified by ligation - mediated pcr carried out as described by snijders et al . . The target dnas (0.5 g/l) were dissolved in 50%-dimethylsuloxide and printed in triplicate onto the glass slides (matsunami glass co. ltd .) Using the affymetrix 417 arrayer (affymetrix). The screenings of both stages were conducted following the procedures described previously . In brief, for labeling dna, test and reference genomic dna (1.25 g each) was cut by bamhi, and labeled by a random priming method with cyanine-5- and cyanine-3-labeled dutp (cy5- and cy3-dutp; perkinelmer life sciences, wellesley, ma, usa). The labeled probes were mixed and centrifuged with microcon column (millipore co., bedford, ma, usa) to purify the probes . Subsequently, human coti dna (120 g; roche diagnostic gmbh, mannheim, germany) was added to the column, and recentrifuged . After the volume of the mixture became less than 20 l, it was transferred to microtubes with 100 l of hybridization solution (50% formamide, 10% dextran sulfate, 1% tween 20, 2 ssc, 10 mm tris - hcl [ph 7.4] and 800 g of yeast t - rna [invitrogen, carlsbad, ca, usa]). The hybridization mixture was then denatured at 70c for 10 minutes, and subsequently incubated at 37c for at least five hours to block repetitive sequences of the labeled probes . Prehybridization was conducted in order to block repetitive sequence binding of target dna on the arrays, and to prevent nonspecific binding of probe dna to the targets . Following the initial incubation (overnight at 37c), the prehybridization solution was removed, and fresh hybridization solution with cy - labeled dna (prepared as described above) was added . Again, hybridization processes were carried out . All of the procedures were conducted using the genetac hybridization station (genomic solutions inc ., ann arbor, mi, usa). Fluorescent images of the hybridized arrays were obtained using a scanarray 5000 confocal laser scanner (perkinelmer life sciences). Fairfax, va, usa) in stage (1) and gene pix (axon instruments, sunnyvale, ca) in stage (2), respectively, were used to quantify the fluorescence of each spot on the array images . Fluorescent ratios of the total integrated cy3 and cy5 intensities for each target (triplicate spots for each target) were calculated, along with the mean ratios of the triplicate spots (if the raw ratio of one of the triplicate spots differed by more than 10% from the other two, the value was automatically excluded from the mean ratio calculation). Normalized ratios were computed by dividing each raw ratio by the mean raw ratios of every autosomally mapped target (therefore, after normalization, the averaged ratio for all targets in one array was 1.0). The spots whose ratios were 2.58 standard deviations (sd) below the mean (1.0), or 2.58 sd above the mean, were marked cnv . To confirm the quality of each analysis, the ratio of x - linked clones was verified . Dna in the plugs was cleaved by restriction enzymes (paci, or sse8387i). The resulting fragments were then separated by pulse field gel electrophoreses (pfge) on 1% pulse field certified agarose (biorad, hercules, ca, usa) with 0.5 tbe (tris - borate - ethylenediaminetetraacetic acid). Using the chef - dr ii system (biorad), electrophoresis was carried out using 6 v / cm at 14c for 22 hrs . The angle of pulse was 120. the switch time was used: ramped from 0.3 seconds to 15 seconds . Southern blot analyses were carried out using conventional, well - described procedures . In brief, after completion of pfge, the dna in the gel was cleaved by uv irradiation and blotted onto nitrocellulose filters (schleiche & schuell, dassel, germany). The filters were prehybridized with human coti dna (48 g / ml, roche diagnostic gmbh) and salmon testis dna (14 g / ml, sigma - aldrich) to decrease the background due to repetitive sequences . Subsequently, the filters were hybridized with whole bac - dna as a probe . Dna probes were labeled with [-32p] dctp (amersham biosciences, piscataway, nj, usa) and preannealed with human coti dna and salmon testis dna (10.5 g / ml). Prehybridization and hybridization were performed overnight at 37c in a solution containing 50% formamide, 10% dextran sulfate, 1% tween 20, 2 ssc, and 10 mm tris - hcl (ph 7.4). After hybridization, the filters were washed at 65c with 1.0 ssc containing 0.1% sds (sodium dodecyl sulfate) and 0.5 ssc containing 0.1% sds . Banding patterns were obtained by either exposure to x - ray film (fuji film, tokyo, japan) or through use of the molecular imager fx (biorad). The qpcr was performed using sybr premix ex taq (takara - bio) and the light cycler system (roche diagnostics), according to the manufacturers' protocols . Primers were designed with primer3 software (http://primer3.sourceforge.net), and the size of pcr products was confirmed by the pattern of restriction enzyme digested fragments using the labchip dna 500 kit on the 2100 bioanalyzer (agilent technologies, waldbronn, germany). Two, 0.5, and 0.125 ng of genome dna from an individual with cnv were used and the quantification of each amplicon was carried out at 45 cycles of pcr . The results were analyzed with light cycler data analysis software using a second derivative maximum model . The main purpose of this paper is to report the accumulation of the data about highly polymorphic cnvs found in 5.0% of the individuals . (the number of cnvs was 11 or more in each bac - spot .) As shown in table 1, 680 polymorphic cnvs were observed on 16 bac - regions . As described before, the results of two bac (rp11 - 259n12 and rp11 - 121a8) were obtained from 133 unrelated individuals examined in stage (2). Southern blot analyses followed by pfge were carried out for the highly polymorphic cnvs . As shown by the typical cases in figures 1 and 2, the patterns of these two bac clones (rp11 - 79f15 and rp11 - 88l18) are shown in figures 1 (rp11 - 79f15) and 2 (rp11 - 88l18) as the typical examples . Since each individual contained a different number of core segmental duplication units, each individual showed bands having different motilities . The results of qpcr conducted for two bac clone regions are described in figure 3 (rp11 - 89b15) and figure 4 (rp11 - 79o18). For the former case, a part of a gene (meox2) was deleted . On the contrary, for the latter case, the copy number of a part of the gene (nsf) increased, but the copy number of the gene (wnt3) did not change . There are many segmental duplications which have already been summarized in a public data base, such as human genome segmental duplication database (tcag database; http://projects.tcag.ca/cgi-bin/variation/gbrowse), ucsc human genome browser (ucsc database; http:/genome.ucsc.edu / index.html), and ncbi map viewer (ncbi database; http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi). The cnv data obtained in our studies are summarized in table 1 in addition to the presence or absence of cnvs already reported in the databases . One bac clone, named rp11 - 115g22, was mapped on two chromosomes, 6 and 15 in tcag database, so it is likely that this clone is present on two discrete chromosomes . On the contrary, however, we accept the reports from the latter two databases and described that this clone was mapped on only chromosome no.15 . With respect to segmental duplications, 10 out of 16 (about 63%) were present in the above databases (ucsc database, and ncbi database). It was noteworthy that the majority of bac clones containing our cnvs were known to overlap to at least one cnv reported in the database . However, that does not mean that our highly polymorphic cnvs are exactly the same as those reported in previous reports, since precise comparisons between our study and the other studies were not carried out . As mentioned before, there was very little information about the cnvs of 45 japanese individuals for which relatively large sizes of population have been systematically screened . We compared our data with the data of japanese including the hapmap project as reported by redon et al . Ten out of 16 cnvs identified in our study were reported in the data reported by redon et al ., although we should emphasize again that the cnvs identified in our bac region are not exactly the same as those reported by redon et al . . Cnvs identified in our study with lower numbers tend to not be identified in redon's report . On the other hand, when our data are compared with redon's oligo - data from affymetrix 500ea array conducted for japanese, only two cnvs were overlapped to our cnvs . As redon et al . Mentioned in their report, the reason appears to be that oligo arrays have some limitations for a complicated genome, such as segmental duplication areas . We summarized the genes and disease - related genes in omim which overlap to the bac - clone region with our cnvs (table 2). In addition to those two categories, mrnas have been also reported in the database, but they are too many to describe here . All bac - clone regions contained at least one mrna, although the functions of a majority of those mrnas are not known yet (data not shown). We examined 213 unrelated japanese using bac - acgh and found a total of 680 cnvs on 16 bac clones . A large fraction of the regions involved in the cnvs observed in our study (i.e., 625 out of 680 (92%), table 1) have been reported previously in other studies listed in the database . A majority (63%) of the cnvs had been found on the bac clones that overlapped with segmental duplication, suggesting the notion that segmental duplication might play a significant role in the creation of cnvs (table 1). In which they reported the sharing of cnvs among several populations, meaning those specific genomic imbalances either predated the dispersal of modern humans out of africa or arose independently in different populations . On the other hand, our cnvs, especially those showing high frequencies, were also identified by redon's work . On the contrary, our cnvs showing low frequency, such as less than about 10% of individuals, were not observed in their work (table 1). That result suggests that those cnvs might be identified if the number of individuals examined by redon et al . Were increased to the level of our study (about 200 individuals). Moreover, as described before, when our data compared with redon's oligo - data conducted for japanese, only two of their cnvs were overlapped to our cnvs . Although the oligo - based method tends to detect smaller cnvs (about a few kilobases), this approach is less effective in tracking cnvs in genomic regions of complex structure, like segmental duplications, that are not sufficiently tagged by oligo targets . On the contrary, the bac platform can only identify larger cnvs (> 40 kb), but this method has some advantage for detecting cnvs present in the regions of segmental duplications . The cnvs in the human genome are often associated with developmental disorders and susceptibility to diseases . Large duplications and deletions have been known to be present within the human genome based initially on cytogenetic observations in the course of etiological studies of congenital malformations (e.g., [1, 2]). The frequency of those duplications and deletions was presumed to be low and, for the most part, directly related to specific genetic disorders . A limited number of studies reported the presence of specific large duplications and deletions that were not apparently related to diseases (e.g.,). Reported that cgh on a bac - dna - based microarray could reliably detect single - copy gene decreases or increases from normal diploidy . Following this, other array platforms, such as cdna, and oligo - nucleotide, have been developed and many data from them have been reported . As we mentioned before, many cnvs were reported to be closely related to disease phenotypes, and recent studies based on advanced molecular technologies, such as genomewide association studies [2729] and next generation sequencing [30, 31], reported that many genes appear to play important roles in the etiology of common diseases . We report our highly polymorphic cnvs in bac clones, which were reported to contain genes, expected to be related to phenotypic heterogeneity of each individual, based on the tcag database . We focused on genes reported in the above database, although many mrna were listed in the database in addition to genes (table 2). Bac clone (rp11 - 90a9) contains two genes: ankyrin repeat domain 34b (ankrd34b) and dihydrofolate reductase (dhfr). A phosphoprotein encoded by ankrd34b is induced during bone marrow commitment to dendritic cells which play an important role in vertebrate immunity . Dhfr genes were reported to be related to various malignancies including lymphoproliferative disorders such as systemic non - hodgkin's lymphoma, primary central nervous system lymphoma (pcnsl), and childhood acute lymphoblastic leukemia . Those two genes are fully overlapped to the bac clone (rp11 - 90a9). It is likely that the cnvs might affect the copy number of those genes . One bac clone (rp11 - 89b15) contains a gene mesenchyme homeobox 2 (meox2). The analyses by qpcr (figure 3) demonstrated that the copy number of gene (meox2) is deleted in the individual having cnvs detected by this bac clone . Meox2 suppressed epithelial cell proliferation in cooperation with tgf - beta1, and mediated induction of the cell - cycle inhibitor gene p21 . Finally, the data from genome wide association study (gwas) reported that this is one of the candidate genes that might be associated with ischaemic stroke . Another bac clone (rp11 - 115g22) was mapped on the chromosomes 15 . (chrna7). That gene was used as a candidate target for examining interactions on the severity of adult attention deficit hyperactivity disorder (adhd). Moreover, it was reported that the gene is one of the candidates for alzheimer's disease . The gene chrna7 is overlapped to the segmental duplication region of bac clone (rp11 - 115g22). Many cnvs were listed in the databases mentioned as above, and those are overlapped to the gene . For those reasons, our cnv appears to be overlapped to the genes, and it might affect the copy number of gene chrna7 . A bac clone (rp11 - 79o18) contains two genes which are n - ethylmaleimide - sensitive factor (nsf) and wingless - type mmtv integration site family, member 3 (wnt3). The qpcr results (figure 4) demonstrated that the copy number of a gene (nsf) increased but no change was observed in the gene (wnt3). That result suggested that the cnv might affect the nsf gene, but not the wnt3 gene . However, there may be an opportunity for the cnvs to become a surrogate marker of wnt3 for the future association study between the cnv and some disease phenotype . The nsf gene is one of the essential components of membrane fusion machinery which is an important homeostatic process in eukaryotic cells . A recent study showed that the nsf gene is a good candidate marker for association studies for genetic risk underlying parkinson's disease . The wnt3 gene's single nucleotide polymorphisms (snps) were used as candidate markers for association studies of hemorrhagic stroke, hypertension, and chronic kidney disease . Upregulation of the wnt gene family, including wnt3, suggested involvement of the wnt's canonical and/or noncanonical signaling pathway in chronic lymphocytic leukemia . A bac clone (rp11 - 79f15) contains two genes: methyl - cpg binding domain protein 3-like 1 (mbd3l1) and cell surface associated mucin 16 (muc16). The protein is localized to discrete areas in the nucleus, and expression appears to be restricted to round spermatids, suggesting that the protein plays a role in the postmeiotic stages of male germ cell development . On the other hand, muc16 is a member of the mucin gene family and encodes cancer antigen 125 (ca125) which is a blood biomarker routinely used to monitor the progression of human epithelial ovarian cancer (eoc), although its potential role in eoc is poorly understood . Maintenance of an intact mucosal barrier, one of whose components is a gene product of muc16, is critical to preventing damage and infection of wet - surfaced epithelia . As we demonstrated by southern blot analysis (figure 1), the polymorphism was caused by segmental duplications in the bac clone . The series of segmental duplications were on the 5-region of two genes (mbd3l1 and muc16). The cnvs do not affect the expression pattern of the genes, but these cnvs might be useful surrogate markers for future studies . Glutamate receptor, ionotropic, (ampa 2). Ampas are ligand - activated cation channels that mediate the fast component of excitatory postsynaptic currents in neurons of the central nervous system . Since the size of the gene (ampa 2) is larger than that of the bac clone (rp11 - 231j7), it is likely that the cnvs affect the copy number of the gene . A bac clone (ctd-2100f13) contains two genes: rio kinase 3 (yeast) (riok3) and niemann - pick disease, type c1 (npc1). Npc1 can have a function in the egress of certain membrane - impermeable lysosomal cargo . The membrane - bound npc1 and soluble npc2 play an important role for the release of cholesterol from lysosomes . As a result of that mechanism, the gene is associated with obesity [5658]. The bac clone (ctd-2100f13) is fully overlapped to both of the two genes (riok3 and npc1). The size of cnvs summarized in the databases shows that the reported cnvs were overlapped to both genes . We assumed that our cnvs may reflect the change of copy number of the genes themselves . Since the genes mentioned are good candidate markers for enabling us to examine the etiology of common diseases and phenotypical heterogeneities among individuals, our highly polymorphic cnvs should be able to become good markers in future studies . We are currently planning to examine the same population using a high - density oligo - array platform to accumulate more cnv data for japanese . The reason, as mentioned before, is that the bac platform and oligo methods complement each other . The oligo platform is known to be more effective in detecting smaller cnvs (around a few kilobases), even though this approach is less effective in tracing cnvs in genomic regions of complex structure that are covered by the bac approach conducted in this study . We expect to construct a more definite japanese cnv database by the combination of bac- and oligo - platform arrays . We conducted population screening for 213 unrelated japanese, and observed 680 highly polymorphic cnvs . The majority of the polymorphic cnvs presented on bac clones that overlapped with regions of segmental duplication, and had been previously reported in other publications . Moreover, it is expected that the cnvs might be good surrogate markers for detecting etiological genes, even if cnvs did not directly affect the genes themselves.

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