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shear has estimated about 9% while donath about 4% of dentigerous cysts to occur in the first decade of life. developmental type of cyst develops in a mature tooth as a result of fluid accumulation. inflammatory type develops in an immature permanent tooth as a result of spread of inflammation from an overlying non - vital primary tooth. larger cysts may cause springiness of the bone, expansion of cortical plates and mild sensitivity without any pain. radiographically, the cyst presents as a well - defined unilocular radiolucency surrounding a crown of unerupted tooth. histologically, the cyst consists of a fibrous wall lined by non - keratinized stratified squamous epithelium of myxoid tissue, odontogenic remnants and rarely, sebaceous cells. the latter approach is preferred for larger cysts and is especially helpful in pediatric patients to conserve the unerupted permanent successors. a 10-year - old female patient was referred to our department in january 2011 with the chief complaint of painless swelling of one - month duration in the left side of the lower jaw. on extra - oral examination, a slight buccal bulge was present in the mandibular left posterior region [figure 1 ] which was felt hard on palpation. intra - oral examination revealed the presence of grossly carious mandibular primary left canine (73) and mandibular primary left first molar (74) with distally tipped mandibular permanent left lateral incisor (32). we advised an orthopantomogram x - ray to the patient which revealed the presence of a well - defined unilocular radiolucency in relation to the roots of 73, 74 and mandibular primary left second molar (75). the radiolucency also involved unerupted mandibular permanent left canine (33), first premolar (34) and second premolar (35). we advised the patient to have a computed tomography (ct) scan of the region. the ct scan showed a well - defined radiolucency involving 33, 34 and 35 with expansion of buccal and lingual cortical plates [figures 3 and 4 ]. the dimensions of the radiolucent lesion were approximately 25.5 36.0 mm. based on clinical and radiological findings, a provisional diagnosis of dentigerous cyst was made. initial preoperative photograph with buccal swelling clinical view showing swelling in relation to 74 and 75 preoperative ct scan showing a large well - defined radiolucent lesion ct scan (occlusal view) showing expansion of buccal and lingual cortical plates extractions of 73, 74 and 75 were done under local anesthesia which led to opening of the cavity. we also got the tissue from the cavity which was sent for histopathological examination showing wall of the dentigerous cyst lined by stratified squamous epithelium having features of inflammation including numerous proliferating blood vessels and mixed inflammatory cells [figure 5 ]. microphotographs showing (a) wall of dentigerous cyst lined by stratified squamous epithelium (shown by arrow h and e, 100), (b) other area of cyst having features of inflammation including numerous proliferating blood vessels and mixed inflammatory cells (h and e, 100) the cortical plates were compressed and the cavity was left open for continuous drainage. a space maintainer was given with an acrylic and wire extension into the cystic cavity to keep the cavity open. space maintainer was removed after six months as there was closure of the cystic cavity as the permanent teeth moved upwards. nine - month follow - up examination in october 2011 revealed the clinical presence of 34 and 35 in the oral cavity [figure 6 ] and almost complete bone healing [figure 7 ]. the extra - oral swelling caused by expansion of the buccal cortical plate also disappeared completely [figure 8 ]. nine months follow - up clinical view with erupted 34 and 35 nine months follow - up ct scan showing almost complete bone healing nine months follow - up photograph with no buccal swelling inflammatory dentigerous cyst (idc) is a type of dentigerous cyst which is found in mixed dentition only. it develops when the inflammation present at the root apex of a non - vital primary tooth spreads to involve the follicle of the unerupted immature permanent successor. in our case the infection present at the root apex of a grossly carious and non - vital 74 spread to involve the follicle of 34 resulting in formation of idc. in the differential diagnosis of idc, an odontogenic keratocyst, unicystic ameloblastoma odontogenic keratocyst and unicystic ameloblastoma occur in the second and third decade of life and are found in the molar region of the lower jaw. radiograph alone can not differentiate the above mentioned lesions so a histopathological examination should be performed wherever possible. however, as suggested by kozelj and sotosek in 1999, leaking out of cystic fluid during an extraction of a primary tooth or during a decompression, respectively, confirm the clinical impression of the cyst. in our case, histopathological examination as well leaking out of the fluid during the extraction of primary teeth confirmed the diagnosis of idc. marsupialization or decompression technique has been advocated by several authors for treating dentigerous cyst in young patients. in this conservative technique, creation of an accessory cavity helps to relieve intra - cystic pressure and accelerate the healing of the cystic lesion. the permanent teeth generally erupt in the oral cavity with or without the need of orthodontic correction. however, the patient should be followed up until the complete eruption of permanent teeth in their designated location. from the foregoing discussion, it is concluded that marsupialization technique is an ideal approach to treat large dentigerous cysts in pediatric patients. however, the follow - up of the patient should be done until the complete eruption of permanent teeth in their right location in the oral cavity. it is observed that, if permanent teeth are left undisturbed then as the cyst heals in due course of time, these permanent teeth erupt in the oral cavity. | dentigerous cyst may be developmental or inflammatory in origin. the latter is found only in mixed dentition with a low frequency. treatment of inflammatory type of dentigerous cyst in children should be done with the aim of saving developing permanent teeth which should not be sacrificed as far as possible. this is a case report of a large inflammatory dentigerous cyst in a 10-year - old female patient treated conservatively by marsupialization method saving all teeth (mandibular permanent left canine, first and second premolars) in relation to the cyst. |
transcatheter procedures for treatment of diseased heart valves in selected patients are of increasing importance, with promising results following transcatheter aortic valve implantation (tavi). the number of catheter - based isolated aortic valve surgeries in germany tripled from around 2,500 procedures in 2009 to around 7,500 procedures in 2011 [1, 2 ] ; 58.6% of these have been performed in patients with an advanced age of 80 to 89 years. without the necessity for open surgery and cardio - pulmonary bypass, these percutaneous procedures are of particular benefit to multi - morbid patients. the demand for alternative, less invasive treatment options, which avoid the use of a cardio - pulmonary bypass, is rapidly growing as the number of patients of advanced age is further increasing due to demographic changes. in 2010, the cohort of seniors aged 65 and over was 13.1% of the total population in the usa and this number is projected to increase to 21.6% in 2025. next to tavi, the catheter based treatment of the atrio - ventricular valves in the beating heart is focus of recent research. the main challenge is that, unlike in the aortic or pulmonary position, no conveniently defined adjacent structures there are no convenient adjacent defined structures for device anchoring. the first experimental off - pump transcatheter stent implantation into the mitral valve via the left atrium implantation via the left atrium into the mitral valve has been reported by ma. in 2005. seven years later, the first - in - human transfemoral transcatheter mitral valved stent implantation was performed by sndergaard and colleagues in copenhagen in june 2012. an 86-year - old male suffering from severe mr was treated and survived for 2.5 days. simultaneously, our group has reported multiple studies, showing the feasibility of successful mitral valved stent implantation via transapical approach with follow up times of up to two months, in which a self - expanding valved stent was implanted in the native mitral position under transesophageal echocardiographic (tee) guidance in the beating heart. the nitinol stent frame (euroflex gmbh, pforzheim, germany), comprised of a ventricular stent body and an atrial element connected at a preset angle, was covered with a polytetrafluoroethylene (ptfe) membrane (zeus inc., orangeburg, sc, usa) to minimize paravalvular leakage (pvl). a tri - leaflet bovine pericardial or native porcine aortic heart valve was sewn into the ventricular body and a ventricular fixation system, consisting of four individual neo - chordae, was attached to the ventricular rim of the stent (figure 1) mitral valved stent for transapical implantation into the beating heart. a : atrial view ; b : ventricular view showing the ventricular fixation system consisting of four neo chordae attached to the ventricular rim of the valved stent. in the in vivo trials this was achieved by an adequate valved stent design, a well functioning delivery system and deployment strategy, and a professional interdisciplinary team. first prototypic designs included a star - shaped atrial element to prevent stent migration into the left ventricle (figure 2a) [7, 10,11,12 ]. iterative changes of this design resulted in a crown shaped atrial element connected to the ventricular body at a preset angle of 45 (figure 2b). in first studies 30 pigs underwent off - pump mitral valved stent implantation with follow up times of 60 min (n=17) and 7 days (n=13). transesophageal echocardiography and computed tomography were used to evaluate stent function and positioning following a standardized protocol. after valved stent deployment, accurate adjustment of the intra - annular position reduced pvl in all animals. no valved stent migration, embolization, systolic anterior movement or left ventricular outflow tract obstruction was observed. these studies proved the feasibility of reproducible deployment of the mitral valved stent that achieved low gradients across the left ventricular outflow tract, and adequate stent function in acute and short term experimental settings. stent mal - deployment and stent fracture were two of the main complications seen throughout these studies. a further study focused on the evaluation and comparison of two different designs of a mitral valved stent. in the first case the stent was designed with a circular crown - shaped atrial element connected to a tube shaped ventricular element while in the second case, the same atrial element was d - shaped to achieve better anatomical alignment (figure 2c). the design with d - shaped atrial element depicting the native annulus showed promising results in hydro - static in vitro testing in relation to reduction of pvl and stent alignment. in vivo, a rotation of the stent in the mitral annulus was observed which caused more severe pvl and prevented these advantages to take effect [data not yet published ]. different prototype designs resulting from iterative development of the mitral valved stent. a : valved stent with star shaped atrial element ; b : valved stent with crown shaped atrial element attached at 45 to the ventricular element, c : valved stent with a d - shaped atrial element for better alignment in the antero - medial area. ten days or one month after successful implantation a cardiac computed tomographic (ct) evaluation was performed following a standard protocol. analysis of the cardiac ct data allowed evaluation of different stent design related and anatomical parameters (figure 3). a : 3d transesophageal echocardiographic image showing the valved stent after successful deployment in the native mitral annulus during systole. b : 3d reconstruction from cardiac computed tomographic data showing correct position of the valved stent within the native mitral annulus. in the group of design - related parameters, the angle between the ventricular and atrial element (atrioventricular junction) of the valved stent showed to be a critical parameter. in the early prototypes this angle was preset to 45. in vivo - evaluation showed great deflections of this preset angle of up to 56.414.5. design iteration of our valved stent included an increase of this angle to 110. analysis of the deformation derived from ct data of these optimized stents revealed a lower degree of deflection [data not yet published ]. reduced deformation lowers the mechanical stresses and hence can increase the long - term durability. correct alignment within the native anatomy of the left heart and good paravalvular sealing was achieved with the latest prototype in our in vivo experiments. this is crucial, as the occurrence of paravalvular leakage (pvl) is directly linked to the patients well - being. of particular importance was that no pvl were found in twelve out of twelve animals directly after implantation in our most recent series of valved stent implantation [data not yet published ]. however, an adaptation of the circular stent shape to the oval anatomical shape of the native mitral annulus was detected in this series leading to central mr in some cases. in - growth of the mitral valved stents into the surrounding tissue was satisfactory after one or more months with similar results in our studies. we consider the tissue in - growth to be of high importance for the secondary stability and long - term performance of the mitral valved stents. studies investigating the biological responses of different intracardiac devices showed complete coverage with endothelial cells after three months [14, 15 ]. therefore we assume that a similar healing process is achievable with our valved stent, since we have also observed good secondary stability up to now (figure 4). gross evaluation two month after successful implantation showing good ingrowth of the valved stent and adequate stent position. a : atrial view showing the ingrowths of the atrial element ; b : ventricular view showing the correct stent position and ingrowth of the ventricular element. histological evaluation was carried out in samples taken at defined regions of interest such as the adhesion on the atrial element and the mitral annulus. movat s pentachrome is a standard staining method for cardiac tissue and was used to differentiate between different collagen types, matrix, fibrin structures and muscle cells. generally, normal myocardial tissue with an endothelial cell layer towards the lumen was detected. in the animals evaluated after 4 weeks or more of instead normal atrial tissue was found in the atrial adhesion with muscle cells and matrix containing mycin and collagen [data not yet published ]. however, before attempting the step towards clinical application of the novel device, more long - term in vivo data are necessary (no central leakage, maintaining left ventricular function). percutaneous implantation of a tricuspid valved stent into the beating heart is investigated in only few studies. these studies either require preliminary tv operations (replacement or conduit implantation) or suggest heterotopic implantation within the vena cava. reported their first steps towards the replacement of the tv using angiographic guidance in 2005 mentioning difficulties of deploying and securing a stent in the tricuspid position. in 2012, pott. suggested a novel approach consisting of two tubular anchoring components positioned within the tricuspid annulus and the superior vena cava, which are connected by elastic elements to provide stability. our group has reported on the feasibility of off - pump replacement of atrioventricular valves under transesophageal echocardiographic (tee) guidance and presented first results of an acute study. in this study, a nitinol stent was developed composed of a right atrial anchoring element and a right ventricular tubular stent that carried a trileaflet bovine pericardial valve. the self - expanding nitinol stent frame was covered with a ptfe membrane and pouch filled with a super absorband polymer (sap) was attached to the ventricular stent body. this pouch was specifically designed for sealing between native tricuspid annulus and nitinol stent frame (figure 5). valved stent for transventricular implantation into the native tricuspid valve in the beating heart after short term in vivo evaluation. the stent was implanted into the native tricuspid annulus of seven pigs in the beating heart using a transventricular approach and tee guidance. short - term evaluation was conducted 1h and 6h after successful implantation following a standard protocol. in six of seven cases the valved stent was correctly deployed and positioned within the native tricuspid annulus and normal hemodynamics were maintained. mild pvl were observed directly after implantation in two cases, but decreased throughout the observation period. this was related to the full expansion of the sap sealing pouch in the time between the two evaluation points. | the development of transcatheter techniques for treatment of severe mitral valve regurgitation in the beating heart is focus of recent research. an off - pump treatment technique poses great benefits, particularly for multimorbid patients, often being non - compliant to the gold standard treatment, being open heart surgery with use of a cardiopulmonary bypass. thereto, two approaches are being followed : transcatheter valve repair and transcatheterimplantation of a valved stent into the native mitral valve annulus. a valved stent has to provide safe and secure fixation within the high pressure system of the left heart. one of the main challenges in the development of such a valved stent is the complex anatomy of the mitral valve, with no clearly defined structures for device anchorage. our group has developed a self - expanding nitinolvalved stent for transapical implantation in the beating heart. during the development process of thevalved stent, different design iterations were conducted to decrease the risk of paravalvular leakages, to enhance the reproducibility and to improve the overall stent performance. this article reviews the major milestones passedin the development process of our mitral valved stent and advances achieved withinthe last years. multiple design iterations lead to a prototype providing secure stent deployment, hig h reproducibility, low paravalvular leakages and only mild stent deformation in the beating heart. in future, further long - term in vivo trials have to be conducted before attempting the step towards clinical application of this novel device. |
the nature of the forces involved in partitioning chromosomes has been an active area of research for more than 50 years. edwin taylor and bruce nicklas were among the first to consider the forces that resist chromosome movement. separate theoretical analyses predicted that 0.1 pn would be required to move a chromosome at 1 m / min when resisted only by viscous cytoplasmic drag (nicklas, 1965 ; taylor, 1965). almost 20 years after publishing his theoretical work, nicklas was able to test the force on a single chromosome during anaphase of meiosis i (nicklas, 1983). using a microneedle to measure the stall force on chromosomes in grasshopper spermatocytes, nicklas found that 700 pn could act on a chromosome (nicklas, 1983). he estimated that the kinetochores tested in these studies were bound by 15 microtubules (nicklas, 1983), suggesting that each microtubule may be capable of generating up to 45 pn of force. in a later study, nicklas determined that 50 pn of force was produced on a chromosome during prometaphase (nicklas, 1988). this calculation was based on observations of chromosome congression and correlations with his previous work. by nicklas own admission, the microneedle assays to measure the force exerted on anaphase chromosomes had a high associated error, and it is unknown whether forces in the hundreds of piconormals would ever be produced at a kinetochore in the absence of a perturbation. regardless, no other work since has provided a more exact measurement, and 700 pn remains the standard reference value for the force that can act at a metazoan kinetochore. as nicklas work suggested, it is likely that the force felt by kinetochores varies throughout the cell cycle and under different types of attachments (discussed later in this paper). in particular, the arrangement of paired sister chromatids attached to opposite spindle poles during metaphase would allow for the greatest tension to be applied to kinetochores. recent work visualizing sister chromatid oscillations during metaphase has observed that at time points immediately before the switch from poleward to antipoleward motion, the poleward - moving kinetochore experiences the highest forces, at least as judged by changes in intra- and interkinetochore distances (dumont., 2012 ; in addition, the antipoleward - moving kinetochore may experience passive forces (inou and salmon, 1995 ; maddox., 2003) however, the magnitude of force during these directional switches and how this force is accommodated continues to be a subject of debate. as the higher order organization of kinetochores remains unknown, it is unclear how the forces from the multiple microtubule interactions at a single kinetochore are combined or what force is experienced by an individual protein within the kinetochore structure. with the discovery of the potentially large forces produced at kinetochores (nicklas, 1983), a major challenge has been to define the mechanisms by which this force is generated. many initial studies focused on the contributions of the microtubule - based motors, dynein and kinesin, that were found to localize to kinetochores (inou and salmon, 1995). the ability of these motors to transport cargoes along microtubules suggested that they might function similarly to move a chromosome within a cell. individual kinesin and dynein motors have been shown to stall under 57 pn of opposing force, termed a load (visscher., 1999 ; gennerich., 2007), and the combined action of multiple motors could generate the forces that nicklas observed. however, subsequent studies have found that chromosome movement can still largely occur in the absence of these motors in fungi (cottingham., 1999 ; grishchuk and mcintosh, 2006). in metazoans, motors, including the kinesin cenp - e and dynein, contribute to chromosome segregation (sharp., 2000 ; an alternative hypothesis was that the microtubules themselves generated the force to move chromosomes (inou and salmon, 1995). several early studies provided evidence that microtubules could direct the movement of isolated chromosomes under conditions that would not permit motor protein function (koshland., 1988 ; coue. this microtubule - derived movement could be caused by forces generated either at the kinetochore by microtubule depolymerization (grishchuk., 2005) or at the spindle poles as a result of poleward flux and microtubule disassembly at the minus end (lafountain. in fact, subsequent work suggested that the stall forces measured by nicklas were a result of minus end microtubule disassembly in equilibrium with the plus end microtubule polymerization caused by the application of tension via the microneedle (lafountain. although it is now generally accepted that microtubules generate the primary forces responsible for chromosome movement, kinetochore - localized motors may generate some force, act as a back - up system when kinetochore capture by microtubules fails (kapoor., 2006), generate tension via the production of the polar ejection forces (mazumdar and misteli, 2005), function to distribute force over additional linkages, and regulate microtubule dynamics (bader and vaughan, 2010 ; al - bassam and chang, 2011). in addition to forces generated either directly or indirectly by the microtubules, a third model proposes that the chromosomes themselves may contribute to the segregation process because of entropic forces that act on the dna (jun and wright, 2010 ; finan., 2011). although such forces would likely be very small, they may assist chromosome distribution, particularly in smaller cells. in support of a primary role for microtubules in generating force at kinetochores, microtubules have been shown to generate pulling force during their depolymerization in vitro (grishchuk., 2005 ; powers., 2009 ; akiyoshi., 2010, gtp - bound tubulin dimers are added to the growing microtubule plus end (desai and mitchison, 1997). the resulting gdp - bound tubulin dimers associate with each other along an individual protofilament and between neighboring protofilaments within the microtubule lattice to maintain a straight microtubule (nogales, 2000 ; nogales and wang, 2006). however, when a microtubule switches to depolymerization, a process termed catastrophe, gdp - bound dimers exposed at the microtubule end lose these stabilizing interactions, causing the protofilaments to peel backward. according to measurements and calculations by grishchuk. (2005), the conformational change that occurs for an individual depolymerizing protofilament can generate a power stroke of 5 pn, suggesting that a depolymerizing microtubule composed of 13 protofilaments could generate as much as 65 pn of force. importantly, to harness this force and ensure proper chromosome movement, it is critical to control microtubule polymerization and depolymerization at kinetochores. the formation of kinetochore microtubule attachments as well as the resulting tension may directly modulate microtubule dynamics by slowing microtubule depolymerization and decreasing the rate of catastrophe (franck., 2007 ; akiyoshi., 2010 ; umbreit., 2012). in addition, microtubule polymerization factors, such as the tog (tumor overexpressed gene) domain proteins xmap215 and clasp, and depolymerases, such as kinesin-13 proteins, which are present both at the kinetochore and on the spindle, also modulate microtubule behavior (bader and vaughan, 2010 ; al - bassam and chang, 2011). although microtubule depolymerization has the capacity to generate force, a key question is how chromosome movement is coupled to microtubule depolymerization. thus far, two models have dominated the literature to explain how kinetochores harness the force from microtubule depolymerization, although these models are not mutually exclusive. the first model, termed the hill sleeve model or (hill, 1985), postulates that the association of the kinetochore with a microtubule is formed by multiple weak interactions that can diffuse equally in either direction. however, because of a large free energy barrier that disfavors the loss of an interaction, this diffusion is biased toward the microtubule minus end as binding sites disappear from the plus end. the second model, termed the forced walk model (molodtsov., 2005), proposes that the kinetochore is coupled to microtubules in such a way that, as the protofilaments peel backward during depolymerization, the coupling protein is pushed along the microtubule. the way in which the microtubule is connected to the kinetochore has important implications for understanding how the force manifests at the kinetochore and remains an important focus for future work. recent studies have focused on how kinetochores and kinetochore proteins harness the energy from microtubule depolymerization. microtubule interface, such as the ndc80, dam1, and ska1 complexes (mcintosh., 2009 ; lampert., 2010 ; tien., 2010 ; schmidt., 2012) for their abilities to track on depolymerizing microtubules, and have attempted to analyze the kinetochore as a whole using partial purifications of kinetochores from saccharomyces cerevisiae (akiyoshi., 2010). although individual protein complexes and isolated yeast kinetochores are able to move with depolymerizing microtubules, studies performed using optical tweezers have found that the tested proteins and complexes are able to withstand less than 10 pn of pulling force before a rupture event is observed (powers., 2009 ; akiyoshi. this is in contrast to the theoretical maximum of 65 pn that a microtubule has been proposed to produce during depolymerization (grishchuk., it is likely that in the context of a kinetochore assembled on a chromosome, the complex architecture of the kinetochore has the capacity to harness and withstand larger forces. thus, the in vivo load - bearing properties of the kinetochore likely depend on a combination of the properties of both the individual protein components and the organization of the entire complex. during mitosis, it is critical that paired sister chromatids attach to opposite spindle poles. when this biorientation fails, this error must be detected and corrected, and a signal to delay cell cycle progression must be produced to prevent chromosome missegregation. (1995) demonstrated that the external application of force to a chromosome using a microneedle could overcome the checkpoint signal generated by an unattached kinetochore. this and other work have supported the model that the tension produced on bioriented sister kinetochores can alter the signaling state of the kinetochore. this tension results in two apparent physical alterations to mitotic chromosome structure : an increase in the distance between paired sister kinetochores and an increase in the distance between the inner and outer kinetochore regions of a single kinetochore. under some conditions, this inter- and intrakinetochore stretch can be uncoupled (maresca and salmon, 2009), and recent research has focused on the importance of intrakinetochore stretch in modulating the signals that monitor attachment state. by measuring the relative spatial positions of the different kinetochore proteins, work from several groups has found that kinetochore structure is altered when chromosomes are bioriented relative to conditions of reduced tension (maresca and salmon, 2009 ; uchida., 2009 ; wan., 2009 ; suzuki., 2011 ; dumont., biorientation results in the separation of inner kinetochore components (such as cenp - a and cenp - c) from outer kinetochore components (such as ndc80 and mis12) as well as changes in the spatial distribution of other proteins within the kinetochore and possibly conformational changes within the proteins themselves. because the generation of tension is dependent on the presence of opposing forces, changes in kinetochore structure in contrast, when one sister kinetochore lacks an attachment to the spindle (monotelic), or if both kinetochores attach to the same pole (syntelic), it is not possible to generate similar opposing forces. however, even in these cases, some force may still be present because of the viscosity of the cytoplasm resisting chromosome movement (nicklas, 1965 ; taylor, 1965) or the action of chromokinesins that generate polar ejection forces (mazumdar and misteli, 2005). it remains unclear how force is exerted on a single kinetochore that simultaneously attaches to opposing spindle poles (merotelic) or how these incorrect attachments are resolved (gregan. the observed structural changes at kinetochores have been assumed to correlate with the presence of tension, but thus far, such studies have not made direct measurements of force or tension. nevertheless, careful quantitative analysis of the dynamic changes in the distances between cenp - c and hec1 or cdc20 during sister chromatid oscillations has supported the model that changes in intrakinetochore distance are force dependent (dumont., 2012). however, these structural alterations may also be the result of changes in the conformation, organization, or localization of proteins within the kinetochore. ultimately, it is important to translate the mechanical signals produced by force at kinetochores into a chemical signal that regulates the activities of kinetochore proteins. a key player in correcting errors in microtubule attachment state is the aurora b kinase. substrates for aurora b show tension - sensitive phosphorylation ; they are highly phosphorylated in the absence of tension and become dephosphorylated upon biorientation (liu., 2009 ; welburn., 2010) the forces generated at kinetochores have been implicated in controlling aurora b signaling by altering the spatial separation between the kinase and its substrates (tanaka, 2002 ; liu., 2009), although other models for tension - sensitive aurora b phosphorylation have also been proposed (sandall., 2006). the key substrates of aurora b are located at the outer kinetochore and can be > 100 nm away from the majority of aurora b, which is localized at the inner centromere, depending on whether the sister kinetochores are under tension (wan., 2009). therefore, structural changes caused by opposing force at kinetochores separate the kinase and its substrates. the increased separation under tension makes aurora b less likely to phosphorylate its now distant substrates (liu. one effect of aurora b phosphorylation on outer kinetochore proteins is to reduce their microtubule binding affinity (cheeseman., 2006 ; welburn., 2010 ; schmidt. thus, it has been proposed that the presence of tension can ultimately stabilize microtubule attachments through changes in kinetochore conformation that cause a decrease in aurora b phosphorylation, which in turn increases the microtubule binding activities of various kinetochore components. in addition to altering the signaling state of kinetochores, changes in force at kinetochores may also have a direct effect on microtubule binding. one recent study suggested that outer kinetochore proteins are force sensitive and show catch slip properties (akiyoshi., 2010), resulting in less frequent detachment under increasing force. chinese finger trap and would allow the attachment to become stabilized as the microtubule pulls on the kinetochore. whether tension affects kinetochore microtubule attachments directly or indirectly, force appears to play an essential role in establishing and signaling biorientation in addition to driving chromosome movement. force is a vector quantity that, when applied to a bond, decreases bond energy barriers, increasing the likelihood of bond breakage. although the kinetochore must function under force to perform its roles properly, this force also represents a challenge with the potential for deleterious consequences to kinetochore function. force could result in protein unfolding or the breakage of protein protein interactions (fig. if a core kinetochore protein unfolded or if protein interactions within the kinetochore were disrupted, the connectivity between centromeric dna and the microtubules would be compromised. the typical force required to unfold a protein or break interactions is in the range of 10100 pn (weisel., 2003 ; lin., 2005 ; kumar and li, 2010). nicklas did not observe an immediate rupture of chromosome spindle attachments even while applying 700 pn on chromosomes, suggesting that the kinetochore is constructed in a way that can withstand high loads. models for force response at kinetochores at both the individual protein level and global scale. (a c) we propose three nonexclusive models for how kinetochores respond to the application of force : kinetochore proteins with elastic properties could serve to absorb some of the force produced by depolymerizing microtubules (a), multiple weak interfaces could form parallel attachments between the depolymerizing microtubule and chromosome such that the force produced by the microtubule would be diffused across multiple connections (b), and additional kinetochore components could serve as dynamic cross - linkers to diffuse force and add interactions between pairs of proteins to strengthen the protein protein interface (c). the kinetochore protein components themselves could have multiple responses at a molecular level including that (1) under pulling forces, the bonds holding together the tertiary and secondary structure of a protein can break, causing the protein to unfold. if reversible, this would provide elastic properties, but if permanent, could lead to loss of functional kinetochore components. (2) the force generated by kinetochores is directed toward the limited number of protein dna interactions formed between the kinetochore proteins and the chromosome. some tension may be relieved as the dna wrapped around adjacent nucleosomes is pulled. this first results in the straightening out of the compact beads on a string structure, but with sufficient pulling force, the nucleosomes would be removed from the dna. (3) protein protein interfaces held together by noncovalent bonds can break under pulling force, but the presence of additional proteins to strengthen interactions could prevent the loss of important interfaces. at kinetochores, rupture events caused by force - dependent protein unfolding or the loss of protein protein interfaces are likely avoided at least in part through the architecture and organization of the kinetochore. previous theoretical work on the effects of force on protein structure and protein protein interactions has highlighted organization and arrangement as key features for facilitating force resistance (leckband, 2000 ; evans, 2001). in a series arrangement, bonds are organized linearly such that the full force is felt by each component. however, in a parallel arrangement, the force is divided over multiple attachments arranged in parallel so that the force felt by each attachment is greatly reduced. the higher order organization of the kinetochore could diffuse the microtubule - generated force over multiple attachments, significantly decreasing the force that is felt by an individual kinetochore protein molecule. although the kinetochore clearly has evolved mechanisms to accommodate potentially large cellular forces, our understanding of the architecture and organization of a kinetochore remains limited. at the level of the minimal molecular path between a microtubule and centromeric dna, the proteins involved appear to be connected linearly (fig. 1 ; gascoigne and cheeseman, 2011 ; gascoigne., 2011 ; bock. however, there are multiple connections formed between the centromere and a single microtubule. for example, as many as 1020 kinetochore - localized ndc80 complexes have been measured as associating with each microtubule in both fungi and vertebrate cells (joglekar., 2006, 2008 ; johnston., 2010 ; the complexities of these connections have proven a hurdle to devising methods to measure the force produced by microtubules on specific kinetochore components or the total force exerted on the kinetochore during normal mitotic processes. in addition to defining the forces that kinetochore proteins experience, the amount of force necessary to break a bond depends on both the loading rate (force / time) and the duration of the applied force (merkel., 1999). for the kinetochore, the extended periods of force experienced during metaphase (in which sister chromatids move under force in one direction for 12 min ; mitchison and salmon, 1992), as well as the rapid changes in force that occur during sister chromatid oscillations, have the potential to result in a high loading rate and extended durations of applied force. as such, it will be important to account for the way that these challenges are accommodated at kinetochores. several calculations have estimated the power output of the grasshopper and yeast spindles (nicklas, 1988 ; bloom, 2008) and provided indirect measures for the spring constant of the kinetochore based on analysis of the chromatin spring constant during anaphase (fisher., 2009). however, as a result of experimental limitations, it has not been possible to precisely determine the force constant and other key force parameters at kinetochores. without knowledge of the force constant, it is not possible to calculate the loading rate experienced by a kinetochore. 2). the kinetochore is assembled on centromeric dna, but if the kinetochore chromatin interface were disrupted, kinetochore function would be lost. one way in which this force could be accommodated is that the force applied through the kinetochore displaces nucleosomes in pericentric regions, alleviating the mechanical stress experienced by the kinetochore itself (bouck and bloom, 2007 ; verdaasdonk., 2012). studies of the chromatin force response in s. cerevisiae have shown that a deformation of chromatin structure occurs in the regions immediately surrounding the centromere during mitosis (pearson., 2001 ; bouck and bloom, 2007) and that there is an increased turnover of nucleosomes in these surrounding regions (verdaasdonk., 2012). these studies have obtained values of between 4 and 20 pn to irreversibly remove a nucleosome from dna, depending on the specific approach and source of nucleosomes that was used (cui and bustamante, 2000 ; bennink., 2001 ; brower - toland., 2002 ; yan., 2007). for these studies, force was applied to the ends of the dna rather than perpendicular to the dna strand as would occur at kinetochores. this difference in the directionality of force may alter the amount of force necessary to remove a nucleosome from chromatin under mitotically applied forces. nucleosome displacement and chromatin stretching in pericentric regions could allow the chromosome to absorb some force. centromere interface. at centromeres, there are two key connections between kinetochore proteins and the underlying dna (gascoigne and cheeseman, 2011 ; gascoigne., 2011). the first occurs through the histone h3 variant, cenp - a, which epigenetically defines the centromere and forms the main site of attachment for cenp - c (fig. the other occurs via the recently identified cenp - t w s x histone fold complex, which forms a heterotetrameric nucleosome - like structure (nishino., although adjacent nucleosomes surrounding the centromere could be displaced in the presence of force without severe consequences, the loss of the interaction of cenp - a or cenp - t with dna would eliminate kinetochore function. both the cenp - a nucleosome and the cenp - t w s x complex are structurally distinct from canonical nucleosomes (sekulic., 2010 ; nishino., 2012), raising the possibility that they may have different force resistance properties. future work characterizing the behavior of these specialized nucleosomes and the other kinetochore components will be important to understand how intrakinetochore and kinetochore dna attachments are maintained in the presence of force. although the roles of force at kinetochores have been a focus of recent work, less is known about how kinetochores are able to accommodate the forces generated at these sites. recent work has isolated kinetochore particles from budding yeast (akiyoshi., 2009) and partially reconstituted kinetochores from xenopus laevis extract on defined templates (guse., 2011). although it is not clear how accurately these assemblies represent functional kinetochores, the reconstitution of kinetochore - like structures in vitro should allow for the analysis of its force resistance properties. at present, it remains unclear which proteins at kinetochores contribute to force resistance and how kinetochores are organized to achieve this. the first path involves an attachment of cenp - a to cenp - c followed by the mis12 complex, which contacts knl1 and the ndc80 complex, with ndc80 completing the connection to the microtubule (fig. the second connection is anchored to centromeric dna by the cenp - t w s x complex, which makes its own direct connection to the ndc80 complex. the available biochemical data suggest that these two connections in their most minimal forms are constructed linearly and that there are two separate pools of ndc80 that make connections to the microtubules from the mis12 complex and cenp - t (bock., 2012 ; this suggests that some parts of the kinetochore might be held together by only a single protein protein interface. however, it is possible that there are interactions between these pathways, either directly or via other protein components (fig. 1 ; gascoigne., if the individual protein protein interactions within each pathway can not withstand the force produced by the depolymerizing microtubule, the current architectural models of the kinetochore may be incomplete. based on the currently available structural details for the kinetochore, several different models could explain how kinetochores withstand cellular forces (fig. it is possible that a subset of kinetochore proteins have elastic properties, such as those suggested by the elongation of cenp - t (suzuki., 2011). elasticity of a protein within a series arrangement would allow it to absorb some energy, thereby decreasing the force passed through the subsequent protein protein interfaces for at least some time, much in the same way that nucleosome displacement in pericentric chromatin could diffuse the force generated at kinetochores (verdaasdonk., 2012). in this model, energy is absorbed by breaking or rearranging bonds within kinetochore proteins rather than between proteins, thereby protecting the key interfaces within the kinetochore. second, the connections between the microtubule and centromere are likely to be arranged in a parallel manner such that they sum to a strong interface. the multiple copies of each core kinetochore protein that are present per microtubule (joglekar., 2006, 2008 third, there may be additional kinetochore proteins that are not part of the linear connectivity between the centromere and microtubule but that strengthen connections between kinetochore components that would otherwise be too weak. for example, the tetrahymena thermophila cilia protein bld10 has recently been proposed to structurally stabilize the basal body under the force generated during cilia beating (bayless., 2012). at kinetochores, proteins could serve a similar role either by serving as dynamic cross - linkers, connecting separate linear pathways, or by reinforcing existing connections by adding contacts between proteins. it is likely that the actual force resistance properties of the kinetochore complex require a combination of all three models. work spanning the last 60 years has shown that the mitotic spindle can generate force that acts on kinetochores. the work we have summarized here provides a preliminary foundation for understanding the consequences of force at kinetochores, but the proposed models will change as more is discovered about kinetochore structure and organization. defining the force resistance properties of the kinetochore will provide a better understanding of how it is able to function in the presence of force and the mechanisms by which it acts during chromosome segregation. as we look toward the future prospects of the field, the advances in the biophysical understanding of focal adhesions (roca - cusachs., 2012) provide an excellent blueprint for generating a detailed molecular picture of a large protein complex that functions under force. for focal adhesions, researchers have defined the pathway between the extracellular matrix and the cytoskeleton, analyzed the force response of each component along this pathway, and defined how cells use mechanosensors to signal to the cell. achieving a similar understanding for the kinetochore will provide key insights into the function of this central cell division structure. | chromosome segregation requires the generation of force at the kinetochore the multiprotein structure that facilitates attachment of chromosomes to spindle microtubules. this force is required both to move chromosomes and to signal the formation of proper bioriented attachments. to understand the role of force in these processes, it is critical to define how force is generated at kinetochores, the contributions of this force to chromosome movement, and how the kinetochore is structured and organized to withstand and respond to force. classical studies and recent work provide a framework to dissect the mechanisms, functions, and consequences of force at kinetochores. |
gynoid lipodystrophy, better known as cellulite, is the most common lipodystrophic disease and is found in 85% of post - adolescent women.14 cellulite usually develops in particular anatomical areas, such as the thighs, buttocks, abdomen, and upper arms, and becomes visible through its classical orange peel appearance, characterized by an irregular, dimpled skin surface with thinning of the epidermis / dermis and the presence of nodular clusters of fat cells.14 it represents not only a cosmetic concern for women, but often becomes a major psychological problem, impairing sporting activities, choice of clothing, and social interaction. the pathophysiology of cellulite is related to various predisposing factors, such as biotype, heredity, ethnic background, body weight, age, hormonal changes, smoking, and genetic predisposition.1,2,46 four main hypotheses regarding the etiopathogenesis of cellulite have emerged over recent decades : a different anatomical conformation of the subcutaneous tissue in women compared with men;7,8 changes in the biomechanical properties of epidermal and dermal tissues;8 excessive hydrophilia of the extracellular matrix increasing interstitial pressure and causing edema of the fatty tissue;9 and alterations in both microvascular and lymphatic circulation resulting in the often painful protrusion of subcutaneous adipose tissue into the lower reticular dermis, causing distinctive mattress - like surface irregularities.10 however, these hypotheses are mutually conflicting and do not consider recent advances in our understanding of the complex physiopathology of the adipose organ.10 for instance, one can not exclude that inflammation also contributes to the formation of cellulite.11,12 nevertheless, various treatments for cellulite have been developed over recent decades, focusing on skin tightening with radiofrequency or lasers, improving blood and lymphatic circulation using both physical treatments and pharmacotherapy, and treating deeper deformities with surgical subcision, laser treatments, ultrasound devices, or liposuction (summarized in table 1). however, there is no single treatment of cellulite that is completely effective.13,14 extracorporeal shock wave therapy (eswt) and radial shock wave therapy (rswt) have been introduced as safe and effective treatment options for cellulite.1523 a shock wave is an acoustic pressure wave that is produced in any elastic medium, such as air, water, or even a solid substance.24,25 shock waves differ from sound waves in that the wave front, where compression takes place, is a region of sudden change in stress and density.24,25 both focused shock waves (eswt) and radial shock waves (rswt) are characterized by a high positive peak pressure (in mpa), a fast initial rise in pressure (approximately a few microseconds or less), a diffraction - induced tensile wave following the positive pressure amplitude that can generate cavitation, and a short life cycle of approximately 1020 seconds (figure 1).2429 extracorporeal shock wave lithotripsy is widely used for stone management in urology.30 eswt and rswt are byproducts of lithotripter technology. since the late 1980s, they have been introduced into treatment for various diseases of the musculoskeletal system, such as plantar fasciopathy, achilles tendinopathy, medial tibial stress syndrome, greater trochanteric pain syndrome, lateral and medial epicondylitis, and calcifying tendonitis of the shoulder.2729,31,32 shock waves have both a direct and indirect effect on treated tissues. the direct effect is the result of the energy of the shock wave being transferred to the targeted tissues. the indirect effect is the result of the creation of cavitation bubbles in the treated tissue.24,25,29 it has been hypothesized that both the direct and indirect effects produce a biological response in the treated tissues.24,25,29 eswt devices share two technical key characteristics of extracorporeal shock wave lithotripsy devices used for stone management, namely the electrohydraulic, electromagnetic, or piezoelectric generation of pressure waves and the generation of focused or so - called defocused pressure waves.29,33 radial shock waves are generated ballistically, ie, by accelerating a bullet that strikes an applicator, transforming the kinetic energy of the bullet into a radially expanding pressure wave (figure 1).29,32,33 in this regard, it is of note that, in several studies on eswt / rswt for cellulite, the therapy was termed acoustic wave therapy (awt)15,17,20,22 or extracorporeal pulse activation therapy (epat).21,22 the terms awt and epat are proprietary names of the manufacturer of the corresponding devices (storz medical, tgerwillen, switzerland ; see also russe - wilfingseder). awt is registered as non - medical electric and electronic apparatus and instruments for the generation and application of shock waves or pressure waves in the fields of cosmetics and beauty care,34 and epat as electronic apparatus and parts of the apparatus for generating and applying pressure or shock waves for use in the fields of cosmetics and beauty care.35 the similarity between awt, epat, and rswt has been addressed in several papers in the literature.21,36,37 unaddressed in the studies on eswt / rswt for cellulite carried out to date1523 is whether the individual clinical outcome of the therapy can be predicted by the patient s cellulite grade at baseline, age, body mass index (bmi), weight, height, and/or age. we hypothesized that the individual clinical outcome of rswt for cellulite can be predicted by the patient s cellulite grade at baseline and the patient s bmi. fourteen caucasian females with cellulite were enrolled in a prospective, single - center, randomized, open - label phase ii study. the mean (standard error of the mean) patient age was 42.42.81 (2357) years. the study was approved by the ethics committee of canton geneva (geneva, switzerland) under registration number ge 08 - 40 and by the swiss agency for therapeutic products (swissmedic, bern, switzerland) under registration number 2009-md-0005. the study is registered with clinicaltrials.gov (nct01974115).38 the mean cellulite grade of the patients at baseline was 2.50.09 (range 23). cellulite grades were determined by clinical inspection of the patients skin (documented by digital photography) and by contact thermography. photographs of the patients were taken before the treatment cycle and at each follow - up using a d80 digital camera system (nikon, tokyo, japan), pocketwizard transceivers (lpa design, burlington, vt, usa), and studiomax iii lighting equipment (photogenic professional lighting, bartlett, il, usa), with standardized lighting settings and distance to the patient at each photographic session. patients were asked to fully contract the buttock muscles each time a photograph was taken. this aimed to fully show and standardize the appearance of the cellulite and thus to avoid any softening effects due to varying muscle tone that might change the visibility of the cellulite. contact thermography was performed using the cell - meter system professional cellulite thermodetector (ips srl, milan, italy) that was applied directly on the skin of the treated areas. the temperature is displayed in a color code, with brown - orange - yellow indicating cold areas (29.5c30.5c) and bluish shades indicating warm areas (32c33.5c). cellulite grades, determined by clinical inspection of the skin, correlated well with the contact thermography data. all patients were treated with radial extracorporeal shock waves using the swiss dolorclast device (electro medical systems, sa, nyon, switzerland) and the swiss dolorclast power+ hand piece with the 36 mm applicator (figure 1). patients were positioned on a treatment table as indicated in figure 2 and the areas of the posterior thigh and the anatomical buttock area were treated. the medial and lateral lines of the thigh served as borders of the treatment area which extended superiorly until the buttock crease and inferiorly 5 cm above the popliteal crease. patients were treated unilaterally with 2 weekly treatments for 4 weeks on a randomly selected side (left or right), totaling eight treatments on the selected side. after application of coupling gel, treatment was performed at 3.54.0 bar, with 15,000 impulses per session, and applied at 15 hz. the condition of each patient s skin was evaluated before treatment, after the last treatment, and at a follow - up visit 4 weeks after the last treatment. at both the last treatment and at follow - up, patients completed a detailed questionnaire with scores for treatment comfort, pain intensity, and satisfaction, while also indicating undesired effects, such as bruising. dependence of the clinical outcome of rswt (calculated as the individual difference in cellulite grades either between baseline and after the last treatment [-1 ] or between baseline and follow - up [-2 ]) on the patients initial cellulite grade at baseline, bmi, weight, height, age, pain during the treatment, feeling of comfort during treatment, and satisfaction at the end of treatment (or at the end of the follow - up period) was tested using spearman s nonparametric rank correlation. because -1 and -2 were each tested against eight variables, an effect was considered statistically significant if its associated p - value was smaller than 0.05/8=0.00625 considering the bonferroni correction for multiple hypothesis testing.39 spearman s nonparametric rank correlation was also used for testing the relationship between -1 and -2. in this case, the effect was considered to be statistically significant if the associated p - value was smaller than 0.05. calculations were performed using graphpad prism version 5.0 for windows (graphpad software, san diego, ca, usa). fourteen caucasian females with cellulite were enrolled in a prospective, single - center, randomized, open - label phase ii study. the mean (standard error of the mean) patient age was 42.42.81 (2357) years. the study was approved by the ethics committee of canton geneva (geneva, switzerland) under registration number ge 08 - 40 and by the swiss agency for therapeutic products (swissmedic, bern, switzerland) under registration number 2009-md-0005. the mean cellulite grade of the patients at baseline was 2.50.09 (range 23). cellulite grades were determined by clinical inspection of the patients skin (documented by digital photography) and by contact thermography. photographs of the patients were taken before the treatment cycle and at each follow - up using a d80 digital camera system (nikon, tokyo, japan), pocketwizard transceivers (lpa design, burlington, vt, usa), and studiomax iii lighting equipment (photogenic professional lighting, bartlett, il, usa), with standardized lighting settings and distance to the patient at each photographic session. patients were asked to fully contract the buttock muscles each time a photograph was taken. this aimed to fully show and standardize the appearance of the cellulite and thus to avoid any softening effects due to varying muscle tone that might change the visibility of the cellulite. contact thermography was performed using the cell - meter system professional cellulite thermodetector (ips srl, milan, italy) that was applied directly on the skin of the treated areas. the temperature is displayed in a color code, with brown - orange - yellow indicating cold areas (29.5c30.5c) and bluish shades indicating warm areas (32c33.5c). cellulite grades, determined by clinical inspection of the skin, correlated well with the contact thermography data. all patients were treated with radial extracorporeal shock waves using the swiss dolorclast device (electro medical systems, sa, nyon, switzerland) and the swiss dolorclast power+ hand piece with the 36 mm applicator (figure 1). patients were positioned on a treatment table as indicated in figure 2 and the areas of the posterior thigh and the anatomical buttock area were treated. the medial and lateral lines of the thigh served as borders of the treatment area which extended superiorly until the buttock crease and inferiorly 5 cm above the popliteal crease. patients were treated unilaterally with 2 weekly treatments for 4 weeks on a randomly selected side (left or right), totaling eight treatments on the selected side. after application of coupling gel, treatment was performed at 3.54.0 bar, with 15,000 impulses per session, and applied at 15 hz. the condition of each patient s skin was evaluated before treatment, after the last treatment, and at a follow - up visit 4 weeks after the last treatment. at both the last treatment and at follow - up, patients completed a detailed questionnaire with scores for treatment comfort, pain intensity, and satisfaction, while also indicating undesired effects, such as bruising. dependence of the clinical outcome of rswt (calculated as the individual difference in cellulite grades either between baseline and after the last treatment [-1 ] or between baseline and follow - up [-2 ]) on the patients initial cellulite grade at baseline, bmi, weight, height, age, pain during the treatment, feeling of comfort during treatment, and satisfaction at the end of treatment (or at the end of the follow - up period) was tested using spearman s nonparametric rank correlation. because -1 and -2 were each tested against eight variables, an effect was considered statistically significant if its associated p - value was smaller than 0.05/8=0.00625 considering the bonferroni correction for multiple hypothesis testing.39 spearman s nonparametric rank correlation was also used for testing the relationship between -1 and -2. in this case, the effect was considered to be statistically significant if the associated p - value was smaller than 0.05. calculations were performed using graphpad prism version 5.0 for windows (graphpad software, san diego, ca, usa). the mean cellulite grade improved from 2.50.09 (range 23) at baseline to 1.570.18 (range 0.252.75) at the end of the treatment (ie, the mean -1 was 0.93 cellulite grades). at the end of the follow - up period, the mean cellulite grade was 1.680.16, ranging between 0.5 and 2.75 (ie, the mean -2 was 0.82 cellulite grades). the individual -1 varied between 0 grades (ie, no improvement) and 1.75 grades, and the individual -2 between 0 grades and 1.5 grades (figure 3). accordingly, compared with baseline, no patient s skin condition worsened during treatment and follow - up. the treatment was well tolerated and no unwanted side effects were observed (note that discomfort during treatment and reddening of the skin up to 24 hours after each treatment session are usual side effects of rswt and were therefore not considered unwanted side effects). no statistically significant (ie, p<0.05/8) correlation was found between -1 or -2 and cellulite grade at baseline, bmi, weight, height, age, pain during treatment, feeling of comfort during treatment, or satisfaction at the end of treatment (or at the end of the follow - up period, figures 4 and 5). for eleven of the 14 patients, the condition of the skin further improved or remained constant during the interval between the last treatment and follow - up (figure 6). however, there was no statistically significant correlation between -1 and -2 (p=0.105). the results of the present study are generally in line with earlier reports of successful treatment of cellulite with rswt in the literature.15,17,2022 rswt can improve the clinical picture by one cellulite grade on average. however, to the authors knowledge, the present study is the first to demonstrate that the individual clinical outcome of rswt for cellulite can not be predicted by the patient s individual cellulite grade at baseline, bmi, weight, height, or age. we hypothesize that the same applies to eswt for cellulite. in our clinical experience, the patient s perception of their individual cellulite grade and consequently their satisfaction with the result of treatment for cellulite varies widely from one patient to another and is truly subjective. normally, patients with low cellulite grades are more demanding and therefore more difficult to manage in their expectations, even if there is an objectively confirmed clinical improvement. this was confirmed in our analysis because patient satisfaction, the most important end point of any treatment for cellulite, did not correlate with -1 or -2. there were patients with -1=1 (ie, improvement by one cellulite grade) who were very satisfied, whereas other patients with -1=1 were not satisfied at all (figure 5e). for the clinical setting, this observation underlines the role of the therapist, who must correctly evaluate the suitability of the candidate for a cellulite treatment and must manage the patient s expectations accordingly. for studies evaluating existing or new cellulite treatments, this observation underscores the crucial importance of applying objective analytical methods, such as contact thermography and standardized photographic documentation (in full muscular contraction), because satisfaction scores may suffer from variations in their consistency. note that individual patient satisfaction scores were either not reported or not correlated with individual objective outcome measures in the studies of eswt / rswt for cellulite published to date.1523 standardized yet easy clinical analysis of the severity of cellulite should include easy, effective, and reproducible measurement tools. in our opinion, clinical evaluation serves for classification of the cellulite grade, double contrast photography as applied in the present study provides a visual contour analysis, and contact thermography measures the superficial blood perfusion of the skin. recoil and elasticity measurements, as applied in some studies of eswt / rswt for cellulite,15,20,21 are helpful in small treatment areas but may considerably vary over the length of a thigh depending on changing quality and thickness of the skin in the respective parts. in recent years, eswt / rswt has become the best studied therapy option for cellulite (table 1). this is most likely due to the fact that eswt / rswt is noninvasive, does not require administration of drugs, and can be easily accomplished within a few minutes per treatment session. it is justified to consider eswt (ie, focused shock waves) and rswt (ie, radial shock waves) as very similar therapeutic options for cellulite. this is due to the fact that the energy signatures of eswt and rswt share fundamental physical characteristics, such as high peak pressure, a fast initial rise in pressure, a low tensile amplitude that can generate cavitation, and a short life cycle. some authors have offered the following physical definition of real shock waves:26,27 a high positive peak pressure, sometimes more than 100 mpa, but more often approximately 5080 mpa ; a fast initial rise in pressure during a period of less than 10 nanoseconds ; a low tensile amplitude (up to 10 mpa) ; a short life cycle of approximately 10 seconds ; and a broad frequency spectrum, typically in the range of 1620 mhz. it is well known that radial shock waves do not fulfill the characteristics set out by this physical definition of real shock waves (see also figure 1).29,40 some eswt devices generate pressure waves that fulfill the characteristics set out by this physical definition of real shock waves, whereas others do not.29,40,41 among those eswt devices that do not produce real shock waves is the electromagnetic duolith device (storz medical)41 that has recently been introduced into eswt for cellulite.16 another device that was used in several studies for treating cellulite is the d - actor 200 (storz medical).15,17,22 the pressure waves generated by this device are termed low - energy radial shockwaves in the literature.42 in contrast, russe - wilflingseder described the d - actor 200 device as a vibrating massage system. regardless of these different descriptions in the literature, the d - actor 200 device is making use of the same construction principle as the swiss dolorclast and accelerates a projectile by means of compressed air. for this reason, the d - actor 200 device generates pressure waves that are very similar to the pressure waves generated by the swiss dolorclast device, including the possibility of generating cavitation (csszr, submitted for publication). because the studies on eswt / rswt for cellulite considerably vary with respect to the level of evidence, shock wave device used, and treatment protocol, they are discussed separately, as follows. in an early pilot study, braun treated 20 patients with severe cellulite measured with a pinch test18 using the electromagnetic dermaselect shock wave device (storz medical). the average age of the patients was 37.25 (range 1956) years and their mean bmi was 29.18 (range 2041.6). each patient received six treatment sessions with 2,400 impulses per session on the left leg (the time interval between treatments, size of the treatment area, and energy flux density of the shock waves were not provided). according to the authors subjective impressions of the treated leg and photographic analyses, a significant improvement in skin surface angehrn treated 21 female patients with cellulite (grade 1, n=5 ; grade 2, n=6 ; grade 3, n=10) using defocused shock waves generated with the electrohydraulic activitor - derma device (switech medical, kreuzlingen, germany). treatment consisted of 12 sessions at intervals of 34 days, treatment of the skin of the lateral left and right thigh with 4,000 impulses per thigh per treatment session, homogeneously distributed over an area of 160 cm per side with an energy flux density of 0.018 mj / mm. bmi was 2024 in ten patients, 2529 in nine patients, 3034 in one patient, and 3540 in one patient. end points were subjective opinion of improvement and collagenometry measurements performed with the high - resolution ultrasound system, collagenoson (minhorst, meudt, germany). at the end of the treatment period, two patients showed clear worsening of collagenometry results compared with baseline, five patients showed some worsening, two patients showed no change, eight patients showed improvement, and four patients showed clear improvement compared with baseline. seven patients evaluated the treatment as not suitable (pain during treatment), six patients assessed it as suitable (no pain during treatment), and eight patients were indifferent. the authors concluded that their results provided evidence that low - energy defocused eswt caused remodeling of the collagen within the dermis of the tested region. al20,21 treated a total of 59 female patients with cellulite grade 2 or 3 with planar or radial shock waves generated with the electromagnetic cellactor sc1 device (storz medical). group 1 (n=15, mean age 44.6 years, mean bmi 24.4) was treated with planar shock waves generated with the c - actor hand piece of the cellactor sc1 device (six treatment sessions at intervals of 34 days, treatment of lateral and medial thigh areas as well as the buttocks, total of 3,200 impulses per treatment session with an energy flux density of 0.25 mj / mm homogeneously distributed over a total area of 2030 cm). group 2 (n=44, mean age 45.5 years, mean bmi 25.3) was treated identically but with eight treatment sessions. end points were the elasticity of the skin measured with the dermalab device (cortex technology, hadsund, denmark) and the structure of the connective tissue in the dermis evaluated with the dermascan ultrasound device (cortex technology) before and after treatment. the mean skin elasticity in group 1 patients was improved by 46% after treatment and by 78% at 3-month follow - up compared with baseline. in group 2, the mean improvement in skin elasticity was 72% after treatment, 95% at 3-month follow - up, and 105% at 6 months after baseline. the structure of the connective tissue also improved between baseline and the 6-month follow - up. statistical analysis was not performed to evaluate the impact of bmi on the results in this study. kuhn presented a case report concerning a 50-year - old woman with grade 3 cellulite on her left thigh treated with the activitor - derma device (four therapy sessions, 800 impulses per session, energy flux density 0.115 mj / mm). based on high frequency, high resolution ultrasound measurements, contact thermography, and histopathologic biopsies, the authors reported some improvement in the epidermis and the extracellular matrix of the dermis.23 sattler compared three treatments for cellulite. group 1 (eleven patients, mean age 40 years, mean bmi 27) was treated with radial shock waves generated with the ballistic d - actor 200 device (a mean of 6.2 treatment sessions, an average of 1,909 impulses per treatment session ; device operated at 2.43.0 bar and a frequency of 15 hz). group 2 (eleven patients, of whom nine were included in the analysis, mean age 34 years, mean bmi 23) was treated with planar shock waves generated with the c - actor hand piece of the electromagnetic cellactor sc1 (a mean of 6.1 treatment sessions, 1,000 impulses per treatment session with an energy flux density of 0.35 mj / mm). group 3 (eight patients, of whom seven were included in the analysis, mean age 40 years, mean bmi 23) was treated with a combined radial and planar shock wave protocol (a mean of 6.4 treatment sessions ; 2,350 radial pulses on average followed by an average of 1,925 planar impulses per treatment session ; radial impulses generated by operating the control unit at 2.63.0 bar ; planar impulses with an energy flux density of 0.35 mj / mm). treatment was focused either on the buttock and dorsal thigh area or on the ventral thigh area, depending on the individual clinical picture. end points were visual impression of the skin (analyzed on photographs), patient satisfaction, and skin elasticity (measured with the dermalab device) 3 months after the last treatment session compared with baseline. analysis of the photographs showed an optimum treatment result for five (46%) patients, a satisfactory treatment result for three (27%) patients, and a not significant treatment result for three (27%) patients (specific criteria for optimum, satisfactory, and not significant were not specified). for patients in groups 2 and 3, the corresponding data were : an optimum treatment result in 1/9 (11%) and 2/7 (29%), respectively ; a satisfactory result in 5/9 (56%) and 4/7 (57%), respectively ; and a not significant result in 3/9 (33%) and 1/7 (14%), respectively. a statistical analysis was not performed. it is of note that the authors did not recognize any change in skin elasticity as a result of shock wave treatment (mean data for group 1, 11.6 mpa at baseline, 10.0 mpa after treatment, and 10.1 mpa at 3-month follow - up ; mean data for group 2, 12.1 mpa at baseline, 10.8 mpa after treatment, and 12.1 mpa at 3-month follow - up ; mean data for group 3, 10.3 mpa at baseline, 10.4 after treatment, and 10.9 at 3-month follow - up). the authors discussed the limitations of their study,15 ie, small numbers of patients, and differences in mean age and mean bmi between the groups, but concluded that treatment for cellulite with radial shock waves might be the best choice (as also performed in the present study). adatto treated 25 women of mean age 42.6 (range 2763) years with a mean bmi of 24 (range 1731) on one leg each with the ballistic d - actor 200 device (a mean of six treatment sessions within 4 weeks with an average of 3,000 impulses per treatment session ; device operated at 2.63.6 bar and with a frequency of 15 hz). the authors compared, for each patient, the treated leg with the untreated leg 1 week and 12 weeks after the last treatment. furthermore, three - dimensional images of the skin structure were recorded using the dermatop system (eotech, paris, france). adatto found that skin elasticity, roughness elevation, and skin depression improved in a statistically significant manner on the treated legs compared with the untreated legs. they concluded that the d - actor 200 device can be used effectively to treat cellulite without any side effects. in a double - blind, randomized controlled trial, knobloch randomly assigned 53 women to either focused shock waves using the electromagnetic duolith device (n=25 ; mean age 41.4 years, mean bmi 24.23.2 kg / m ; six sessions of eswt every 12 weeks, with 2,000 impulses at 4 hz, and an energy flux density of 0.35 mj / mm) or sham treatment (n=28 ; mean age 45.0 years, mean bmi 25.34.5 kg / m ; six treatment sessions every 12 weeks, with 2,000 impulses and an energy flux density of 0.01 mj / mm). in addition to eswt or sham - eswt, all patients underwent specific gluteal strength exercise training. among other measurements, the primary end point was score on the photonumeric cellulite severity scale (css) determined by two blinded, independent assessors. eswt reduced the mean css from 10.93.8 at baseline to 8.34.1 at 12 weeks after the last treatment, whereas sham - eswt did not (css at baseline 10.03.8 ; css 12 weeks after the last treatment 10.13.8). the authors concluded that the combination of eswt and gluteal strength training was superior to gluteal strength training and sham - eswt in moderate to severe cellulite in terms of css in a 3-month perspective. it remains unknown why females with documented cellulite grade 0 according to nrnberger and mller,7 ie, no cellulite, were eligible for and enrolled in this study. furthermore, the authors described that they performed an intention - to - treat analysis because seven sham - treated women were lost to follow - up. however, they did not describe which of the various available methods for handling missing data in clinical trials they applied.43 russe - wilflingseder randomly assigned 16 women with cellulite (mean age 42.77.4 years, mean bmi 22.51.85 kg / m) to either radial shock waves using the d - actor 200 device (n=11 ; eight treatments once a week ; 1,000 impulses at 23 bar air pressure applied using a di15 deep impact transmitter (storz medical, tgerwilen, switzerland) ; 2,500 impulses at 35 bar applied by the d - actor transmitter d20-s ; frequency of shock waves not provided) or sham treatment (n=5 ; treatment protocol identical to the rswt protocol but using a placebo hand piece that did not emit shock waves). clinical outcome was assessed by a patient satisfaction questionnaire, weight control, measurements of thigh circumference, visual appearance of the skin in standardized photographs, and an analysis of images taken with a specially designed three - dimensional imaging system. patients were investigated at baseline, before the last treatment, and at 1 and 3 months after the last treatment. by combining the results of four efficacy criteria at the two follow - up visits, the authors found a statistically significant improvement in the skin of women treated with radial shock waves but not for those treated with placebo. the authors concluded that radial shock wave treatment is safe and efficient for patients with cellulite. this is in line with the results of the present study. finally, a study by ferraro warrants mention. the authors treated 37 women and 13 men with the proshock ice device (promoitalia, milan, italy) in five different areas : abdomen (five women, nine men), ankles (three women, one man), arms (five women, three men), buttocks (six women), and thighs (18 women). the authors described the proshock ice device as a combination of a controlled cooling system (freezing probe) and a shock wave generator (shock probe) with pressure variable from 50 to 500 bar, and with impulses that have a duration of 8 mseconds.44 unfortunately, it remains unclear what this actually means, given that radial shock wave devices are usually operated with an air pressure of 15 bar, have a maximum pressure of 100 bar (10 mpa), and a duration of approximately 20 seconds.29,40 ferraro applied tissue - specific (fat edematous cellulite, fibrous cellulite) treatments (freezing probe, shock probe) for 2060 minutes every 15 days for 8 weeks (an average of 3.73 treatment sessions per patient). in addition to evaluations of each patient s individual subjective impression of the effect and objective clinical data such as skin - fold thickness and hepatic markers, the authors investigated skin biopsies of treated and untreated tissue to detect apoptosis, laminin, and collagen. the results showed statistically significant reductions in circumference of the treated body regions (abdomen, on average 6.86 cm ; ankles, on average 2.25 cm ; arms, on average 2.75 cm ; buttocks, on average 5 cm ; thighs, on average 5.78 cm) with no change in body weight. microscopic investigation of the skin biopsies showed signs of dying fat cells (adipocytes) and an inflammatory process in the treated tissue. ferraro discussed their method as a noninvasive alternative to conventional liposuction for patients who require only small or moderate removal of adipose tissue and cellulite or who are not suitable candidates for surgical approaches to body contouring.44 several studies have demonstrated that cellulite can be treated effectively and safely with eswt and rswt. the main conclusion of the present study is that the individual clinical outcome of treatment with shock waves for cellulite can not be predicted by the patient s cellulite grade at baseline, age, bmi, weight, or height. several questions regarding eswt / rswt for cellulite remain open and should be addressed in future studies. for instance, the striking difference between the results reported by christ,21 and those reported by sattler, regarding treatment - related changes in skin elasticity, require an independent reanalysis. the higher efficacy of rswt relative to eswt in treating cellulite15 should also be investigated. presumably, the most important task will be to unravel the molecular and cellular mechanisms of shock waves in skin and fat tissue. in this regard, it is of note that several potential mechanisms have been proposed in the literature, comprising improved microcirculation, apoptosis of fat tissue, and improved lymph circulation (table 3). many of these mechanisms may be secondary to the activation of c nerve fibers in the skin by shock waves and the release of substance p.45,46 substance p is one of the body s neurotransmitters for pain and heat,47 and is responsible for causing slight discomfort during and after shock wave treatment.29 capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance p.48 a recent study showed an age - related decrease in thrombomodulin - positive cells and vascularity in the skin, and demonstrated that topic application of capsaicin to the skin may boost factor xiiia - positive dendrocytes, thrombomodulin - positive cells, and the blood vessel network of the skin.49 | backgroundextracorporeal shock wave therapy has been successfully introduced for the treatment of cellulite in recent years. however, it is still unknown whether the individual clinical outcome of cellulite treatment with extracorporeal shock wave therapy can be predicted by the patient s individual cellulite grade at baseline, individual patient age, body mass index (bmi), weight, and/or height.methodsfourteen caucasian females with cellulite were enrolled in a prospective, single - center, randomized, open - label phase ii study. the mean (standard error of the mean) cellulite grade at baseline was 2.50.09 and mean bmi was 22.81.17. all patients were treated with radial extracorporeal shock waves using the swiss dolorclast device (electro medical systems, s.a., nyon, switzerland). patients were treated unilaterally with 2 weekly treatments for 4 weeks on a randomly selected side (left or right), totaling eight treatments on the selected side. treatment was performed at 3.54.0 bar, with 15,000 impulses per session applied at 15 hz. impulses were homogeneously distributed over the posterior thigh and buttock area (resulting in 7,500 impulses per area). treatment success was evaluated after the last treatment and 4 weeks later by clinical examination, photographic documentation, contact thermography, and patient satisfaction questionnaires.resultsthe mean cellulite grade improved from 2.50.09 at baseline to 1.570.18 after the last treatment (ie, mean -1 was 0.93 cellulite grades) and 1.680.16 at follow - up (ie, mean -2 was 0.82 cellulite grades). compared with baseline, no patient s condition worsened, the treatment was well tolerated, and no unwanted side effects were observed. no statistically significant (ie, p<0.05) correlation was found between individual values for -1 and -2 and cellulite grade at baseline, bmi, weight, height, or age.conclusionradial shock wave therapy is a safe and effective treatment option for cellulite. the individual clinical outcome can not be predicted by the patient s individual cellulite grade at baseline, bmi, weight, height, or age. |
secure a wiretrol 5 ul glass capillary tube onto glass micropipette puller and adjust heater and solenoid settings to pull pipette with a smooth, shallow taper. attach a source of positive air pressure onto the end of the pulled micropipette and gently lower the tip of the pipette onto a metal grating surface at a 45 angle to create a beveled tip. positive air pressure helps to clear glass debris from the inside of the pipette. clean the end of the beveled tip with an ethanol - moistened tissue. the tip should have a smooth bevel with an internal opening diameter of ~100 um. smaller diameters may be used but often result in clogging of the pipette with fluorescent beads. backfill pipette with mineral oil until half - full, then insert grease - dipped plunger into back of pipette. advance meniscus of mineral oil to the pipette tip by manually pushing in the plunger. secure the micropipette and plunger onto a micromanipulator, then screw the micromanipulator pipette holder onto a small stationary arm with adjustable height. frontload the pipette with the fluorescent microbead solution, consisting of 50% fluorescent microbead stock solution, 45% water, and 5% glycerol. glycerol is added to increase the density of the solution so that when deposited onto the wholemount the microbeads sink onto the surface. place the micromanipulator in a safe place where the needle will not be accidentally broken and proceed with wholemount dissection. to prepare for wholemount dissection, warm sufficient quantity of l-15 leibovitz media to 37c. you will need approximately 10 ml per animal you plan to dissect. also gather all supplies you will need for dissection and fixation by the stereomicroscope : scissors, toothed large forceps, smooth fine forceps, sharpoint 22.5 microsurgical stab knife, dissecting dish, paper towel, biohazard bag, and a 24 well plate on ice filled with 4% paraformaldehyde with or without 0.1% triton x-100. triton x-100 is used to decrease the surface tension of the pfa solution, which decreases the incidence of shearing the wholemount surface when immersing it in this solution. pour 5 - 10 ml of the warmed media into a dissecting dish placed under a fluorescent stereomicroscope. dissecting dishes are prepared by pouring an elastic polymer, called sylgard 184, into a 6 cm plastic dish and letting the polymer solution cure for 1 week under vacuum, then thoroughly rinsing dishes in large volumes of water before use. usually, we let the dishes soak in water in a 1 l beaker for 1 week. the animal is sacrificed by cervical dislocation and the head is cut off.note : if desired, blood may be cleared from the vasculature by perfusing the animal with normal saline prior to dissecting out the brain. a midline incision is made, posterior to anterior, along the scalp to reveal the skull. a series of 4 cuts in the skull are made to open the cranium : one cut spans the two orbits anterior to the olfactory bulbs, the next two cuts are inferior to the cerebellum and separate the cranium from the skull base, and the final cut runs posterior to anterior along the mid - sagittal suture. the cranial flaps are gently retracted and the brain is extracted and placed into the dissecting dish. if you wish additionally to examine the olfactory bulbs, simply fix them by immersion in 4% pfa overnight and you may subsequently prepare them for sectioning and staining. divide the brain along the interhemispheric fissure. a coronally - oriented cut is then made at the posterior - most aspect of the interhemispheric fissure, allowing the caudal hippocampus to be visualized in cross - section. the hippocampus, which forms the medial wall of the lateral ventricle at this position, must then be released from the overlying cortex, which forms the dorsal - lateral wall of the ventricle. first, the knife is inserted into the small ventricular space between the cortex and hippocampus dorsally, and a cut is made in the cortex where it reflects ventrally, away from the midline, to join the hippocampus. after this cut is made, the cortex can be slowly peeled away from the hippocampus to reveal the lateral ventricle moving from dorsal to ventral. this maneuver is expedited by cutting off a wedge of cortex at the corner where the hippocampus was released. after reaching the ventral - most extent of the lateral ventricle at this position, you may either visualize or feel where the cortex again wraps around, this time reflecting back medially, to join the hippocampus. another cut must be made in this position to completely release the hippocampus or medial wall of the lateral ventricle from the cortex or lateral wall of the lateral ventricle. it will then be easy to pull the hippocampus away from the cortex, medially and anteriorly, to open the lateral ventricle widely. continue to gently pull the hippocampus anteriorly using small strokes of the forceps and knife to retract the medial and lateral walls apart. first, to increase your exposure to the lateral ventricle and in particular, the lateral wall and svz, dissect away the cortex. the cortex is cleanly dissected away by visualizing the interface between the corpus callosum and the vz / svz. simply cut along this interface staying on the callosal side to avoid damaging the svz. in order to continue retracting the medial wall away from the lateral wall, two more cuts are needed : one cut dorsally where the lateral wall, medial wall, and cortex all converge, and one cut ventrally where the lateral wall, medial wall, and thalamus converge. with these cuts made, further gentle retraction on the medial wall proper lighting is essential throughout the procedure and especially in the next step where the medial and lateral walls are separated anteriorly. at this anterior position in the lateral ventricle, adjust the lighting such that shadows cast between the two walls reveal this reflection point, which appears like a valley between the two walls. finally, completely expose the lateral wall by removing any overhanging cortex dorsally and the thalamus ventrally. if preparing wholemounts for immunostaining, carefully transfer the wholemount, ventricle side up, from the dissection dish into a 24-well plate filled with 4% pfa with or without 0.1% triton - x100 for an overnight fixation at 4c. for fixation - sensitive antigens, wholemounts may be fixed for shorter periods of time. then proceed to section 4 on immunostaining wholemounts. if preparing wholemounts for ependymal flow analysis, transfer the wholemount to a clean dissection dish filled with fresh, 37c leibovitz medium and proceed to the next section. immobilize the wholemount on a clean dissecting dish using 2 insect pins, one in the thalamus and one in anterior - dorsal corner of the wholemount. make sure the height of the adjustable arm is maximally elevated to avoid breaking the needle against the dissection dish. carefully dip the tip of the needle in media in an ependorf tube to clean off microbeads that are present on the exterior tip of the needle. if these are not cleaned away, they may subsequently be deposited on the wholemout surface inadvertently during needle positioning and reduce the overall quality of the movie. position the needle tip over the dorsal surface of the lateral wall and lower the arm to bring the needle tip into the medium. the needle should be lowered until it is just above the lateral wall surface. with the needle in position, if you will be making a recording of the ependymal flow, begin acquiring images at this time. once the initial bolus of beads has been cleared off the surface by ependymal flow, additional rounds of bead ejection can be performed. wholemounts dissected for immunostaining are immersion - fixed overnight in 4% pfa with or without 0.1% triton - x100 at 4c. the use of triton - x100 is preferred for antigens that tolerate this treatment, but can be left out in cases where staining quality is diminished by detergent. the following morning, pfa is aspirated from the 24-well plate and the wholemounts are washed 3 times for 5 minutes each in 0.1 m pbs with or without 0.1% triton - x100. as before, the use of triton - x100 is preferred, but not required, for all washes in this protocol. throughout this protocol, exchanging solutions over the wholemount requires careful aspiration of the solution from the side of the well. then, the well is refilled with solution using a transfer pipette angled such that the solution washes over the side of the well, not directly onto the wholemount. take care to keep the ventricle side of the wholemount facing up at all times. we prefer to exchange solutions over 1 wholemount at a time to prevent tissue from drying. after washing off the pfa, wholemounts are incubated for 1 hour at room temperature in blocking solution, containing 10% normal goat or donkey serum in 0.1 m pbs with or without triton - x100. if using triton - x100 for your staining, you may choose to use either 2% or 0.5% triton - x100 in the blocking solution. we use 2% triton - x100 when staining for antigens that require deeper antibody penetration into the tissue, such as those antigens located in the svz. however, when staining for antigens located closer to the surface of the lateral wall, such as antigens found in the apical surface of ependymal cells, we use 0.5% triton - x100. in addition, triton - x100 can be left out for cell - surface or other antigens that are removed or altered by detergent. next, remove the blocking solution and add primary antibodies diluted in the same blocking solution and incubate for 24 or 48 hours at 4c. choice of the incubation period depends on the antigen, similar to choice of 0.5% or 2% triton. for antigens located on the surface of the wholemount, 24 hour incubation suffices. however, for antigens located deeper, such as in the svz, 48 hour incubation periods provide better results.for example, to study the apical surface and basal bodies of cells lining the lateral ventricle wall, stain with antibodies to -catenin, to label the cell membrane, and -tubulin, to label basal bodies. dilute mouse anti--catenin antibodies (1:500) and rabbit anti--tubulin antibodies (1:1000) in 0.1 m pbs containing 10% normal goat serum and 0.5% triton - x100. incubate at 4c for 24 hours.to stain the adult neural stem cells, or type b1 cells, stain the lateral wall with gfap antibody. dilute mouse anti - gfap antibodies (1:500) in 0.1 m pbs containing 10% normal goat serum and 2% triton - x100. primary antibodies are washed off initially by 2 quick rinses in pbs with or without 0.1% triton - x100. dilute secondary antibodies in the same blocking solution used for primary antibodies and add to wholemounts to incubate for the same length of time as for primary antibodies at 4c.for example, for staining for -catenin and -tubulin : dilute alexa fluor 488 goat anti - mouse antibodies (1:400, recognizes mouse anti--catenin) and alexa fluor 594 goat anti - rabbit antibodies (1:400, recognizes rabbit anti--tubulin) in 0.1 m pbs containing 10% normal goat serum and 0.5% triton - x100. gfap immunostaining : dilute alexa fluor 488 goat anti - mouse antibodies (1:400, recognizes mouse anti - gfap) in 0.1 m pbs containing 10% normal goat serum and 2% triton - x100. secondary antibodies are washed off the wholemount using the same washes performed for primary antibodies. if desired, nuclear counter - staining can be performed at this point by incubating in dapi diluted in pbs for 30 minutes at room temperature and then washing one time in pbs. for high - resolution confocal imaging, following immunostaining the wholemounts needed to be sub - dissected to preserve only the lateral wall of the lateral ventricle as a sliver of tissue 200 - 300 um thick. separating the lateral wall from the underlying striatum allows it to be mounted onto a slide and covered with a coverslip in a flat manner. return to the stereomicroscope with immunostained wholemounts and the following tools and equipment : smooth fine forceps, sharpoint 22.5 microsurgical stab knife, dissecting dish, microscope slides and coverslips, 0.1 m pbs, and aquamount mounting medium. transfer the wholemount from the 24-well plate to the dissecting dish containing 0.1 m pbs being careful to keep the ventricle side up. first, completely remove the dorsal cortex of the wholemount from posterior to anterior by cutting precisely along the line where the corpus callosum meets the lateral wall. this is recognized as the interface between the callosal white matter and the pink - appearing svz. then make a long horizontally - oriented cut across the ventral aspect of the wholemount. this cut surface will provide a platform onto which you can stabilize the wholemount during the next step in the dissection. with the dorsal surface of the wholemount facing up, you will be able to visualize the thickness of the svz from anterior to posterior along the lateral wall. note that this view was made possible by the intial removal of the cortex allowing the underlying striatum and svz to be seen. the svz is identified as the thin band of tissue extending from the ventricular surface to the striatum. the svz has a homogeneous pink appearance while the striatum is infiltrated by cords of white matter. once you have identified the interface of the svz and striatum, carefully begin cutting at this interface at the anterior - most aspect of the lateral wall, advancing the knife from dorsal to ventral. to do this accurately, stabilize the wholemount with your forceps used as two pins. you can also use your forceps to slightly turn the wholemount to visualize the blade advancing from dorsal to ventral across the lateral wall. the key to this dissection is for the resulting sliver of tissue to be very flat. this means that as you slice off the svz from anterior to posterior across the lateral wall, the orientation of the cuts you make must remain parallel to the ventricular surface at all times. rather than thinning out your dissection to cut off only the svz at this point, it is important that as you advance posteriorly, the thickness of the tissue being dissected remains the same. after completely separating the lateral wall from the underlying striatum, carefully remove all other surrounding tissues from this sliver that are not part of the ventricular wall. then pick up this sliver from below using your forceps and position it in the center of a microscope slide. apply a few drops of aquamount directly onto the wholemount and gently place a coverslip centered over this, trying not to introduce bubbles onto the surface of the tissue. the weight of the slide will ensure that the aquamount disperses evenly and will produce a refined flattening of the lateral wall surface. the amount of aquamount used and the size of the coverslip depend on the age of the tissue being dissected. for embryonic and early postnatal tissues, we prefer 1 drop of aquamount and a 22 " x 30 " coverslip. heavier coverslips may distort the tissue and more aquamount will interfere with imaging quality because the confocal lasers will be less able to penetrate a thin layer of aquamount residing between the coverslip and the tissue surface. for later postnatal and adult tissues, we use 4 drops of aquamount and a 24 " x 60 " coverslip. the slides are then stored flat in a slide book at 4c for 1 - 2 days before imaging to allow the coverslips to settle. wholemount approaches have provided several key insights into the germinal activity of the adult svz. the network of chains of migrating young neurons in the svz was first observed after wholemounts of the lateral wall of the lateral ventricle were immunostained with antibodies to polysialylated neural cell adhesion molecule (psa - ncam). these chains of migrating neuroblasts can also be seen after immunostaining wholemounts with doublecortin antibodies (figure 1). remarkably, the network of chains has a stereotyped pattern, with two general streams of cells, one running dorsally over and one running ventrally around the adhesion point. wholemounts of the svz also provide a comprehensive view of the proliferative activity of progenitors in this region, as seen with ki67 staining in figure 2. interestingly, two recent studies suggest a close interaction between dividing svz cells and the local vasculature (figure 2). when examined under high power confocal microscopy, the en - face view provided by wholemounts allows a unique perspective of the apical surface of cells lining the ventricular system. this en - face perspective has recently revealed that svz type b1 cells, the adult neural stem cells, are part of a mixed neuroepithelium with non - dividing differentiated ependymal cells. the apical surface of type b1 cells contacts the lateral ventricle and is surrounded by large apical surfaces of ependymal cells in a pinwheel configuration (figure 3, arrows indicate b1 apical surfaces). furthermore, close examination of the apical surface of ependymal cells has revealed that the translational position and rotational orientation of their basal bodies are indicators of their planar polarity. this patch is displaced from the center of the apical surface in the downstream " direction with respect to csf flow (translational polarity) ; within this patch, each basal body is rotated about its long axis such that the basal foot, an accessory of the basal body, points in the direction of flow (rotational polarity). importantly, videomicrographs of the ependymal flow assay can be used to directly compare the flow in a specific region of the lateral wall to the orientation of ependymal cell basal bodies in that region (figure 4). in addition to providing a panoramic perspective of the largest germinal region in the adult brain, with higher power imaging, wholemounts allow a more complete and detailed analysis of individual cellular morphologies in the svz. high power confocal imaging of gfap immunostaining on wholemounts has revealed that type b1 cells, in addition to their short ventricle - contacting apical process, have a long basal process in contact with blood vessels (figure 5). this cytoarchitecture had not been appreciated previously in coronal sections because the basal process runs mostly parallel to the ventricular wall. serial sectioning therefore cuts individual cells into small fragments, making it nearly impossible to reconstruct a cell s complete morphology, or to understand its relationship to other cell types in the svz. the wholemount approach has several advantages over classical sectioning techniques, both in providing panoramic views with low power microscopy and a complete perspective of individual cells with high power microscopy. this technique will continue to be an important complement to future studies of this adult brain germinal zone. tiled confocal images reconstruct a lateral wall wholemount that was stained with antibodies to doublecortin, which labels migrating neuroblasts throughout the svz. there are two general streams of migration, one running dorsally over and one running ventrally around the adhesion point, indicated by the asterisk (). this lateral wall wholemount was immunostained with antibodies for ki67, to label dividing cells in green, and antibodies against mouse immunoglobulins, to label the vasculature in red. because this wholemount was not perfused with saline prior to staining, the endogenous mouse igg molecules remain within blood vessels and are stained by secondary anti - mouse antibodies. recent work suggests that dividing svz precursors (green) are located in close proximity to blood vessels (red) { shen, 2008 # 6523}{tavazoie, 2008 # 6522}. arrows indicate anterior (a) and dorsal (d) directions. high power confocal image of a wholemount immunostained for -catenin, to label cell membranes in green, and -tubulin, to label basal bodies in red, reveals the planar organization of these epithelial cells. type b1 cells, the adult neural stem cells, have a small apical surface with a single basal body, indicated by arrows. the apical surface of these cells is surrounded by the large apical surface of ependymal cells in a pinwheel configuration. ependymal cells have planar polarity indicated by the position of their multiple basal bodies on the apical surface. neighboring ependymal cells have their basal body clusters located on the same side of the apical surface (downward and leftward in this region), corresponding to the direction of csf flow { mirzadeh, 2010 # 6573}. scale bar = 10 m. composite image created by merging 100 sequential frames from a movie taken during the ependymal flow assay. fluorescent microbeads deposited dorsal and posterior to the adhesion area were propelled by ependymal cilia in two oriented streams, one over and one under the adhesion, towards the foramen of monro. each flow line depicts the position of a single bead at consecutive points in time. gfap+ type b1 cells have a long basal fiber with end - feet on blood vessels. maximum projection of a high power confocal stack taken from a lateral wall wholemount immunostained with gfap antibodies to label svz astrocytes. this staining labels adult neural stem cells, or type b1 cells, which have an apical ending on the ventricular surface, and as shown here, a long gfap+ basal fiber that ends on blood vessels (arrows). blood vessels are stained here because the secondary antibody used to visualize mouse anti - gfap antibodies recognize endogenous mouse igg within the vasculature. secure a wiretrol 5 ul glass capillary tube onto glass micropipette puller and adjust heater and solenoid settings to pull pipette with a smooth, shallow taper. attach a source of positive air pressure onto the end of the pulled micropipette and gently lower the tip of the pipette onto a metal grating surface at a 45 angle to create a beveled tip. positive air pressure helps to clear glass debris from the inside of the pipette. clean the end of the beveled tip with an ethanol - moistened tissue. the tip should have a smooth bevel with an internal opening diameter of ~100 um. smaller diameters may be used but often result in clogging of the pipette with fluorescent beads. backfill pipette with mineral oil until half - full, then insert grease - dipped plunger into back of pipette. advance meniscus of mineral oil to the pipette tip by manually pushing in the plunger. secure the micropipette and plunger onto a micromanipulator, then screw the micromanipulator pipette holder onto a small stationary arm with adjustable height. frontload the pipette with the fluorescent microbead solution, consisting of 50% fluorescent microbead stock solution, 45% water, and 5% glycerol. glycerol is added to increase the density of the solution so that when deposited onto the wholemount the microbeads sink onto the surface. place the micromanipulator in a safe place where the needle will not be accidentally broken and proceed with wholemount dissection. to prepare for wholemount dissection, warm sufficient quantity of l-15 leibovitz media to 37c. you will need approximately 10 ml per animal you plan to dissect. also gather all supplies you will need for dissection and fixation by the stereomicroscope : scissors, toothed large forceps, smooth fine forceps, sharpoint 22.5 microsurgical stab knife, dissecting dish, paper towel, biohazard bag, and a 24 well plate on ice filled with 4% paraformaldehyde with or without 0.1% triton x-100. triton x-100 is used to decrease the surface tension of the pfa solution, which decreases the incidence of shearing the wholemount surface when immersing it in this solution. pour 5 - 10 ml of the warmed media into a dissecting dish placed under a fluorescent stereomicroscope. dissecting dishes are prepared by pouring an elastic polymer, called sylgard 184, into a 6 cm plastic dish and letting the polymer solution cure for 1 week under vacuum, then thoroughly rinsing dishes in large volumes of water before use. usually, we let the dishes soak in water in a 1 l beaker for 1 week. the animal is sacrificed by cervical dislocation and the head is cut off.note : if desired, blood may be cleared from the vasculature by perfusing the animal with normal saline prior to dissecting out the brain. a midline incision is made, posterior to anterior, along the scalp to reveal the skull. a series of 4 cuts in the skull are made to open the cranium : one cut spans the two orbits anterior to the olfactory bulbs, the next two cuts are inferior to the cerebellum and separate the cranium from the skull base, and the final cut runs posterior to anterior along the mid - sagittal suture. the cranial flaps are gently retracted and the brain is extracted and placed into the dissecting dish. if you wish additionally to examine the olfactory bulbs, simply fix them by immersion in 4% pfa overnight and you may subsequently prepare them for sectioning and staining. divide the brain along the interhemispheric fissure. a coronally - oriented cut is then made at the posterior - most aspect of the interhemispheric fissure, allowing the caudal hippocampus to be visualized in cross - section. the hippocampus, which forms the medial wall of the lateral ventricle at this position, must then be released from the overlying cortex, which forms the dorsal - lateral wall of the ventricle. first, the knife is inserted into the small ventricular space between the cortex and hippocampus dorsally, and a cut is made in the cortex where it reflects ventrally, away from the midline, to join the hippocampus. after this cut is made, the cortex can be slowly peeled away from the hippocampus to reveal the lateral ventricle moving from dorsal to ventral. this maneuver is expedited by cutting off a wedge of cortex at the corner where the hippocampus was released. after reaching the ventral - most extent of the lateral ventricle at this position, you may either visualize or feel where the cortex again wraps around, this time reflecting back medially, to join the hippocampus. another cut must be made in this position to completely release the hippocampus or medial wall of the lateral ventricle from the cortex or lateral wall of the lateral ventricle. it will then be easy to pull the hippocampus away from the cortex, medially and anteriorly, to open the lateral ventricle widely. continue to gently pull the hippocampus anteriorly using small strokes of the forceps and knife to retract the medial and lateral walls apart. once the resistance to this retraction begins to increase, first, to increase your exposure to the lateral ventricle and in particular, the lateral wall and svz, dissect away the cortex. the cortex is cleanly dissected away by visualizing the interface between the corpus callosum and the vz / svz. simply cut along this interface staying on the callosal side to avoid damaging the svz. in order to continue retracting the medial wall away from the lateral wall, two more cuts are needed : one cut dorsally where the lateral wall, medial wall, and cortex all converge, and one cut ventrally where the lateral wall, medial wall, and thalamus converge. with these cuts made, further gentle retraction on the medial wall allows the anterior - most extent of the lateral ventricle to be opened. proper lighting is essential throughout the procedure and especially in the next step where the medial and lateral walls are separated anteriorly. at this anterior position in the lateral ventricle, adjust the lighting such that shadows cast between the two walls reveal this reflection point, which appears like a valley between the two walls. finally, completely expose the lateral wall by removing any overhanging cortex dorsally and the thalamus ventrally. if preparing wholemounts for immunostaining, carefully transfer the wholemount, ventricle side up, from the dissection dish into a 24-well plate filled with 4% pfa with or without 0.1% triton - x100 for an overnight fixation at 4c. for fixation - sensitive antigens, wholemounts may be fixed for shorter periods of time. then proceed to section 4 on immunostaining wholemounts. if preparing wholemounts for ependymal flow analysis, transfer the wholemount to a clean dissection dish filled with fresh, 37c leibovitz medium and proceed to the next section. immobilize the wholemount on a clean dissecting dish using 2 insect pins, one in the thalamus and one in anterior - dorsal corner of the wholemount. make sure the height of the adjustable arm is maximally elevated to avoid breaking the needle against the dissection dish. carefully dip the tip of the needle in media in an ependorf tube to clean off microbeads that are present on the exterior tip of the needle. if these are not cleaned away, they may subsequently be deposited on the wholemout surface inadvertently during needle positioning and reduce the overall quality of the movie. position the needle tip over the dorsal surface of the lateral wall and lower the arm to bring the needle tip into the medium. the needle should be lowered until it is just above the lateral wall surface. with the needle in position, if you will be making a recording of the ependymal flow, begin acquiring images at this time. once the initial bolus of beads has been cleared off the surface by ependymal flow, additional rounds of bead ejection can be performed. wholemounts dissected for immunostaining are immersion - fixed overnight in 4% pfa with or without 0.1% triton - x100 at 4c. the use of triton - x100 is preferred for antigens that tolerate this treatment, but can be left out in cases where staining quality is diminished by detergent. the following morning, pfa is aspirated from the 24-well plate and the wholemounts are washed 3 times for 5 minutes each in 0.1 m pbs with or without 0.1% triton - x100. as before, the use of triton - x100 is preferred, but not required, for all washes in this protocol. throughout this protocol, exchanging solutions over the wholemount requires careful aspiration of the solution from the side of the well. then, the well is refilled with solution using a transfer pipette angled such that the solution washes over the side of the well, not directly onto the wholemount. take care to keep the ventricle side of the wholemount facing up at all times. we prefer to exchange solutions over 1 wholemount at a time to prevent tissue from drying. after washing off the pfa, wholemounts are incubated for 1 hour at room temperature in blocking solution, containing 10% normal goat or donkey serum in 0.1 m pbs with or without triton - x100. if using triton - x100 for your staining, you may choose to use either 2% or 0.5% triton - x100 in the blocking solution. we use 2% triton - x100 when staining for antigens that require deeper antibody penetration into the tissue, such as those antigens located in the svz. however, when staining for antigens located closer to the surface of the lateral wall, such as antigens found in the apical surface of ependymal cells, we use 0.5% triton - x100. in addition, triton - x100 can be left out for cell - surface or other antigens that are removed or altered by detergent. next, remove the blocking solution and add primary antibodies diluted in the same blocking solution and incubate for 24 or 48 hours at 4c. choice of the incubation period depends on the antigen, similar to choice of 0.5% or 2% triton. for antigens located on the surface of the wholemount however, for antigens located deeper, such as in the svz, 48 hour incubation periods provide better results.for example, to study the apical surface and basal bodies of cells lining the lateral ventricle wall, stain with antibodies to -catenin, to label the cell membrane, and -tubulin, to label basal bodies. dilute mouse anti--catenin antibodies (1:500) and rabbit anti--tubulin antibodies (1:1000) in 0.1 m pbs containing 10% normal goat serum and 0.5% triton - x100. incubate at 4c for 24 hours.to stain the adult neural stem cells, or type b1 cells, stain the lateral wall with gfap antibody. dilute mouse anti - gfap antibodies (1:500) in 0.1 m pbs containing 10% normal goat serum and 2% triton - x100. primary antibodies are washed off initially by 2 quick rinses in pbs with or without 0.1% triton - x100. dilute secondary antibodies in the same blocking solution used for primary antibodies and add to wholemounts to incubate for the same length of time as for primary antibodies at 4c.for example, for staining for -catenin and -tubulin : dilute alexa fluor 488 goat anti - mouse antibodies (1:400, recognizes mouse anti--catenin) and alexa fluor 594 goat anti - rabbit antibodies (1:400, recognizes rabbit anti--tubulin) in 0.1 m pbs containing 10% normal goat serum and 0.5% triton - x100. incubate at 4c for 24 hours.for gfap immunostaining : dilute alexa fluor 488 goat anti - mouse antibodies (1:400, recognizes mouse anti - gfap) in 0.1 m pbs containing 10% normal goat serum and 2% triton - x100. secondary antibodies are washed off the wholemount using the same washes performed for primary antibodies. if desired, nuclear counter - staining can be performed at this point by incubating in dapi diluted in pbs for 30 minutes at room temperature and then washing one time in pbs. for high - resolution confocal imaging, following immunostaining the wholemounts needed to be sub - dissected to preserve only the lateral wall of the lateral ventricle as a sliver of tissue 200 - 300 um thick. separating the lateral wall from the underlying striatum allows it to be mounted onto a slide and covered with a coverslip in a flat manner. return to the stereomicroscope with immunostained wholemounts and the following tools and equipment : smooth fine forceps, sharpoint 22.5 microsurgical stab knife, dissecting dish, microscope slides and coverslips, 0.1 m pbs, and aquamount mounting medium. transfer the wholemount from the 24-well plate to the dissecting dish containing 0.1 m pbs being careful to keep the ventricle side up. first, completely remove the dorsal cortex of the wholemount from posterior to anterior by cutting precisely along the line where the corpus callosum meets the lateral wall. this is recognized as the interface between the callosal white matter and the pink - appearing svz. then make a long horizontally - oriented cut across the ventral aspect of the wholemount. this cut surface will provide a platform onto which you can stabilize the wholemount during the next step in the dissection. with the dorsal surface of the wholemount facing up, you will be able to visualize the thickness of the svz from anterior to posterior along the lateral wall. note that this view was made possible by the intial removal of the cortex allowing the underlying striatum and svz to be seen. the svz is identified as the thin band of tissue extending from the ventricular surface to the striatum. the svz has a homogeneous pink appearance while the striatum is infiltrated by cords of white matter. once you have identified the interface of the svz and striatum, carefully begin cutting at this interface at the anterior - most aspect of the lateral wall, advancing the knife from dorsal to ventral. to do this accurately, stabilize the wholemount with your forceps used as two pins. you can also use your forceps to slightly turn the wholemount to visualize the blade advancing from dorsal to ventral across the lateral wall. the key to this dissection is for the resulting sliver of tissue to be very flat. this means that as you slice off the svz from anterior to posterior across the lateral wall, the orientation of the cuts you make must remain parallel to the ventricular surface at all times. rather than thinning out your dissection to cut off only the svz at this point, it is important that as you advance posteriorly, the thickness of the tissue being dissected remains the same. after completely separating the lateral wall from the underlying striatum, carefully remove all other surrounding tissues from this sliver that are not part of the ventricular wall. then pick up this sliver from below using your forceps and apply a few drops of aquamount directly onto the wholemount and gently place a coverslip centered over this, trying not to introduce bubbles onto the surface of the tissue. the weight of the slide will ensure that the aquamount disperses evenly and will produce a refined flattening of the lateral wall surface. the amount of aquamount used and the size of the coverslip depend on the age of the tissue being dissected. for embryonic and early postnatal tissues, we prefer 1 drop of aquamount and a 22 " x 30 " coverslip. heavier coverslips may distort the tissue and more aquamount will interfere with imaging quality because the confocal lasers will be less able to penetrate a thin layer of aquamount residing between the coverslip and the tissue surface. for later postnatal and adult tissues, we use 4 drops of aquamount and a 24 " x 60 " coverslip. the slides are then stored flat in a slide book at 4c for 1 - 2 days before imaging to allow the coverslips to settle. wholemount approaches have provided several key insights into the germinal activity of the adult svz. the network of chains of migrating young neurons in the svz was first observed after wholemounts of the lateral wall of the lateral ventricle were immunostained with antibodies to polysialylated neural cell adhesion molecule (psa - ncam). these chains of migrating neuroblasts can also be seen after immunostaining wholemounts with doublecortin antibodies (figure 1). remarkably, the network of chains has a stereotyped pattern, with two general streams of cells, one running dorsally over and one running ventrally around the adhesion point. wholemounts of the svz also provide a comprehensive view of the proliferative activity of progenitors in this region, as seen with ki67 staining in figure 2. interestingly, two recent studies suggest a close interaction between dividing svz cells and the local vasculature (figure 2). when examined under high power confocal microscopy, the en - face view provided by wholemounts allows a unique perspective of the apical surface of cells lining the ventricular system. this en - face perspective has recently revealed that svz type b1 cells, the adult neural stem cells, are part of a mixed neuroepithelium with non - dividing differentiated ependymal cells. the apical surface of type b1 cells contacts the lateral ventricle and is surrounded by large apical surfaces of ependymal cells in a pinwheel configuration (figure 3, arrows indicate b1 apical surfaces). furthermore, close examination of the apical surface of ependymal cells has revealed that the translational position and rotational orientation of their basal bodies are indicators of their planar polarity. this patch is displaced from the center of the apical surface in the downstream " direction with respect to csf flow (translational polarity) ; within this patch, each basal body is rotated about its long axis such that the basal foot, an accessory of the basal body, points in the direction of flow (rotational polarity). importantly, videomicrographs of the ependymal flow assay can be used to directly compare the flow in a specific region of the lateral wall to the orientation of ependymal cell basal bodies in that region (figure 4). in addition to providing a panoramic perspective of the largest germinal region in the adult brain, with higher power imaging, wholemounts allow a more complete and detailed analysis of individual cellular morphologies in the svz. high power confocal imaging of gfap immunostaining on wholemounts has revealed that type b1 cells, in addition to their short ventricle - contacting apical process, have a long basal process in contact with blood vessels (figure 5). this cytoarchitecture had not been appreciated previously in coronal sections because the basal process runs mostly parallel to the ventricular wall. serial sectioning therefore cuts individual cells into small fragments, making it nearly impossible to reconstruct a cell s complete morphology, or to understand its relationship to other cell types in the svz. the wholemount approach has several advantages over classical sectioning techniques, both in providing panoramic views with low power microscopy and a complete perspective of individual cells with high power microscopy. this technique will continue to be an important complement to future studies of this adult brain germinal zone. tiled confocal images reconstruct a lateral wall wholemount that was stained with antibodies to doublecortin, which labels migrating neuroblasts throughout the svz. there are two general streams of migration, one running dorsally over and one running ventrally around the adhesion point, indicated by the asterisk (). this lateral wall wholemount was immunostained with antibodies for ki67, to label dividing cells in green, and antibodies against mouse immunoglobulins, to label the vasculature in red. because this wholemount was not perfused with saline prior to staining, the endogenous mouse igg molecules remain within blood vessels and are stained by secondary anti - mouse antibodies. recent work suggests that dividing svz precursors (green) are located in close proximity to blood vessels (red) { shen, 2008 # 6523}{tavazoie, 2008 # 6522}. arrows indicate anterior (a) and dorsal (d) directions. high power confocal image of a wholemount immunostained for -catenin, to label cell membranes in green, and -tubulin, to label basal bodies in red, reveals the planar organization of these epithelial cells. type b1 cells, the adult neural stem cells, have a small apical surface with a single basal body, indicated by arrows. the apical surface of these cells is surrounded by the large apical surface of ependymal cells in a pinwheel configuration. ependymal cells have planar polarity indicated by the position of their multiple basal bodies on the apical surface. neighboring ependymal cells have their basal body clusters located on the same side of the apical surface (downward and leftward in this region), corresponding to the direction of csf flow { mirzadeh, 2010 # 6573}. scale bar = 10 m. figure 4. the ependymal flow assay. composite image created by merging 100 sequential frames from a movie taken during the ependymal flow assay. fluorescent microbeads deposited dorsal and posterior to the adhesion area were propelled by ependymal cilia in two oriented streams, one over and one under the adhesion, towards the foramen of monro. each flow line depicts the position of a single bead at consecutive points in time. gfap+ type b1 cells have a long basal fiber with end - feet on blood vessels. maximum projection of a high power confocal stack taken from a lateral wall wholemount immunostained with gfap antibodies to label svz astrocytes. this staining labels adult neural stem cells, or type b1 cells, which have an apical ending on the ventricular surface, and as shown here, a long gfap+ basal fiber that ends on blood vessels (arrows). blood vessels are stained here because the secondary antibody used to visualize mouse anti - gfap antibodies recognize endogenous mouse igg within the vasculature. most studies of neurogenesis in ventricular and subventricular zones have relied on classical sectioning techniques to examine the microanatomy and cellular relationships in these regions. here we describe an alternative technique, first used to analyze the network of migratory chains of neuroblasts generated in the svz, then used to study regeneration of the svz progenitor population following anti - mitotic treatment, and most recently used to study the precise apical and basal cell - cell interactions of adult svz neural stem cells. interestingly, this technique has revealed that the neural stem cells, or type b1 cells, of the adult svz are part of a mixed neuroepithelium with differentiated non - dividing ependymal cells. en - face imaging using wholemounts has shown that this mixed neuroepithelium has pinwheel architecture consisting of the apical endings of type b1 cells surrounded by large apical surfaces of ependymal cells. this en - face analysis has clarified our understanding of the lineage of neural stem cells in embryonic and adult brains as consisting of cells with apical endings at the ventricle surface and basal processes contacting a vascular niche. wholemounts also facilitate the identification of neural stem cells via their ventricle - contacting apical process. as more specific markers for these stem cells are found, wholemounts will be an integral part of identifying and analyzing neural stem cell behavior. wholemounts of the lateral ventricle walls also provide the ideal perspective for studying the planar polarity of ependymal cells. ependymal cells are multiciliated cells lining the ventricles that function to propel csf in a coordinated manner. with the wholemount technique, the entire ependymal epithelium is exposed en - face and can be stained and studied comprehensively from its anterior to posterior and dorsal to ventral boundaries. furthermore, ependymal flow assays performed on acutely dissected, live wholemounts robustly demonstrate the planar polarized flow generated by ependymal cilia. interestingly, wholemount studies have also suggested that ependymal - generated csf flow establishes gradients of chemorepellents that guide the migration of young neurons in the svz. wholemount approaches that initially identified the network of migratory neuronal chains are therefore continuing to provide insights into mechanisms regulating chain migration. analysis of the vz and svz by wholemount imaging adds a new approach for both future studies and a way to clarify our understanding of existing studies. for example, a recent study suggested that neural stem cells in the adult svz were cd133+/cd24- cells in contact with the ventricle. based on their immunostaining in sections, these authors claimed that these cells were a subpopulation of multiciliated ependymal cells. however, in our study using the wholemount approach, which gives a more comprehensive view of the entire ependymal epithelium, we found that all ependymal cells express cd24 and the only ventricle - contacting cells that were cd133+/cd24- were a subset of the type b1 cells. furthermore, the wholemount technique promises to be useful in future studies examining the recently described mosaic organization of neural stem cells in the adult brain. several studies have shown that neural stem cells in the adult brain are not a homogeneous population, but are regionally specified and normally produce only specific subtypes of olfactory bulb interneurons. these studies have proposed that different subpopulations of neural stem cells may be distinguished either by the expression of specific transcription factors and/or by their regional localization along the dorsal - ventral and anterior - posterior extents of the lateral wall. as more molecular markers of the regionally specified subpopulations of adult neural stem cells are identified, wholemount imaging should provide a comprehensive view of the parcelation of these different progenitor domains along the ventricular wall. the wholemount dissection and imaging techniques presented here may also be used to analyze the ventricular walls in the embryo. the dissection of the embryonic lateral wall is performed, step - by - step, in the same manner. there are only slight differences in the level of difficulty ; the embryonic ventricles are relatively larger making the dissection easier, but the tissue is softer making manipulation more difficult. in particular, a similar exposure of the lateral ventricle can be used in embryos to dissect the cortical wall of the ventricle to study cortical neurogenesis. recent evidence suggests that asymmetric centrosome inheritance maintains radial glia at the ventricular surface during cortical neurogenesis. en - face imaging of radial glial apical surfaces may provide insights into how centrosomes within these dividing cells are asymmetrically inherited. there are, however, a few elements in the dissection that are key to better results : 1) lighting adjusting the illumination of the sample to create shadows provides invaluable contrast during the dissection of tissue that is otherwise relatively homogeneous, 2) using the forceps like two insect pins the forceps in this technique are never used to pinch together or pick up tissue, but are used as maneuverable pins that can be continually readjusted to stabilize the tissue while cutting, 3) a balance of gentle retraction and cutting the knife should not only be used to cut but also to provide gentle retraction to separate the medial and lateral walls, remembering that the majority of this dissection is actually performed through gentle retraction with only intermittent cutting. | the walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. the subventricular zone (svz) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. traditionally, these studies have relied on classical sectioning techniques for histological analysis. here we present an alternative approach, the wholemount technique, which provides a comprehensive, en - face view of this germinal region. compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the svz. this approach has recently revealed that the adult neural stem cells, or type b1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. in addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. with recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche. |
anthocyanins belong to a diverse group of secondary metabolites of the phenylpropanoid class, the flavonoids, which are found in different plant species. they represent some of the most important natural pigments, which are responsible for the wide range of red to purple colors present in many flowers, fruits, seeds, leaves, and stems. besides having great economical relevance, flower and fruit pigments play an important ecological role in the animal attraction for pollination and seed dispersal, wich is a spectacular example of coevolution between plants and animals [13 ]. the biosynthetic pathway of anthocyanins has been well characterized biochemically and genetically in species with different floral morphology, pigmentation pattern, and pollination syndromes such as petunia hybrida [4, 5 ], matthiola, dianthus, eustoma, gerbera, zea mays [10, 11 ], antirrhinum majus, and ipomoea [13, 14 ]. a representation of a general anthocyanin biosynthetic pathway is shown in figure 1. briefly, the pathway is initiated with chalcone synthase (chs) catalyzing the stepwise condensation of three molecules of acetate residues from malonlyl - coa with one molecule of 4-coumaroyl - coa to form the basic structure of flavonoids (tetrahydroxychalcone), which is rapidly isomerized to the colorless naringenin by chalcone isomerase (chi). dihydroflavonol 4-reductase (dfr), which is a specific enzyme for the anthocyanin synthesis, catalyses the production of leucoanthocyanidins from dihydroflavonols, which can be hydroxylated on the 3 or 5 position of the b - ring by flavonoid 3-hydroxylase (f3h) to produce dihydroquercetin or by flavonoid 35-hydroxylase (f35h) to form dihydromyricetin. subsequently, leucoanthocyanidin oxidase / anthocyanidin synthase (ldox / ans) is responsible for the formation of the anthocyanidins from the colorless leucoanthocyanidins. gt enzymes (o - glucosyltransferases) represent the final step in anthocyanin biosynthesis : anthocyanidins are converted in differentially decorated anthocyanin molecules [15, 16 ]. biochemical approaches have demonstrated that all anthocyanin pigments are derived from one of three aglycones : pelargonidin, cyaniding, and delphinidin. the main determinants of the apparent color of these pigments are the hydroxylation and methylation patterns, as well as the number and type of sugars on the beta ring of the flavonoid molecule [1, 3, 1719 ]. at least, two groups of genes are required for anthocyanin biosynthesis : the first group is represented by the structural genes encoding enzymes for the production of the flavonoid precursors, as well as those involved in the formation of particular (decorated) anthocyanin molecules. the second group includes the genes encoding regulatory factors that control the expression of structural genes which are mainly orenestrated by complexes formed by myb and basic helix - loop - helix (bhlh) transcription factors that include wdr (wd40 repeats) proteins [2, 4, 15, 16, 2023 ]. some passiflora species have economical importance due to the production of fruits (passionfruit) or use as ornamentals. nevertheless, a large number of passiflora species are rare and/or endangered, as the environment of their diversity center has been increasingly degraded by human activities. an enormous floral diversity is observed among passiflora species, including variation in color, size, morphology, and fusion of floral organs. these and other floral characteristics, including evolutionary innovations such as the presence of coronal filaments and an androgynophore, are indicative of the wide range of pollination syndromes found in the genus. wide passionflowers may be pollinated by insects (bees and wasps), hummingbirds, and bats. the most striking feature of floral variation among passionflowers is the wide range of pigmentation patterns of the corona filaments. most of the floral pigments in passiflora are different types of anthocyanin molecules [25, 26 ]. among all passiflora species, p. edulis deg and p. suberosa l. are of particular interest, because they are model passiflora species for which expressed sequences tags (ests) were produced within the frame of the passioma project. these flowers are about 812 cm wide, and their coronas contain multiple series of purplish filaments with white tips. the flowers of p. suberosa l. are small (2 - 3 cm wide) and show two morphologically distinct series of corona filaments : the outer series is greenish, and the inner series is formed by smaller purple filaments. we are particularly interested in the characterization of genes involved in the anthocyanin biosynthetic pathway of these two passiflora species. with this aim, we searched for putative passiflora genes responsible for flower pigmentation, using the key proteins known to be involved in the different enzymatic steps of anthocyanin biosynthesis as baits to search for expressed sequences tags (ests) in the passioma database. the clustered expressed sequence tags (ests) from the passioma project database were used as a primary source of data for our analyses. these sequences were assembled from ests obtained from the sequencing of several p. edulis or p. suberosa cdna libraries, made from floral buds at different developmental stages (see for details on library construction, sequencing, and database structure). nucleotide sequences and their respective deduced amino acid sequences from genes known to be involved in anthocyanin biosynthesis (see figure 1) were obtained from the national center for biotechnology information (ncbi ; http://www.ncbi.nlm.nih.gov/). searches for putative homolog sequences in the passioma database were conducted using the tblastn module that compares the consensus amino acid sequence with a translated nucleotide sequences database. we generally used arabidopsis thaliana or petunia hybrida as query consensus sequences as the anthocyanin biosynthesis pathways in these model species are more thoroughly studied at the molecular level [3032 ]. all sequences in the passioma database that exhibited a significant alignment (e - value lower than 105) with the query were retrieved from the passioma database. the clusterization of all reads identified using a given query sequence was performed using the cap3 algorithm from the bioedit software. the novel cluster consensus sequences obtained were reinspected for the occurrence of conserved motives using interproscan and were compared to ncbi databases using blast. sequences that did not show the main motives present in the query sequence were discarded. the obtained alignments were eventually corrected by hand and imported into the molecular evolutionary genetics analysis (mega) software. phylogenetic trees were obtained using parsimony and/or genetic distance calculations (in the later case using pairwise deletion option and with the poisson correction model). neighbor - joining and bootstrap (with 10,000 replicates) trees were also constructed. the cdna libraries of the passioma project were obtained from mrna extracted from floral buds at different developmental stages, and it is expected that all est sequences correspond to genes expressed during passiflora flower development. this sequence search detected a total of 75 passiflora est sequences, 34 of them corresponding to p. edulis sequences and 41 of them corresponding to sequences derived from p. suberosa libraries. when submitted to the cap3 algorithm and detailed comparison of their deduced amino acid sequences, the number of valid clusters was reduced to 15, potentially corresponding to 15 different genes. when the validated amino acid sequences obtained from the passioma database were compared to other plant protein sequences in the public databases this was expected, as passiflora and these genera belong to the same order (malpighiales) and are considered to be closely related. we obtained assembled est sequences corresponding to genes of the following genes families : chs, dfr, gt, gst, myb, and wd40 (see table 1). therefore, we used 15 passiflora assembled sequences from the passioma database and a selected set of genes from divergent plant species from the public databases to explore their evolutionary relationships. the obtained sequence comparison alignments allowed the construction of phylogenetic trees for each of these families of genes involved in the different enzymatic steps of the anthocyanin pathway. the similarities among all genes identified in this study and those reported from other plant species were assembled in table 1 and ranged from 70% (pacepe3030g03.g ; representing a putative member of the gst, glutathione s - transferase superfamily) to 96% (pacepe3007g07.g ; potentially encoding a wd40 protein). some of these gene sequences showed significant similarity to elements required for early or late steps of the pathway ; others putatively encode regulatory proteins involved in the control of the spatial and temporal patterns of pigmentation, while others are responsible for intracellular transport of the anthocyanin molecules. the role of each of these genes in the anthocyanin biosynthesis and the probable implications for the understanding of the passiflora flower pigmentation are presented in the discussion. we have found 5 passiflora assembled sequences (5 putative genes) encoding enzymes of the chs family : pacepe3010g11.g, pacepe3014b06.g, pacepe3007g06.g, pacepe3023h10.g and paceps7017d03.g. these sequences are expected to encode proteins with 231, 158, 254, 237, and 222 amino acids, respectively. the deduced chs proteins showed more than 80% similarity to chss of other plant species (table 1). to determine the phylogenetic relationship of different chss, we aligned protein sequences from a diverse range of plant species (moss, ferns, gymnosperms and angiosperms), cyanobacterium (synechococcus sp.) and passiflora representatives of the chs superfamily (figure 2). one of these monophyletic clades (highlighted in figure 2) contains all the anther - specific chs - like genes (ascls ; [40, 41 ]). the remaining sequences, including three passiflora members, were clustered in the other sister clade together with all chs genes from seed plants. a single passiflora cdna sequence of 850 bp encoding a predicted protein of 204 amino acids showed significant e - value (1e) and 94% similarity to a populus dfr sequence (table 1). figure 3 shows an alignment of the deduced amino acid sequence of the passiflora dfr with some other plant sequences containing an nadp - binding domain, considered the region of substrate preference of dfr enzymes [42, 43 ]. additionally, the passiflora dfr showed an aspartic acid residue at position 134, as it is observed for the petunia and populus proteins, whereas gerbera and some lotus dfr show an asparagine residue at the same position (figure 3). we adopted the terminology suggested by shimada and coworkers to designate the conserved motifs present in the dfr sequence. a neighbor - joining tree was constructed based on the alignment dfr sequences shown in figure 3. the monocots and eudicots dfrs were positioned separately. while monocot dfr genes formed one clade, the eudicot dfr sequences diverged into two clades. clearly, asn - type dfrs are found in a larger number of species. on the other hand, asp - type dfrs we identified two passiflora est clones, pacepe3030g03.g and paceps7021h02.g, encoding proteins with sequence similarity to ricinus communis glucosyltransferases (table 1). the first cdna sequence contained an orf specifying a 124 amino acid protein, and the second cdna encoded a protein of 200 amino acid residues. these putative passiflora gt proteins were compared with those gt enzymes described by kovinick and colleagues and retrieved from the ncbi database. the obtained phylogenetic tree resulted in five clades, according to their in vitro substrate specificities. phylogenetic analysis revealed that the passiflora sequences were positioned within the cluster ii proteins (figure 5). comparison of these deduced gst protein sequences with those in the genbank database revealed homology with multifunctional gsts from populus, ricinus, and glycine spp (see table 1). phylogenetic relationships among the putative passiflora gsts and family members of other plant species were established (figure 6). based on sequence similarity, the five passiflora putative gsts were grouped into three clades. pacepe3018f08.g, paceps4006h06.g, and paceps7023b03.g are type i gsts, pacepe3007a05.g is a type ii gst, and pacepe3013h01.g is a type iii gst. we could not find any putative homologs to chalcone isomerases (chi), flavanone 3-hydroxylases (f3h), and anthocyanidin synthases (ans ; see figure 1) in the passioma database. three est sequences were identified corresponding to a putative flavonoid 3-o - hydroxylase (f3h) gene, and one sequence was found that showed significant homology to genes encoding flavonoid 3 - 5-o - hydroxylases (f35h ; data not show). as these sequences were incomplete at their 5 end, they were not considered in our analyses. based on the searches in the passioma database, we identified one potential homolog for an myb transcription factor of the r2r3 class. the p. suberosa cdna clone paceps7022e07.g encodes a protein of 132 amino acids showing 91% similarity to the ricinus communis r2r3 myb. on the other hand, pacepe3007g07.g is a putative p. edulis wd40 gene of 886 bp encoding 291 amino acid residues showing 96% similarity to an r. communis, wd40 (table 1). figure 7 shows an alignment of the deduced paceps7022e07.g protein sequence with 17 other plant anthocyanin - related r2r3-myb, indicating the presence of a conserved dna - binding domain, designated as the r2r3 domain. all sequences analyzed also contained a second conserved amino acid motif in the r3 repeat (red box), important for the interaction between myb and bhlh proteins in arabidopsis. the four specific residues required for this interaction in maize are also indicated by the arrows in figure 7. the third conserved motif appears to be andv (blue box) in the r3 repeat of all eudicot r2r3-myb proteins related to anthocyanin biosynthesis. a phylogenetic tree of selected plant r2r3-myb transcription factors, including paceps7022e07.g, was constructed using the alignment of the conserved r2r3 repeats (figure 8). the passiflora sequence was placed within the clade including zmc1 (zea mays), phph4 (petunia hybrida), vvmyb5a, and vvmyb5b (vitis vinifera), which are known to be involved in the regulation of the anthocyanin pathway in these species [4951 ]. sequence comparison of selected plant wd40 proteins with the sequence obtained from p. edulis indicated that the four wd repeats are highly conserved among all species analyzed (figure 9). phylogenetic analysis of these amino acid sequences confirmed that p. edulis wd40 grouped together with ricinus communis wd40 and found to be more related to other dicot proteins (figure 10). flavonoid pathway results in the production of a range of flavonoid compounds, including anthocyanins (figure 1). chs is the first enzyme in the phenylpropanoid pathway and is encoded by members of a plant - specific multigene family of polyketide synthases. nevertheless, genes belonging to the chs family have been recently described to occur in some microorganisms (azotobacter vinelandii ; and neurospora crassa ;) and, therefore, indicate chs functions might have evolved previous to the divergence of land plants. thus, the biological functions of some of the chs superfamily members are clearly important to plant adaptation. chs proteins are collectively linked to the biosynthesis of different plant products with diverse functions such as uv protection, defense against pathogens, pigment biosynthesis, and pollen fertility [54, 55 ]. sequence analysis indicated that two passiflora chs deduced proteins belong to a small distinct group of chalcone synthases that includes angiosperm and gymnosperms homologs to anther - specific chalcone synthase - like genes (ascls ; highlighted in figure 2). furthermore, all ascls form a monophyletic clade. recently, ascls transcripts were detected within the tapetum cells during microspore stage in wheat. these genes apparently have important roles in anther development and in pollen fertility [40, 41, 56 ]. the remaining three passiflora chss were clustered together in a sister clade containing all seed plant chs genes. these include chsa and chsj genes, known to be expressed in floral tissues, and involved in floral pigmentation in petunia [30, 31, 57 ]. moreover, two nonchalcone genes, divergent from the typical chss, formed a separate clade. the sypks gene from cyanobacterium encodes an enzyme of the thiolase superfamily, whereas the function of the ppchs11 gene (from physcomitrella patens) may resemble more the most recent common ancestor of all plant chss than do other members of the plant chs superfamily. we do not have identified putative genes encoding chi enzymes. besides the general limitations and drawbacks of the est - based approach, another possible explanation may be because the rapid isomerization of chalcone to form narigen and the fact that even in the absence of a functional chi enzyme, chalcone can spontaneously isomerize to form naringenin. dfr is an enzyme catalysing the reduction of three dihydroflavonols : dihydromyricetin (dhm), dihydroquercetin (dhq), and dihydrokaempferol (dhk) into colorless leucoanthocyanidins. these are further converted to delphinidin, cyaniding, and pelargonidin (figure 1). the synthesis of three different anthocyanidins is mainly determined by the enzymes activities of two hydroxylases : f3oh and f35oh. the first converts dhk to dhq and f35oh converts dhk to dhm. in some plant species, dfr displays distinct substrate specificity in according to the hydroxylation pattern of anthocyanin molecule. a hypothesis to determine substrate specificity was proposed based on the amino acid sequence alignment of petunia dfr with others plants. naturally, petunia hybrida does not produce orange flowers, because the dfr enzyme can not use dihydrokaempferol as substrate to produce pelargonidin, due to an aspartic acid residue at the 134th position [30, 42 ], as it was also observed for passiflora (figure 3), thus converting dihydroquercetin to leucocyanidin and, more efficiently, the reduction of dihydromyricetin to leucodelphinidin [30, 59 ]. on the other hand, some gerbera genotypes have an asparagine residue at this same position and can utilize three dihydroflavonols as substrates of dfr, consequently producing orange to red colored flowers [9, 30 ]. thus, the flower color is partly determined by alteration of a single amino acid that changes the substrate specificity of the dfr enzyme. almost all anthocyanidins undergo several modifications, which vary across species and involve enzymes of the glucosyltransferase, methyltransferase, and acyltransferase families. the most common is glycosylation of the 3-position of anthocyanidins (represented in figure 1) to produce stable anthocyanin molecules [15, 30, 31, 60 ]. udp - glucose : flavonoid 3-o - glucosyltransferase (3gt) belongs to a large multigene glucosyltransferases (gts) family, representing the final step in anthocyanin biosynthesis. in this work, we adopted the classification of the gts into clusters according to kovinic and colleagues. cluster ii includes gts with multiples substrates preferences, generally for chalcones, flavones and flavonols but not anthocyanidins. cluster iv glycosylates flavonol and isoflavonol substrates and cluster v have anthocyanin 5-o and/or flavonol 7-o - ugt enzymes. our results indicated that the obtained passiflora glucosyltransferase gene sequences were grouped in cluster ii, together with other family members that show a high catalytic specificity for more than one class of flavonoid substrates (figure 5). dicgt5 (from dianthus caryophyllus) glycosylates a chalcononaringenin 2-o - glucosyltransferase, whereas the beta vulgaris gt has a favonoid-7, 4-o - betanidin-5-o - glucosyltransferase activity. both gts have non - anthocyanidin substrate specificity. despite these results, obviously neither gt substrate specificity, nor in vivo function of the passiflora gts can be predicted solely based on amino acid sequence similarities and must be experimentally determined. anthocyanin biosynthesis has been demonstrated to occur predominantly in the cytosol, but these pigments are exclusively accumulated in the vacuole of epidermal cells. transport of pigments to the vacuoles requires a glutathione s - transferase and a specific carrier protein localized in the vacuolar membrane. plants gsts are classified on the basis of sequence identity into four classes : phi, tau, theta, and zeta. the two small zeta and theta classes include gsts from animals and plants, while the phi and tau classes are plant - specific. phan2 (from petunia), zmbz2 (from maize), and attt19 (from arabidopsis) are gst proteins involved in anthocyanin transport [3032, 6365 ]. to characterize their phylogenetic relationships, the deduced amino acid sequences from the passiflora putative gsts were compared with other plant gst sequences, including the ones mentioned above. figure 6 shows that the passiflora gsts are included into three different clades : three sequences were positioned in the same clade of phan9 and attt19 (phi class), whereas one sequence was grouped together with zmbz2 (tau class ;). although of these known proteins belong to distinct gst clades, they perform similar functions [6365 ]. interestingly, pacepe3007a05.g was clustered with carnation (dianthus caryophyllus) gst type ii (zeta class) which is associated to petal senescence in response to ethylene [67, 68 ]. at the moment, we can classify the passiflora gsts into type i (phi), type ii (zeta), and type iii (tau). at least, four of them might be involved in the anthocyanin pathway and pacepe3007a05.g might be related to other biological processes related to flower development such as those observed for the carnation gst. in all analyzed species, the spatial and temporal expression of the structural genes of the anthocyanin biosynthetic pathway is controlled by regulatory genes, which interfere with the intensity and pattern of anthocyanin biosynthesis. mybs, basic helix - loop - helix (bhlh) transcription factors and wd40 proteins form a transcriptional complex for the activation of the structural genes [4, 12, 20, 47, 69, 70 ]. mybs and bhlhs proteins are coded by large multigene families, and those associated with anthocyanin biosynthesis are characterized by a conserved dna - binding domain consisting of two imperfect repeats (named r2r3), and a specific bhlh domain, respectively. these two gene families have been extensively studied in model plants such as arabidopsis and maize [48, 49, 71 ]. a multiple sequence alignment of the r2r3 domains of selected myb proteins known to be involved in anthocyanin biosynthesis regulation, and the deduced amino acid sequence of paceps7022e07.g confirmed the presence of the conserved r2r3-myb domain in this p. suberosa sequence (figure 7) as well as that of a second conserved domain in the r3 repeat (red box, figure 7), which is known to be necessary for the interaction between myb and bhlh transcription factors [48, 49 ]. additionally, a third motif in the r3 repeat (andv, blue box in figure 7) represents a conserved motif shared among all eudicot mybs involved in the anthocyanin biosynthesis. the phylogenetic tree obtained using the alignment shown in figure 7 is presented in figure 8 and indicates lhmyb6 and lhmyb12 clustered outside the eudicot clade. these two genes regulate anthocyanin biosynthesis in the flowers of lily (lilium hybrid), a monocot. one clade is formed exclusively by eudicot anthocyanin regulators (phan2, atpap1, atpap2, amrosea1, and amrosea2 ; [12, 71, 7477 ]. curiously, one regulator of the anthocyanin in maize (a monocot), zmc1 was positioned in the same clade of other dicot members such as phph4 (from petunia), vvmyb5a, and vvmyb5b (from vitis), as well as the passiflora r2r3-myb sequence. vvmyb5a and vvmyb5b genes are involved in the regulation of anthocyanin biosynthesis during grape berry development. wd40 proteins are highly conserved and can be found in organisms that do not biosynthesize anthocyanins as algae, fungi, and animals [78, 79 ]. in plants, as an example, the arabidopsis transparent testa glabra 1 (ttg1), which is a wd40 protein, is involved in regulating trichome formation, anthocyanin biosynthesis, seed coat pigmentation, and seed coat mucilage production. a common feature of wd40 repeat proteins is that they facilitate protein - protein interactions between the myb and bhlh proteins [22, 79 ]. the alignment of the passiflora wd40 protein sequence with other known wd40s from different plant species revealed the presence of conserved wd40 motifs in the c - terminal region (figure 9). the results indicated that the monocot sequences zmpac1 and oswd clustered together, whereas the eudicot wd40s known to function as anthocyanin regulators were grouped into a different clade, with passiflora wd40 being closely related to the ricinus communis protein (rcwd, table 1 and figure 10). although wd40 proteins are required to regulate anthocyanins and proanthocyanidin together with myb and bhlh transcription factors, their potential involvement in other biological processes is enormous, therefore, it is premature to say what functions pacepe3007g07.g might perform in passiflora. the fact that no putative homologs to bhlh transcription factors were found in the passioma database may reflect the high degree of novelty of most of the libraries of the passioma project indicating that full gene expression spectra was not completely achieved. perhaps a more deep sequencing effort would reveal that such homologs are indeed expressed in passiflora flowers, as these elements are generally essential to myb - wd40 protein complex stability [3032 ]. we took the first steps toward the understanding of the molecular processes involved in the biosynthesis of anthocyanins in passiflora that could account for the differences in pollinator preferences found in the genus. we identified 15 putative coding sequences derived from two distinct passiflora species (p. edulis and p. suberosa) expressed in developing flower buds and potentially involved in the anthocyanin biosynthetic pathway. comparisons of deduced amino acid sequences from the 15 passiflora cdnas with selected sequences from other plant species revealed strong similarity with genes that encode key elements involved in the biosynthesis (8 sequences), transcriptional regulation (2 sequences), and transport (5 sequences) of anthocyanin molecules. needed research concerning the determination of temporal and spatial expression patterns of all these passiflora putative anthocyanin - related genes presented here are already ongoing in our group. we expect that future work on the manipulation of their expression patterns, using transgenic approaches, will help us to unravel important aspects relating anthocyanin biosynthesis, flower pigmentation, and flower pollination in rapidly changing tropical environments. | most of the plant pigments ranging from red to purple colors belong to the anthocyanin group of flavonoids. the flowers of plants belonging to the genus passiflora (passionflowers) show a wide range of floral adaptations to diverse pollinating agents, including variation in the pigmentation of floral parts ranging from white to red and purple colors. exploring a database of expressed sequence tags obtained from flower buds of two divergent passiflora species, we obtained assembled sequences potentially corresponding to 15 different genes of the anthocyanin biosynthesis pathway in these species. the obtained sequences code for putative enzymes are involved in the production of flavonoid precursors, as well as those involved in the formation of particular (decorated) anthocyanin molecules. we also obtained sequences encoding regulatory factors that control the expression of structural genes and regulate the spatial and temporal accumulation of pigments. the identification of some of the putative passiflora anthocyanin biosynthesis pathway genes provides novel resources for research on secondary metabolism in passionflowers, especially on the elucidation of the processes involved in floral pigmentation, which will allow future studies on the role of pigmentation in pollinator preferences in a molecular level. |
antimicrobial resistance is a troubling and growing public health threat. in addition to resistance mechanisms that can develop at the cellular level, the propensity of bacteria to form biofilms further protects them from environmental assaults, including antibiotics and the host immune system. in fact, bacterial surface - attachment and subsequent biofilm formation are considered important hallmarks of the capacity of microbial communities to cause persistent infections. understanding factors that contribute to bacterial aggregation during biofilm maturation is critical to the study of microbial physiology and ecology, as well as to the advancement of new treatments for chronic infections and novel strategies to prevent biofilm - associated problems. thus far, the primary optical tool for studying biofilms has been laser scanning microscopy, from single - photon, two - photon to multi - photon excitation microscopy. although these imaging techniques have reified our current view of complex and heterogeneous biofilm structures, new optical tools are still desired for characterization and manipulation of biofilms, especially since their structures vary over time and under different environmental conditions. optical tweezers with fine - shaped light beams provide excellent tools for trapping and manipulating bacteria as well as micro- and nano - scale particles. over the past decades, optical tweezers have been routinely used for single - molecule force spectroscopy to understand the mechanics of biological processes. recently, they have also been employed to study bacterial aggregation, as well as to better characterize bacterial motility and flagellar rotation. in conventional single - beam gradient optical tweezers (figure 1a), however, a rod - shaped object or bacterium (such as a bacillus thuringiensis (b. thuringiensis) cell) tends to align preferentially towards the propagation direction of a trapping beam, preventing direct in - plane observation and manipulation of the trapped object. in single - molecule force measurements, a molecule of interest is often tethered to a surface at one end and attached to a trapped dielectric bead at the other end, or both ends are attached to simultaneously trapped beads in dual - beam (dumbbell) optical tweezers. at the single - cell level, a dual - beam optical trap (figure 1b) is also necessary for orientation and manipulation of individual cells in space. the need for active control of single or numerous trapped objects has motivated the development of multi - trap optical tweezers, including dynamic holographic tweezers and those created with complex beam - shaping techniques. nevertheless, available methods for optical trapping and manipulation of bacteria still have substantial limitations in quantitative characterization of bacterial motility and intercellular interaction. for example, dual - beam optical tweezers rely on a pair of perpendicularly polarized beams and significant user control of each trapping beam : a rod - shaped bacterium has to be trapped and flipped by one of the beams first, and then the user needs to manually control the other trap to orient the cell into the desired observing plane. in addition, the use of optical tweezers to stretch a bacterial cell directly or to break up cellular clusters still remains a challenge. in this work, we describe an optical tweezer - based assay for the study of bacterial adhesion, relying on a tug - of - war (tow) design from novel shaping of light (figure 1c). instead of using two separate traps under independent control, we split a single beam into a pair of elongated trapping beams propagating with a diverging angle. this tow design has the following advantages over the conventional single- or dual - beam optical tweezers. first, it allows for stable in - plane trapping of a rod - shaped object with a single control implemented at will, without any mechanical movement or phase - sensitive interference. second, and more importantly, such tow tweezers can apply a tunable lateral pulling force on the trapped object, and the strength of the pulling force can be varied by changing the trapping beam intensity from femto- to piconewton levels. as an example, we employed the tow tweezers to trap, stretch and even break apart sinorhizobium meliloti (s. meliloti) cellular clusters aggregated under different conditions. we estimated the force needed for disassembly of adhesive s. meliloti cells, and found that the strength of bacterial adhesion is dependent on the growth medium. we believe this technique can bring about new avenues of exploration for optical manipulation and biophotonics. first, we discuss the design and demonstration of the tow optical tweezers. conceptually, the design of tow optical tweezers relies on nontrivial shaping of a gaussian beam into two elongated (stripe - like) beams with opposite transverse momenta. when applied in the optical tweezers setting, pulling forces arise on both sides of the trapped object, as in a tow duel. in practice, the size, separation, and propagation direction of the two beams can be varied at will, as implemented by encoding the holographic information onto a labview - assisted spatial light modulator (slm), thus allowing interactive control of the directions and magnitudes of the pulling forces (supplementary information). to better visualize the intensity distribution and structure of the resulting tow beam, a technique for volumetric representations of holographic optical traps was used. by acquiring a stack of two - dimensional images of the trapping beam near the focal plane, experimentally recorded data are replotted in figure 1d, in which a composite picture shows the side - view of the beam structure, along with the calculated vector field distribution of the intensity gradient. this design of the tow tweezers, although still based on the holographic principle, leads to an effective optical tool for trapping rod - shaped objects. distinct from conventional dual - beam or holographic tweezers, the intensity landscaping in the tow tweezers manifests a strong intensity gradient in the central pulling region, with maximal momenta oriented in opposing directions as a result of the synergistic action of optical scattering and gradient forces. in addition, the two elongated beams in the tow tweezers provide a better match to the bacterial morphology, enabling stable trapping of a rod - shaped bacterium even at low power levels, thus reducing the effects of photodamage on the trapped cell. the vector field of the intensity gradient of the trapping beam in figure 1 represents a useful description of the contribution from the gradient force that normally dominates in optical tweezers. by reshaping the trapping beam (thus the force distribution), tow tweezers can be optimized to trap rod - shaped objects of different sizes and compositions, including silica rods (used as a proof of principle), escherichia coli (gram - negative bacterium about 2 m in length) and b. thuringiensis (gram - positive bacterium with cell length ranging from 5 to 15 m). in fact, tow tweezers can be reconfigured to trap asymmetrically shaped particles with lengths varying from micrometre to sub - millimetre. as an example, we show in the bottom panels of figure 1d experimental snapshots obtained from the tow trapping and self - aligning of a dividing, rod - shaped b. thuringiensis cell. after entering the trap, the bacterium is reoriented onto the observing plane and stretched from two ends, unable to escape the trap. to illustrate the feasibility and potential application of the tow design for biofilm study, we used tow optical tweezers to disassemble clusters of s. meliloti cells and to show that the strength of cell cell adhesion depends on the growth medium. s. meliloti is a gram - negative soil bacterium capable of establishing endosymbiosis with compatible host plants. host interactions and shares with related bacteria, including those that act as pathogens, many critical factors that regulate cellular differentiation and organelle development. recent analysis revealed that a common laboratory strain of s. meliloti, rm1021, possesses a nonsense mutation in the podj gene, which encodes a conserved polarity factor that influences various cell envelope - associated functions. correcting the mutation and restoring the gene to that seen in environmental isolates of s. meliloti (supplementary information) resulted in a strain that develops robust biofilms in select liquid medium. this podj strain forms cellular clusters that resemble those of related alphaproteobacteria, suggesting that the strain synthesizes an adhesive organelle at one pole of the cell, similar to the holdfast of caulobacter crescentus and unipolar polysaccharide of agrobacterium tumefaciens. the amount of biofilms formed by the podj strain depends on the growth medium and correlates with the extent of cellular aggregation observed (figure 2). specifically, the strain forms large cellular clusters and heavy biofilm in peptone yeast extract (pye) medium, while the clusters tend to be smaller and the biofilm lighter in tryptone yeast extract (ty) medium. there is no or very weak biofilm formation and aggregation in lysogeny broth (lb) medium. thus, the degree of biofilm formation appears to reflect the strength of intercellular attachment. with the tow optical tweezers, we can administer an adhesive strength assay by directly trapping and stretching a cellular cluster to estimate the underlying force. typical experimental results are presented in figure 3. as shown in the left panels of figure 3a3f, cells attached to one another in the ty medium could be trapped by the tow tweezers, stretched gradually from two ends, and, most importantly, broken apart eventually. note that the whole disassembly process does not need any tethering or mechanical movement, nor does it require recalibration of trapping power with beam positions. tuned merely by varying the trapping beam power at the focal point and/or slm - controlled angle and the spacing of the tow beams. (for the results shown in figure 3, the length of the two stripe - like beams was about 1.5 m each, while the spacing between the two beams was about 5 m). the beam power used to break apart the s. meliloti clusters in ty medium was only about 20 mw. this simply can not be achieved with conventional dual - trap tweezers or an optical stretcher created with two counter - propagating beams. by reconfiguring the tow beams, even an asymmetrically shaped cellular cluster in ty medium in contrast, s. meliloti clusters formed in pye medium remained intact even when the power of the tow beam was increased to more than 5 times higher (figure 3 g and 3h), indicating stronger adhesion among the cells. from our estimate, the pulling force required to break up a s. meliloti cluster in ty medium should be at least 5 pn from each side of the tow tweezers. a much stronger force would be needed in pye medium for similar disassembly. these results illustrate that the tow tweezers allow us to characterize quantitatively the effects of growth media on cell cell adhesion, thus facilitating the elucidation of environmental factors that affect bacterial aggregation and biofilm formation. direct measurement of the forces from the tow tweezers acting on the bacteria is challenging since the forces involved in separating and trapping bacterial cells are of vastly varying magnitudes. in order to have a better understanding of the lateral forces in the tow optical trap, we utilized a few different measurement techniques. theoretically, in contrast to the familiar case of a spherical particle in the single - beam trap, precise calculation of stiffness and strength of compound traps such as the tow tweezers is very complicated. in fact, only recently have theoretical models been put forth for the study of trapping forces with asymmetrically shaped particles, and for shape - induced force fields in general. the non - conservative nature of the optical force fields certainly manifests itself directly in the stiffness of trapped aspherical objects, such as a rod - shaped bacterium. first, to substantiate that an outward pulling or splitting force indeed exists in the tow trap, we used the method of particle image velocimetry to estimate the magnitude and direction of the flow of suspended polystyrene beads driven by the tow tweezers. to illustrate the concept, the two beams (diverging in the x - direction) constituting the tweezers have a large separation of about 5 m at the trapping plane, and the beads have an average size of about 500 nm. as seen in figure 4a and 4b, the tow tweezer behave as two micropumps for a thin sample of aqueous suspension of the beads : the beads flow away from the central region along two opposite directions due to the scattering force exerted by the diverging beams (supplementary movie 3). the time - averaged velocity of the particle flow is replotted in figure 4b, where arrowed lines mark the particle flow velocity distribution. clearly, this diagram of hydrodynamic particle flow illustrates a transverse momentum leading to pulling in opposite directions, giving rise to the tow action mediated by our judiciously shaped optical beam shown in figure 1d. moreover, since the hydrodynamic driving of particles infers the direct relation to the force, it also provides information about the force distribution (magnitude and direction) from the tow tweezers. as seen in figure 4a and 4b, the pulling forces drive the particles towards the two sides rather than the central region between the two traps. second, to obtain an estimate of the magnitude of the trapping forces from the tow tweezers, we analysed the time - dependent positions of a single s. meliloti cell trapped by only one side of the tow tweezers using the established method of optical potential analysis. this position distribution of the trapped cell is obtained by extracting data from video microscopy using a particle tracking software. from the occupancy probability and by employing the boltzmann statistics theorem, the potential energy, and thus, the force distribution in space can be deduced. figure 4c shows the position distribution of the cell when it is trapped by only one arm of the tow tweezers. as seen in figure 4c, force distribution in the transverse y - direction (that is, perpendicular to the pulling direction) is not notably different from that resulting from a standard gaussian trap ; in contrast, in the x - direction, the distribution becomes highly asymmetric. in other words, even when just one arm of the tow tweezers is present, the bacterium experiences a net force in a preferred direction (that is, along the pulling direction). although the bacterium is not necessarily stably trapped at the point where the peak force is applied, we can still obtain a value for the peak force by fitting experimental data to the theoretical model. the results from a theoretical estimate of the force are also plotted in figure 4c for comparison, which gives a force of about 0.35 pn per 0.1 m. with this method, the peak force from one arm of the tweezers is estimated to be at least 5 pn for a rod - shaped cell ~ 1.5 m in length when displaced to the centre of the tow trap. of course, when both traps are present under the tow action as shown in figure 3a3c for breaking up the s. meliloti cluster, the actual pulling forces from both sides could be much larger than this estimated value. third, we provided a theoretical analysis of the forces mediated by the optical landscape of the tow tweezers. since the beam shaped by the slm is strongly focused by an objective lens to achieve a high - field gradient, it is necessary to use a rigorous vectorial electromagnetic (em) treatment to facilitate the modelling. as such, the debye wolf integral was used to construct the field in the focal plane. following a similar method used previously for slm - assisted beam shaping, the radiation in the focal plane was calculated as the integral of spherical vectors waves emanating from the objective lens. to simplify the calculation, we treated the trapped bacterium approximately as a spherical particle with a size of 1 m and a refractive index of 1.38. the forces acting on the particle by the tow beams were calculated via the t - matrix method, derived from the generalized lorenz mie theory : at each point of the trapping plane, the incident em field around the particle was calculated based on the actual holograms used experimentally on the slm and the optical parameters of the system, and the scattered field was then calculated via the t - matrix. by comparing the incident and scattered fields, the forces on the particle a typical calculated force distribution around such a particle at the trapping plane is plotted in figure 4d, showing a clear pulling effect on the trapped particle : the optical force is particularly strong in the middle of the tow duel where particles will be strongly pushed away from the centre of the beam. in order to categorize the forces at work in the tow duel, this corresponds to the amount of power required to provide a particular trapping force for a given particle. our analyses show that, under the experimental conditions, the peak q - value of the tow beam is 1.5 times larger than that of a similarly positioned gaussian trap. in other words, in tow tweezers, a trapped particle will experience a pulling force 1.5 times greater that in gaussian beam - based tweezers. in addition, the tow beam has a stronger force differential between the positive and negative sides of the trap when compared with a gaussian equivalent, leading to enhanced lateral pulling forces. nevertheless, due to the elongated shaping of the tow beams, the intensity threshold for bacterial photodamage could be higher as compared with that for gaussian traps. the range of forces that the trap can exert is bounded on the lower end by the thermal force (in the low femtonewton regime, depending on temperature), and on the upper end by undesirable photodamage of bacterial cells (in the high piconewton regime, which typically occurs at a relatively high power level, depending on the species and the trapping wavelength). finally, to highlight the difference between conventional dual - beam tweezers and our tow tweezers, two approaches were used. one was to compare the position distribution of a s. meliloti cell trapped by only one side of the dual - traps, and the other was to compare the stability of a micro - rod trapped by both sides with two different tweezers systems. in both cases, a strongly oscillating environment was provided to test the stability of the traps and to simulate ambient perturbation for motile bacteria. although both types of tweezers can trap and hold a rod - shaped object or a bacterial cluster in the observing plane, the tow system has obvious advantages. first, unlike in a single trap based on a symmetric gaussian beam, a trapped object in one side of the tow beam has a preferred direction of displacement due to its asymmetrically shaped intensity profile (figure 5a5d). as shown in figure 5b and 5d, under a periodic perturbation (for example, the sample is oscillated sinusoidally), a trapped s. meliloti cell moves around its central equilibrium position evenly in the gaussian trap, but it shows up more in a preferred direction in the tow trap, indicating the pulling effect from the latter configuration. second, to stretch the trapped object, at least one of the gaussian - beam - based dual traps has to be moved laterally, while in the tow tweezers, one does not need to translate the objective lens, and the object still experiences a constant stretching force due to the asymmetric intensity gradient. third, an object trapped by tow tweezers exhibits increased stability and resistance to ambient perturbation when compared against conventional dual - beam tweezers. to better illustrate the advantage in trap stability, a rigid silica micro - rod was used as a test object instead of a bacterial cell to prevent any possible damage- or deformation - induced effects. experimental results are presented in figure 5e and 5f. in figure 5e, the position distributions of the trapped rod in both the dual - beam and the tow tweezers are plotted, where the occupancy probability is obtained by taking 10 000 snapshots from a recorded video of the micro - rod in the trap. clearly, in the tow tweezers, the micro - rod is better confined in the y - direction (that is, the direction perpendicular to the stretching direction) than in the x - direction, whereas in the dual traps based on the symmetric gaussian beams there is no such difference. thus, when applied to a bacterial cell or a cellular cluster, the tow tweezers give rise to a stable trapping in the y - direction along with the flexibility to move around its equilibrium position in the x - direction, which offers an advantage for stretching. in figure 5f, the sample experiences sinusoidal oscillation in three dimensions, as driven by a piezoelectric transducer (pzt)-actuated vibration control, and the cutoff amplitude and frequency of oscillation at which the silica rod can no longer stay in the trap are plotted for comparison. from these results stretched in the transverse x - direction, is much more stably trapped in the y- and z - directions in the tow tweezers than it is in the dual gaussian beam tweezers. clearly, both approaches coherently show that a rod - shaped object exhibits better stability when being trapped and stretched by the tow tweezers, as compared with the conventional dual - beam tweezers. in summary, we have demonstrated that judiciously shaped light beams can stretch and even break apart bacterial clusters, leading to a simple assay for cellular adhesion. in particular, we have shown that our specially designed tow optical tweezers can be used as an effective tool for evaluating s. meliloti cell adhesion under different growth conditions. we have estimated the optical forces needed to disassemble s. meliloti flocs and determined that the trapping stability of tow tweezers exceeds that of conventional dual - beam tweezers. this work represents another successful example of using static optical forces and novel beam shaping to perform diagnostic mechanical tests at the cellular level, and the technique can be readily adopted for studying the mechanical properties and dynamics of various living cells. since cellular adhesion has a crucial role in biofilm development, our technique suggests exciting possibilities of developing new optical tools for investigating biofilm formation and related biomedical applications. finally, this technique might be employed in single - molecule force microscopy, for example, to stretch dna molecules without the need for positional calibration of paired traps. | bacterial biofilms underlie many persistent infections, posing major hurdles in antibiotic treatment. here we design and demonstrate tug - of - war optical tweezers that can facilitate the assessment of cell cell adhesion a key contributing factor to biofilm development, thanks to the combined actions of optical scattering and gradient forces. with a customized optical landscape distinct from that of conventional tweezers, not only can such tug - of - war tweezers stably trap and stretch a rod - shaped bacterium in the observing plane, but, more importantly, they can also impose a tunable lateral force that pulls apart cellular clusters without any tethering or mechanical movement. as a proof of principle, we examined a sinorhizobium meliloti strain that forms robust biofilms and found that the strength of intercellular adhesion depends on the growth medium. this technique may herald new photonic tools for optical manipulation and biofilm study, as well as other biological applications. |
bracket bonding has some drawbacks including low strength of bracket bond to tooth structure and subsequently high risk of debonding, high risk of plaque accumulation and consequent development of white spot lesions around brackets [14 ]. prevention of enamel demineralization around orthodontic brackets is a major challenge in fixed orthodontic treatment. evidence shows that the population of cariogenic bacteria significantly increases during the course of orthodontic treatment [79 ]. higher counts of mutans streptococci and lactobacilli have been reported in the oral cavity following placement of fixed orthodontic appliances. at a ph of 0.05). the mean shear bond strength of bracket to enamel in the four groups (mpa) pairwise comparison of the four groups in terms of shear bond strength curcnps : curcumin nanoparticles the mean shear bond strength of bracket to enamel in the four groups with 95% confidence interval the frequency of adhesive remnant index (ari) scores number & percentage in the four groups curcnps : curcumin nanoparticles biofilm test results : the mean colony count for each bacterial strain in the four groups is shown in table 4. the results of post hoc test for s. mutans count revealed that s. mutans colony count in all concentrations of curcnps was significantly lower than that compared to the control group (plain composite) and reached zero (all p0.999). the colony count of s. sanguinis significantly decreased in all three concentrations of curcnps compared to the control group (all p0.999). the colony count of l. acidophilus significantly decreased in all three concentrations of curcnps compared to the control group (all p<0.001). however, l. acidophilus colony count was not significantly different among the three groups with 1%, 5% and 10% concentrations of curcnps (p=0.968 for the comparison of 1% and 5%, p=0.884 for the comparison of 1% and 10% and p=0.992 for the comparison of 5% and 10%). disc agar diffusion test results : in the agar diffusion test, no growth inhibition zone was noted around composite discs in any group. eluted component test : analysis of the results of eluted component test for assessment of bacterial proliferation at three different time points by two - way anova revealed that the interaction effect of time and concentration was not significant for s. mutans (p=0.985), s. sanguinis (p=0.980) or l. acidophilus (p=0.955). the effect of concentration was not significant either for s. mutans (p=0.486), s. sanguinis (p=0.794) or l. acidophilus (p=0.954). assessment of the effect of time revealed that a significant reduction in colony count only occurred at 30 days for all microorganisms (p<0.001). descriptive values of colony count for each bacterial strain in the four groups (cfus / mm) researchers have long been in search of methods to prevent enamel demineralization during orthodontic treatment without adversely affecting the bond strength. addition of nanoparticles to composite resin has been documented as an effective strategy to prevent enamel demineralization. however, aside from their antimicrobial efficacy, their effects on physical and mechanical properties of orthodontic composites must be evaluated. optimal antimicrobial activity of curcumin has been documented against enterococcus faecalis and s. mutans. the cariostatic effect of curcumin is mediated by prevention of bacterial adhesion to enamel and destruction of bacterial cell wall via disrupting the peptidoglycan layer. moreover, despite its strong antimicrobial activity, curcumin is non - toxic and safe ; fernandes, reported that cell viability of human gingival fibroblasts was not affected by exposure to curcumin in short or long - term. they added that curcnps have higher solubility and bioavailability than curcumin extract emulsion and can better pass through the cell membrane ; as the result, curcnps have greater efficacy in lower dose. thus, in the current study, curcnps were added to orthodontic composite to assess their antimicrobial efficacy and effect on sbs of brackets to enamel. shear bond strength testing is commonly used for assessment of bond strength of brackets to enamel. to simulate the thermal and physical stresses applied to teeth in the oral environment, we performed thermocycling according to a previous study. also, we used bovine teeth due to their easy availability. selection of central incisors was due to the fact that they have a smoother surface than other teeth and allow optimal adaptation of bracket to tooth structure ; this increases the accuracy of measurement of sbs. the results of the current study revealed that by addition of curcnps up to 5%, the sbs of transbond xt composite to enamel did not change significantly and was within the clinically acceptable range of 68mpa. however, the sbs in 10% curcnp group was significantly lower than that in the control group. since no previous study was found on the effect of curcnps on bond strength and other properties of composites, we compared our findings with the results of other studies on some other nanoparticles. poosti, assessed the effect of incorporation of tio2 nanoparticles on sbs of composite and reported that 1% concentration of tio2 yielded a bond strength value similar to the control group ; this finding was similar to our results ; however, they used tio2 instead of curcnps. moreover, they mixed the nanoparticles with composite using a mixer while we mixed them manually. also, they stored the samples at 37c for 24 hours while we performed thermocycling ; the latter further decreases the bond strength. akhavan, reported an increase in bond strength following addition of 1% silver nanoparticles / hydroxyapatite while bond strength decreased following the addition of 5% and 10% silver nanoparticles / hydroxyapatite. in terms of dose - dependent results, however, their results regarding 1% concentration were different from our findings, which is attributed to the main differences between the two studies including the type of nanoparticles used and type of teeth. they reported that increased bond strength in 1% group was due to the ability of silver nanoparticles / hydroxyapatite to enhance the adhesion at the interface of restorative material - enamel via increasing the mechanical strength of the adhesive layer and reinforcing the supporting structures. however, it should be noted that increased bond strength is not always optimal and if it exceeds a certain amount, it can cause enamel damage at the time of debonding. moreover, it should be noted that addition of silver nanoparticles even in concentrations as low as 1% can cause significant discoloration of composite, which compromises esthetics. the ari score is an important parameter in selection of orthodontic adhesive by clinicians. in the current study, no significant difference was noted among the four groups in terms of ari scores, which was in agreement with the results of a previous study. the current study also assessed the antimicrobial activity of transbond xt composite containing 1%, 5% and 10% concentrations of curcnps against s. mutans, s. sanguinis and l. acidophilus. initiation of caries mainly depends on the activity of s. mutans while lactobacilli (mainly l. acidophilus) are responsible for progression of caries. presence of s. sanguinis in the oral cavity decreases the population of s. mutans and these two are in equilibrium. biofilm inhibition test was carried out to assess the antimicrobial activity of composites since it has been shown that bacteria in the form of biofilm are four times more resistant to antibacterial agents compared to planktonic form. the current results showed that addition of curcnps to composite significantly decreased the bacterial count of all three strains compared to the control group in all three concentrations. the results for s. mutans were highly favorable since s. mutans colony count in presence of all concentrations of curcnps decreased to zero. this indicates low minimum inhibitory concentration and minimum bactericidal concentration of curcnps against s. mutans. this finding is clinically significant since s. mutans is the main cariogenic microorganism in the oral cavity. on the other hand, l. acidophilus showed higher resistance, which may be due to its role in progression of caries and formation of a very strong biofilm. in contrast to our findings, mirhashemi, showed that only 10% concentration of nano - zinc oxide / nano - chitosan significantly decreased all three microorganisms ; in their study, 5% concentration of nanoparticles was ineffective on l. acidophilus and 1% concentration had no effect on any microorganism. difference between the results of the two studies is due to difference in type of nanoparticles used. these findings indicate the superior antimicrobial activity of curcnps compared to other nanoparticles tested in the above - mentioned studies. in the current study, the antimicrobial effects due to release of nanoparticles in composite samples were assessed by disc agar diffusion test. this test is important because white spot lesions are often formed around brackets (and not beneath them) ; thus, an ideal antimicrobial agent for addition to orthodontic composite must be able to diffuse into the environment. thus, curcnps do not have non - contact (long - distance) antimicrobial activity. zinc nanoparticles also have low solubility similar to curcnps and aydin sevinic and hanley showed that despite optimal antimicrobial activity, zinc nanoparticles did not form growth inhibition zone in disc diffusion test. growth inhibition zone was noted around 10% concentration of chitosan / zinc oxide nanoparticles in the study by mirhashemi. eluted component test shows the antimicrobial activity of a solution containing nanoparticles released from composite discs over time and indicates the substantivity of antimicrobial activity. the results of eluted component test in the current study revealed a significant reduction in all three bacterial colony counts only at 30 days, irrespective of the concentration of curcnps, which indicates low solubility and low diffusion of curcnps in an aqueous environment. similarly, mirhashemi, showed that l. acidophilus colony count only decreased at 30 days following exposure to chitosan / zinc oxide nanoparticles. thus, considering the low solubility and poor diffusion of curcnps, further studies must focus on its use along with a nano - carrier to enhance its diffusion in the environment and improve its release profile. considering the significant effect of 1% concentration of curcnps on antimicrobial property of orthodontic composite and no significant difference between 1% concentration and control group in terms of sbs, as well as the insolubility and low diffusion of curcumin, we suggest adding another soluble nanoparticle with high diffusion to 1% concentration of curcnps. | objectives : this study sought to assess the effect of curcumin nanoparticles (curcnps) on antimicrobial property and shear bond strength (sbs) of orthodontic composite to bovine enamel.materials and methods : in this in vitro, experimental study, 1%, 5% and 10% curcnps were added to transbond xt composite. stainless steel brackets were bonded to 48 sound bovine incisors in four groups (n=12) using composite containing 0% (control), 1%, 5% and 10% curcnps. the bracket - tooth sbs was measured by a universal testing machine. the adhesive remnant index (ari) score was calculated after debonding using a stereomicroscope. also, 180 discs were fabricated of the four composites ; 108 were subjected to eluted component test, 36 were used for disc diffusion test and 36 were used for biofilm test to assess their antimicrobial activity against streptococcus mutans, streptococcus sanguinis and lactobacillus acidophilus.results:the highest and lowest sbs belonged to control and 10% curcnp groups, respectively. the difference in sbs was significant among the four groups (p=0.008). the sbs of control group was significantly higher than that of 10% curcnps (p=0.006). the four groups were not significantly different in terms of ari score (p>0.05). growth inhibition zones were not seen in any group. in biofilm test, the colony counts of all bacteria significantly decreased by an increase in percentage of curcnps. colony count significantly decreased only at 30 days.conclusions:at 1% concentration, curcnps have significant antimicrobial activity against cariogenic bacteria with no adverse effect on sbs. however, insolubility of curcnps remains a major drawback. |
hysterosalpingography (hsg), also called uterosalpingography, is a fluoroscopic imaging method that uses an iodinated contrast media to investigate endometrial - uterine morphology and fallopian patency in women with infertility and repeated abortions. hsg can identify many lesions, including hyperplasia, polyps, fibroids, scarre - synechiae, and mullerian anomalies. fallopian occlusion due to infection, scarring, ectopic pregnancy, diverticula, tubal ligation, closure devices, and reopening interventions can be evaluated by hsg. hsg is easy, safe, useful, and cost - effective with excellent diagnostic and therapeutic outcomes. however, a few complications, including radiation exposure, vasovagal attack, uterine injury, vaginal bleeding, infection, hypersensitivity, and intravasation might be observed during or after the procedure. hysteroscopy like hsg is a useful screening test for the evaluation of infertility through analysis of the uterine cavity. intravasation is the passage of contrast media into the veins due to local or systemic abnormalities. it can be observed with uterophlebography ; however, this technique can create reticular patterns and multiple thin lines that ultimately lead to false assumptions in diagnosis. prevention of intravasation during hsg is critical for procedural safety and may be related to predisposing factors, including endometrial vascularity and permeability. the variability between clinical and basic research on the determination of intravasation suggest the need for a classification to reduce misdiagnosis. to the best of our knowledge, the main preprocedural (leukocytes, menometrorrhagia, secondary infertility, ectopic pregnancy, abortus, polycystic ovaries, endometriosis, interventions) and procedural (pain, scheduling, endometrial - uterine nature, spillage) parameters associated with intravasation and classification of intravasation have not yet been evaluated. our report represents the first classification of intravasation since the work of rindfleisch in 1910 using bismuth. the primary aim of the present study was to compare differences in patients whose hsg scans show no intravasation with patients whose hsg scans show intravasation and to assess the predisposing factors of intravasation. the secondary goal was to describe clinical- and imaging - based novel grading of intravasation. by eliminating predisposing factors the present study protocol was planned in accordance with the declaration of helsinki and was approved by our institutional ethics board. our study included 569 women (mean age 31.1 6.0 (19 - 49) years) who underwent hsg for infertility and repeated abortions between 2008 and 2011 in our center. it is a retrospective study of the hsg scans based on the complication - related grouping, the women without intravasation were assigned to the control group (n = 528) and those with intravasation to the study (n = 41) group. women with increased serum -human chorionic gonadotropin, vaginal bleeding, and hypersensitivities to the contrast medium were excluded. hsg was scheduled between the 3 and 13 days of the menstrual cycle to ensure that menstruation had ended and the women were not pregnant. thus, the women were grouped as follows, post - menstrual (p1 : 3 - 5), mid - follicular (p2 : 6 - 10), and preovulatory (p3 : 11 - 13) periods [figure 1 ]. hsg was performed by an experienced radiologist (ad) as described in four gradual steps in the supine position. speculum was inserted to display the cervix and tenaculum was applied after topical lidocaine (10% xylocaine ; astra zeneca, mississauga, on, canada). leech wilkinson cannula was positioned in the cervical canal before obtaining first image as described. hydrosoluble iodized contrast medium (omnipaque ; nycomed, amersham, uk) 15 ml was slowly administered with fluoroscopic guidance. a second image was obtained at the early phase to evaluate contour irregularity or small filling defects in the endometrial cavity. a third image was obtained when the endometrial cavity distended to evaluate uterine morphology and tubal patency. intravasation was observed to be higher in the post - menstruation (p1) and preovulation (p3) phases than in the mid - follicular (p2) phase. the aim of hsg imaging was to answer the critical clinical questions - the cause of infertility and abortion, prior to the intervention. these questions concerned presence or absence of the venous intravasation and its type (using a novel classification described by authors). all images were reviewed by two radiologists (ad and ab) and two gynecologist (hs and ng), and were grouped by consensus into two (without and with intravasation) groups based on clinical and imaging characteristics. intravasation severity score [table 1 ], was designed based on qualitative and quantitative parameters, including loss of contrast media, systemic hypersensitivity reactions, misdiagnosis, peritoneal spillage, occurrence, extension of zonal location, and visualized urine bladder. intravasation severity score on imaging, intravasation has varied appearance from a reticular pattern to linear pattern seen as multiple thin lines. intravasation severity score included four levels : level 0, no intravasation ; level 1, mild intravasation limited to the myometrium ; level 2, moderate intravasation restricted within the parametrial - adnexial veins occurring slowly ; and level 3, severe intravasation extending from the myometrial - parametrial to the paracaval veins occurring immediately. to apply this tool, we devised a schema divided into four independent levels based on easily identifiable landmarks as (0) endometrium, (1) myometrium, (2) parametrial, and (3) parailiac veins [figure 2 ]. schematic view of the intravasation severity score (iss) based on regional landmarks for intravasations : (a) level 0 : endometrium (none) ; level 1 : myometrium (mild) ; level 2 : parametrium (moderate), and level 3 : parailiac (severe). images show severe (level 3) intravasation in internal iliac veins occurring immediately (thin arrows), endometrial bulging (black arrows), myometrial enhancement (m), and patent tubes (double arrows) with loculated peritoneal spillage (), and notable urine bladder (u) visualization. the statistical package for social sciences (spss) software package for windows (spss version 18.0 ; chicago, il, usa) was used for statistical analysis. continuous (demographic) data were expressed as the median (range, minimum value maximum value). variables (clinical and procedural data) were analyzed using the chi - squared test and compared using the mann - whitney u - test and student 's t - test. our study included 569 women (mean age 31.1 6.0 (19 - 49) years) who underwent hsg for infertility and repeated abortions between 2008 and 2011 in our center. it is a retrospective study of the hsg scans based on the complication - related grouping, the women without intravasation were assigned to the control group (n = 528) and those with intravasation to the study (n = 41) group. women with increased serum -human chorionic gonadotropin, vaginal bleeding, and hypersensitivities to the contrast medium were excluded. hsg was scheduled between the 3 and 13 days of the menstrual cycle to ensure that menstruation had ended and the women were not pregnant. thus, the women were grouped as follows, post - menstrual (p1 : 3 - 5), mid - follicular (p2 : 6 - 10), and preovulatory (p3 : 11 - 13) periods [figure 1 ]. bowel preparation was recommended the night before the procedure to improve diagnostic quality. hsg was performed by an experienced radiologist (ad) as described in four gradual steps in the supine position. speculum was inserted to display the cervix and tenaculum was applied after topical lidocaine (10% xylocaine ; astra zeneca, mississauga, on, canada). leech wilkinson cannula was positioned in the cervical canal before obtaining first image as described. hydrosoluble iodized contrast medium (omnipaque ; nycomed, amersham, uk) 15 ml was slowly administered with fluoroscopic guidance. a second image was obtained at the early phase to evaluate contour irregularity or small filling defects in the endometrial cavity. a third image was obtained when the endometrial cavity distended to evaluate uterine morphology and tubal patency. intravasation was observed to be higher in the post - menstruation (p1) and preovulation (p3) phases than in the mid - follicular (p2) phase. the aim of hsg imaging was to answer the critical clinical questions - the cause of infertility and abortion, prior to the intervention. these questions concerned presence or absence of the venous intravasation and its type (using a novel classification described by authors). all images were reviewed by two radiologists (ad and ab) and two gynecologist (hs and ng), and were grouped by consensus into two (without and with intravasation) groups based on clinical and imaging characteristics. intravasation severity score [table 1 ], was designed based on qualitative and quantitative parameters, including loss of contrast media, systemic hypersensitivity reactions, misdiagnosis, peritoneal spillage, occurrence, extension of zonal location, and visualized urine bladder. intravasation severity score on imaging, intravasation has varied appearance from a reticular pattern to linear pattern seen as multiple thin lines. intravasation severity score included four levels : level 0, no intravasation ; level 1, mild intravasation limited to the myometrium ; level 2, moderate intravasation restricted within the parametrial - adnexial veins occurring slowly ; and level 3, severe intravasation extending from the myometrial - parametrial to the paracaval veins occurring immediately. to apply this tool, we devised a schema divided into four independent levels based on easily identifiable landmarks as (0) endometrium, (1) myometrium, (2) parametrial, and (3) parailiac veins [figure 2 ]. schematic view of the intravasation severity score (iss) based on regional landmarks for intravasations : (a) level 0 : endometrium (none) ; level 1 : myometrium (mild) ; level 2 : parametrium (moderate), and level 3 : parailiac (severe). images show severe (level 3) intravasation in internal iliac veins occurring immediately (thin arrows), endometrial bulging (black arrows), myometrial enhancement (m), and patent tubes (double arrows) with loculated peritoneal spillage (), and notable urine bladder (u) visualization. the statistical package for social sciences (spss) software package for windows (spss version 18.0 ; chicago, il, usa) was used for statistical analysis. continuous (demographic) data were expressed as the median (range, minimum value maximum value). variables (clinical and procedural data) were analyzed using the chi - squared test and compared using the mann - whitney u - test and student 's t - test. intravasation was classified as level 0 (n = 528 ; 92.8%), level 1 (n = 12 ; 2.1%), level 2 (n = 18 ; 3.2%), and level 3 (n = 11 ; 1.9%). all patients were divided into two groups : those without intravasation (level 0 : n = 528, 92.8%) and with intravasation (from level 1 to level 3 ; n = 41, 7.2%). intravasation was evaluated using the demographic data and clinical data noted prior to hsg procedure. no significant difference was observed between groups regarding age (30.9 6.0 years vs. 32.0 6.6 years, p = 0.182). intravasation was associated with an increased leukocyte count (6.8 2.4 vs. 8.2 2.5, p < 0.02), painful procedure (p < 0.04), women with vaginal itching and nonspecific pelvic pain using visual analog pain scale (vas) score during hsg (3.8 1.8 vs. 7.3 2.7, p < 0.04), menometrorrhagia (p < 0.001), secondary infertility (p = 0.019), ectopic pregnancy and abortus (p < 0.001), and polycystic ovarian disease, endometriosis, recently removed fibroids, and hydatidiform mole (p < 0.001) [figure 3 and table 2 ]. images show mild (level 2) intravasation with myometrial reticular enhancement (m), parametrial veins (arrows), patent tubes (double arrows), loculated peritoneal spillage (), and visible urine bladder (u). intravasation was higher during post - menstrual (p1) and preovulatory (p3) than middle follicular (p2) periods (p < 0.001), women with endometrial notch and synechia or bulging (p < 0.001) [figure 4 ], mullerian anomalies (p < 0.001), and loculated peritoneal spillage (p < 0.001). mullerian anomalies consist of hypoplasia / agenesis (1.2% vs. 0.0%), arcuate (16.5% vs. 2.1%), septate (4.6% vs. 0.5%), bicornuate (6.1% vs. 0.7%) [figure 5 ], unicornuate (0.9% vs. 0.4%) [figure 6 ], and didelphus (0.2% vs. 0.2%) uterus without and with intravasation during hsg, respectively were detected according to the american fertility society (afs) classification. no statistically significant difference was detected between the control and intravasation groups regarding the tubal patency due to increased pressure (p = 0.172). periprocedural and postprocedural imaging and clinical data (a and b) 39-year - old women with recent operated myoma, endometrial notch and synechiae (arrows). images show severe (level 3) intravasation with endometrial bulging (arrow), involving myometrium (m), parametrial and paracaval veins (arrows), patent tubes (double arrows), and minimal peritoneal spillage (). (a and b) 36-year - old women with communicating unicornuate uterus (american fertility society (afs) iia. images show mild (level 1) intravasation endometrial bulging (arrow), myometrial enhancement (m), patent tubes (double arrows), and minimal peritoneal spillage (). images show moderate (level 2) intravasation endometrial bulging (black arrow), myometrial enhancement depicting a fundal lobulation (f), patent tubes (double arrows), and peritoneal spillage (). intravasation was classified as level 0 (n = 528 ; 92.8%), level 1 (n = 12 ; 2.1%), level 2 (n = 18 ; 3.2%), and level 3 (n = 11 ; 1.9%). all patients were divided into two groups : those without intravasation (level 0 : n = 528, 92.8%) and with intravasation (from level 1 to level 3 ; n = 41, 7.2%). intravasation was evaluated using the demographic data and clinical data noted prior to hsg procedure. no significant difference was observed between groups regarding age (30.9 6.0 years vs. 32.0 6.6 years, p = 0.182). intravasation was associated with an increased leukocyte count (6.8 2.4 vs. 8.2 2.5, p < 0.02), painful procedure (p < 0.04), women with vaginal itching and nonspecific pelvic pain using visual analog pain scale (vas) score during hsg (3.8 1.8 vs. 7.3 2.7, p < 0.04), menometrorrhagia (p < 0.001), secondary infertility (p = 0.019), ectopic pregnancy and abortus (p < 0.001), and polycystic ovarian disease, endometriosis, recently removed fibroids, and hydatidiform mole (p < 0.001) [figure 3 and table 2 ]. images show mild (level 2) intravasation with myometrial reticular enhancement (m), parametrial veins (arrows), patent tubes (double arrows), loculated peritoneal spillage (), and visible urine bladder (u). intravasation was higher during post - menstrual (p1) and preovulatory (p3) than middle follicular (p2) periods (p < 0.001), women with endometrial notch and synechia or bulging (p < 0.001) [figure 4 ], mullerian anomalies (p < 0.001), and loculated peritoneal spillage (p < 0.001). mullerian anomalies consist of hypoplasia / agenesis (1.2% vs. 0.0%), arcuate (16.5% vs. 2.1%), septate (4.6% vs. 0.5%), bicornuate (6.1% vs. 0.7%) [figure 5 ], unicornuate (0.9% vs. 0.4%) [figure 6 ], and didelphus (0.2% vs. 0.2%) uterus without and with intravasation during hsg, respectively were detected according to the american fertility society (afs) classification. no statistically significant difference was detected between the control and intravasation groups regarding the tubal patency due to increased pressure (p = 0.172). periprocedural and postprocedural imaging and clinical data (a and b) 39-year - old women with recent operated myoma, endometrial notch and synechiae (arrows). images show severe (level 3) intravasation with endometrial bulging (arrow), involving myometrium (m), parametrial and paracaval veins (arrows), patent tubes (double arrows), and minimal peritoneal spillage (). (a and b) 36-year - old women with communicating unicornuate uterus (american fertility society (afs) iia. images show mild (level 1) intravasation endometrial bulging (arrow), myometrial enhancement (m), patent tubes (double arrows), and minimal peritoneal spillage (). images show moderate (level 2) intravasation endometrial bulging (black arrow), myometrial enhancement depicting a fundal lobulation (f), patent tubes (double arrows), and peritoneal spillage (). in the present study, we found that intravasation can be observed during hsg in women with certain clinical symptoms (preprocedural) like increased leukocytes, vaginal itching, nonspecific pelvic pain, menometrorrhagia, secondary infertility, ectopic pregnancy and abortus, polycystic ovarian disease, endometriosis, recently removed fibroids, hydatidiform mole, and subclinical urinary infections. intravasation was seen more frequently in women who experienced pain during hsg procedure, who were in post - menstrual and preovulatory phase and also in women with predisposing factors such as endometrial notch and synechiae or bulging, mullerian anomalies, and loculated peritoneal spillage. no association was found between tubal occlusion (increased pressure) and intravasation. to avoid and minimize complications as well as potential pitfalls, this novel classification (particularly in subsumed conditions) the contrast transits from the uterine cavity directly to myometrial vessels with subsequent draining to the pelvic veins. overall, complications of hsg are not so infrequent. in addition, complications may be accompanied by intravasation, which may involve hypersensitivity, bleeding, and infection. venous intravasation, passage of contrast media, or fluid into the veins from the endometrium can cause pulmonary embolism along with systemic side effects. this variability (misdiagnosis) might be due to the fact that the staging of intravasation has not been done before. to the best of our knowledge, our report represents the first classification of intravasation since the work of rindfleisch in 1910. we defined a novel classification system for intravasation with four levels : level 0, no intravasation ; level 1, mild intravasation limited to the myometrium (leading to false assumptions in diagnosis and confused with adenomyosis) ; level 2, moderate intravasation restricted within the parametrial - adnexial veins and occurring slowly ; and level 3 severe intravasation extending from the myometrial - parametrial to the paracaval veins and occurring immediately. endometrial histologic dating is related to endometrial maturation, which is assessed by luteinizing hormone, follicle - stimulating hormone, and estradiol levels during menstrual cycles. endometrium is thin in the early proliferative phase and is an advantage that helps facilitate imaging. hsg should be scheduled between the cessation of menstruation and before ovulation, yet early enough so that sufficient time exists to clear blood and menses - related residue. moreover, performing hsg during the first 10 days of menstruation is not reliable for unsuspected pregnancy in women with irregular menstruation. microvascular blood flow increases in the early follicular and luteal phases, which reflect preparation for menstrual bleeding, and vascular permeability increases during menstruation. in another study, we found an increased association between intravasation and scheduling of hsg when it is done during the early postmenstrual and the late preovulatory period. discomfort and a painful procedure may be related to spasms caused by cervical fixation and contrast application during hsg. prostaglandin inhibitors can be used to reduce pain and pseudoimages. pelvic discomfort and unusual lingering pain during hsg might be related to intravasation and may require prompt intervention. although intravasation was historically associated with an increased risk of venous embolus due to the used contrast agents, negative side effects have been reduced since hsgs are now performed with hydrosoluble contrast media. hydrosoluble contrast media are associated with less complications and good radiographic quality as compared to the liposoluble contrast media. for this reason, the hydrosoluble media achieved popularity for use with hsg. we did not report systemic effects caused by intravasation due to the use of hydrosoluble contrast media. endometrial and tubal tuberculosis can cause infertility as a consequence of the immunosuppression of the endemic areas. hsg has been reported to demonstrate tubal irregularity, multiple small diverticula in the isthmic portion of the tube wall as salpingitis isthmica nodosa often associated with tubal contraction, hydrosalpinx, synechiae, distortion, peritubal adhesions, and intravasation. a recent paper reported that the treatment of the suspected inflammation beforehand is better than undertreatment to reduce complications of hsg. uterine malformations are related to secondary infertility, repetitive abortion, endometrial injury, and complicated delivery. in a population - based study, the prevalence of mullerian anomalies was reported to be 3%. moreover, mullerian anomalies prevalence was reported as 5 - 10% and 25% in patients with recurrent first- and second - trimester abortus, respectively. the higher incidence of abortus risk among patients with mullerian anomalies was demonstrated as well. although intravasation can occur in patients during hsg, there are some predisposing factors such as uterine anomalies. we found an association between mullerian anomalies and intravasation as a result of increased predisposing factors. most of the studies reported that tubal occlusion might be associated with intravasation due to increasing intrauterine pressure. however, recent studies of the effectiveness of tubal closure devices reported no intravasation during hsg. although a relatively rare event, an awareness of uterine intravasation can prevent potential misinterpretation of hsg. this is a complication and potential pitfall during hsg procedure as the intravasation can mimic intraperitoneal spillage in the occluded tube. we did not observe intravasation in all occluded tubes or as a result of increased pressure. periprocedural complications reported anecdotally during hysteroscopy including venous intravasation, possible anaphylactic or hypertonic reaction for irrigation solution, pulmonary edema from fluid overload, and air embolism, are similar to those seen with hsg. recent uterine and endometrial interventions, repetitive curettage due to placental remnants, and missed or medical abortion might be related to intravasation. the prevalence of asherman 's syndrome, related to secondary amenorrhea following abortion and curettage, was reported to be 1.5 - 43%. endometrial synechie / notch associated filling defects and asymmetrical disturbance of pressure are facilitating factors for venous intravasation.[332. in accordance with the literature, we hypothesize an association between recent uterine intervention and intravasation as a result of increased permeability. hysteroscopy and related interventions carry a risk for intravasation and fluid overload due to increased permeability, opened vessels, and distention / irrigation ; all of which require increased pressure. this method uses a glycine solution to irrigate and distend the endometrial cavity which carries a dilutional hyponatremia risk as a result of the fluid intravasation. additionally, a study reported that the endometrial laser ablation influenced fluid or gas intravasation. administration of a warm isotonic solution with a pressure below 70 mmhg was shown to minimize intravasation. furthermore, the possibility of intravasation and the hazards of cooling of laser heads has been recognized. with increasing experience, proponents of the hsg procedure appear to be achieving its potential as a less invasive and safer alternative to hysterectomy. venous intravasation, a well - described complication during hsg, is a prototype of hysteroscopic interventions whereby contrast and fluids transit from the endometrial cavity through the myometrial, pelvic, and paracaval veins. first, the present study was a hospital - based, cross - sectional study with a limited number of cases. second, we used leech wilkinson cannulation (not a balloon catheter) and compared them because the study was retrospective. some limitations of our study have to be considered. first, the present study was a hospital - based, cross - sectional study with a limited number of cases. second, we used leech wilkinson cannulation (not a balloon catheter) and compared them because the study was retrospective. in conclusion, we found that intravasation might be related to certain variables, including preprocedural or procedural predisposing factors, which include menometrorrhagia, secondary infertility, abortus, endometriosis, mullerian anomalies, recent uterine interventions, and painful procedure. scheduling of hsg during the middle follicular period, eliminating of predisposing factors, and using of hydrosoluble contrast media was shown to minimize or prevent intravasation. radiologists and gynecologists should be familiar with the technique, interpretation, and intravasation for safer hsg or related procedures. clarification of the mechanism of intravasation might refine current hsg techniques and facilitate future studies focusing on the prevention and management of intravasation. | objectives : presently, hysterosalpingography (hsg) is used as a means to evaluate women with infertility and repetitive pregnancy loss. venous intravasation is a complication and potential pitfall during hsg and analogous procedures including hysteroscopy. the aim of our study was to assess the venous intravasation and to obtain critical information for more secure and more accurate procedures. in particular, the primary goal of the present study was to compare hsg without and with intravasation to identify differences seen on hsg and to assess the predisposing factors of intravasation. the secondary goal was to describe clinical- and imaging - based novel classification of intravasation.materials and methods : this study included a patient cohort of 569 patients who underwent hsg between 2008 and 2011 at our center in the absence (control group) or presence (study group) of intravasation. intravasation classified from level 0 (no intravasation) to level 3 (severe intravasation) was compared with preprocedural (demographic and clinical) and procedural (hsg) data. data were analyzed using statistical package for social sciences (spss) statistical software.results:of the 569 patients undergoing hsg, 528 showed no intravasation and 41 (7.2%) patients showed intravasation when associated with preprocedural (leukocytes, menometrorrhagia, secondary infertility, ectopic pregnancy, abortus, polycystic ovaries, endometriosis, and interventions) and procedural (pain, scheduling, endometrial - uterine nature, and spillage) parameters. moreover, intravasation was lower in women with smooth endometrium, triangular uterus, and homogeneous peritoneal spillage. no association was found between age, tubal patency, increased pressure, and intravasation.conclusions:using a novel classification method, intravasation can be observed in women during hsg and associates with preprocedural and procedural predisposing factors in subsumed conditions. this classification method will be useful for improving the efficiency and accuracy of hsg and related procedures by minimization of severe complications caused by intravasation. |
since its clinical introduction in the 1980s, the high - field magnetic resonance (mr) scanners has been progressively developed to improve the signal - to noise ratio (snr) and contrast susceptibility for the better human brain images (1, 2). the current use of higher - field mr scanners for brain tumors is limited to 3.0 t because of technical and safety issues (3). there have been several reports of contrast - enhanced mr imaging (mri) using high - field magnets such as 7.0 t for the visualization of brain tumors in the rodent brain tumor animal model and some cases of human brain tumors (4, 5, 6). to our knowledge, however there was no report about the contrast - enhanced 7.0 t mri taken both before and after surgery in a patient with a glioblastoma. hereby we reported a case of small cell glioblastoma in a 45-yr - old female patient with contrast - enhanced 7.0 t brain mri taken before surgery and at the time of recurrence in comparison of 1.5 t and 3.0 t brain mri. a 45-yr - old female patient was admitted with one - month history of headache and progressive left hemiparesis (grade iv / v) on january 8th, 2010. t2-weighted imaging (t2-wi) and pre- and post - contrast t1-weighted imaging (t1-wi) obtained at 1.5 t (fig. 1a - a ', d - d ') and 3 t (fig. 1b - b ', f - f ') demonstrated a mass lesion in her right frontal lobe. she had taken 7.0 t mri (magnetom 7.0 t, siemens) one day before initial surgery (fig. 1c - c ', e - e ', g - g '). she underwent right fronto - parietal craniotomy and the tumor was subtotally resected (fig. the tumor was composed of less pleomorphic small astrocytic cells but had high mitotic rate (6/10hpf), vascular endothelial hyperplasia and necrosis. on fluorescence in situ hybridization study, neither 1p nor 19q deletion was noted but epidermal growth factor receptor (egfr) gene amplification was robust (fig she underwent concomitant conventional radiation therapy with temozolomide (75 mg / m / day) for 6 weeks. but she had refused to receive booster temozolomide against medical advice. a follow - up 1.5 t brain mri, taken at 8 months after initial surgery demonstrated tumor recurrence in the right frontal lobe adjacent to the anterior horn of the right lateral ventricle, remote from the original site (fig. 3a, b, c). 7.0 t mri (magnetom 7.0 t, siemens, erlagen, germany) was performed again one day before second surgery (fig. her recurrent brain tumor was histologically confirmed the same as the previous one, a small cell glioblastoma with egfr gene amplification. after surgery she was treated with six cycles of avastin (10 mg / kg / day) and irrinotecan (120 mg / m / day) every 2 weeks but succumbed to death due to tumor progression at 15 months after her initial surgery. we received the permission from the korean food and drug administration and the institutional review board of seoul national university hospital and the neuroscience research institute of gacheon medical center (irb number : 0802 - 046 - 234). a written consent was obtained from the patient. the 7.0 t mri (magnetom 7.0 t, siemens, erlagen, germany) at neuroscience research institute of gacheon university of medicine and science was used for imaging of the patient. the 7.0 t magnet, with a clear bore of 90 cm, is equipped with a water cooled gradient and rf coils. the gradient system operates at 2,000 v/650 amp with gradient amplitude of 40 mt / m, a maximum slew rate of 200 mt / m / ms, and a minimum gradient rise time of 200 microseconds. high resolution t2-weighted images were acquired as the following scanning parameters : tr=1,180 ms, te=17.1 ms, thickness=1.5 mm, gap=1.5 mm, flip angle=30, number of slices=20, voxel size=0.250.251.5 l, and matrix size=7041,024. t1-weighted mri was scanned before and after injection of a contrast agent. berlin, germany) was used as the contrast agent by 0.2 ml / kg (0.01 mm / kg). pulse sequence used was 3d mprage (magnetization prepared rapid gradient echo) and the followings are the scanning parameters : tr=4,000 msec, te=4.39 msec, ti=1,100 msec, thickness=0.8 mm, flip angle=10, number of slice=240, voxel size=0.40.40.8 mm, and matrix size=384384. the 3.0 t and 1.5 t preoperative mri were performed using a quadrature transmit - receive head coil. the 3.0 t mri protocol included the following : axial unenhanced and enhanced t1-weighted (tr=600 msec ; te=8.0 msec ; flip angle=90) 3-dimensional spoiled gradient acquisitions with a section thickness of 5.0 mm, field of view of 24 cm, and matrix size of 384192 and t2-weighted acquisitions (tr=4,500 msec ; te=104.4 msec) in the axial planes with a section thickness of 5 mm, field of view of 24 cm, and matrix size of 448256. the 1.5 t mri protocol included the following : axial unenhanced and enhanced t1-weighted (tr=400 msec ; te=8.0 msec ; flip angle=65) 3-dimensional spoiled gradient acquisitions with a section thickness of 5 mm, field of view of 24 cm, and matrix size of 256192 and t2-weighted acquisitions (tr=4,000 msec ; te=115.2 msec) in the axial planes with a section thickness of 5 mm, field of view of 24 cm, and matrix size of 256256. 7.0 t axial images were co - registered to 1.5 t axial images using a software, ondemand3d (cybermed, seoul, korea). co - registration was automatically performed by an algorithm based on the mutual information method. locations of ac and pc, and tumor margins were compared in both image sets five times with the program of ondemand3d (cybermed, seoul, korea). enhanced anatomical details of the brain tumor were provided by 7.0 t mri. the t2-weighted image (t2-wi) of 7.0 t brain mri (fig. 1c) provided a sharper delineation of the central necrosis and hemorrhage in the peripheral parts of the tumor bed with detailed anatomical information compared with the t2-wis of 1.5 t (fig. irregular areas of heterogeneous signal intensity in the tumor area were best displayed on t2-wi of 7.0 t brain mri, presumably representing hemorrhage or tumor microvasculature (fig. 1a ', b ', and c ' which represent the four - times magnified vision of marked areas in fig. 1a, b, and c, respectively, shows fine intratumoral structures such as cyst walls or small vessels in t2-wi of 7.0 t mri (fig. 1c ') compared with those of the 1.5 t (fig. 1a ') and 3.0 t (fig. 1b ') brain mris. in the t1-wi of 7.0 t mri (fig. 1e), we could clearly see heterogeneous component of the brain tumor clearly delineated from surrounding compressed brain cortex whereas only low signal intensity regions are identified in the t1wi of 1.5 t brain mri (fig. 1d ', and e ' which represent the four - times magnified vision of marked areas in fig. 1d and e, respectively, reveals better contrast between solid and cystic components, and more detailed intratumoral stuctures in t1-wi of 7 t brain mri (fig. 1d '). the contrast- enhanced t1-wi of 7.0 t brain mri (fig. 1 g) showed a clearly defined rim - enhancing mass lesion with sharp margin from the surrounding compressed brain cortex whereas only low signal intensity regions are identified in the contrast - enhanced t1-wi of 3 t brain mri (fig. 1f ' and g ' which represent the four - times magnified vision of marked areas in fig. 1f ' and g ', shows the better contrast between enhancing and non - enhancing portion in the contrast - enhanced t1wi of 7.0 t brain mri (fig. 1 g ') than that of 3.0 t brain mri (fig. 1f '). contrast - enhanced 7.0 t brain mri safely performed without any distortion caused by craniofix2 (aesculap inc. 3). in comparison with 1.5 t brain mri (fig. 3a, b, c), enhanced anatomical details of the brain tumor were provided by 7.0 t mri (fig. 3d, e, f) as the same as the preoperative 7.0 t mri. we found that there was no significant discrepancy of the location of the ac and pc as well as the margin of the tumor in the right frontal lobe in the co - registered images of the 1.5 t mri (fig. 7.0 t brain mri with contrast enhancement was safely performed before surgery (fig. 1c, e, g). enhanced anatomical details of the brain tumor were provided by 7.0 t mri. the t2-weighted image (t2-wi) of 7.0 t brain mri (fig. 1c) provided a sharper delineation of the central necrosis and hemorrhage in the peripheral parts of the tumor bed with detailed anatomical information compared with the t2-wis of 1.5 t (fig. irregular areas of heterogeneous signal intensity in the tumor area were best displayed on t2-wi of 7.0 t brain mri, presumably representing hemorrhage or tumor microvasculature (fig. 1a ', b ', and c ' which represent the four - times magnified vision of marked areas in fig. 1a, b, and c, respectively, shows fine intratumoral structures such as cyst walls or small vessels in t2-wi of 7.0 t mri (fig. 1b ') brain mris. in the t1-wi of 7.0 t mri (fig. 1e), we could clearly see heterogeneous component of the brain tumor clearly delineated from surrounding compressed brain cortex whereas only low signal intensity regions are identified in the t1wi of 1.5 t brain mri (fig. 1d ', and e ' which represent the four - times magnified vision of marked areas in fig. 1d and e, respectively, reveals better contrast between solid and cystic components, and more detailed intratumoral stuctures in t1-wi of 7 t brain mri (fig. 1d '). the contrast- enhanced t1-wi of 7.0 t brain mri (fig. 1 g) showed a clearly defined rim - enhancing mass lesion with sharp margin from the surrounding compressed brain cortex whereas only low signal intensity regions are identified in the contrast - enhanced t1-wi of 3 t brain mri (fig. 1f ' and g ' which represent the four - times magnified vision of marked areas in fig. 1f and g, respectively. 1f ' and g ', shows the better contrast between enhancing and non - enhancing portion in the contrast - enhanced t1wi of 7.0 t brain mri (fig. 1 g ') than that of 3.0 t brain mri (fig. 1f '). contrast - enhanced 7.0 t brain mri safely performed without any distortion caused by craniofix2 (aesculap inc. 3). in comparison with 1.5 t brain mri (fig. 3a, b, c), enhanced anatomical details of the brain tumor were provided by 7.0 t mri (fig. 3d, e, f) as the same as the preoperative 7.0 t mri. we found that there was no significant discrepancy of the location of the ac and pc as well as the margin of the tumor in the right frontal lobe in the co - registered images of the 1.5 t mri (fig. we have demonstrated in this case that contrast - enhanced 7.0 t mris were safely taken before surgery and at the time of recurrence in a patient with a small cell glioblastoma. in the past, there were several anecdotic reports of high field mri of 7.0 t or higher performed in a small group of normal subjects or patients (2, 4, 5, 6, 7, 8). thomas. (2) described the in vivo 7.0 t mri of higher signal - to - noise and novel contrast to provide enhanced scrutiny of hippocampal anatomy with their micro - venous structures in six normal subjects. (7) compared the 7.0 t mri with conventional 1.5 t mri in twelve consecutive patients with clinically definite multiple sclerosis. they have reported that ultra - high - field imaging of patients with multiple sclerosis at 7.0 t mri was well tolerated and provided better visualization of multiple sclerosis lesions in the gray matter. there have been several reports of contrast - enhanced mri using high - field magnets such as 7.0 t for the visualization of brain tumors in the rodent brain tumor animal model and human brain tumors (2, 4, 5, 6, 7, 8). (4) evaluated the growth and vascularity of implanted gl261 mouse gliomas by using 7.0 t mri with conventional t1- and t2-wi and dynamic, contrast - enhanced t2-wi in 34 c57bl6 mice at different stages of tumor development. lupo. (5) assessed the feasibility of g generalized autocalibrating partially parallel acquisition (grappa)-based susceptibility - weighted imaging (swi) technique at 7.0 t in healthy volunteers and 11 brain tumor patients. they suggested that unique forms of contrast in 7.0 t swi may be useful for assessing response to both radiation and antiangiogenic therapies for patients with brain tumors (5). (6) reported 7.0 t mr findings of astrocytic brain tumors (who grades ii - iv) in comparison with 1.5 t mri. they described that 7.0 t mri offers more detailed depiction of tumor microvascularity and necrosis within intracranial gliomas because of higher achievable spatial resolution and increased sensitivity for susceptibility contrast compared with 1.5 t mri (6). (8) reported pre- and post - contrast 7.0 t mr findings of twenty - three patients with brain tumors (who grades i - iv) in comparison with 1.5 t mri. they described that the t2-weighted images from 7.0 t brain mri revealed detailed microvasculature and the internal contents of supratentorial brain tumors better than that of 1.5 t brain mri. for brain tumors located in parasellar areas or areas adjacent to major cerebral vessels, flow - related artifacts were exaggerated in the 7.0 t brain mris. for brain tumors adjacent to the skull base, susceptibility artifacts in the interfacing areas of the paranasal sinus and skull base hampered the aquisition of detailed images and information on brain tumors in the 7.0 t brain mris in the study (8). however, there was no report about the 7.0 t mri with contrast enhancement taken both before and after surgery in a patient with a glioblastoma until now. our study demonstrated that the contrast - enhanced t1-wi of 7.0 t mri, using 3d mprage sequence can show detailed brain imaging of a small cell glioblastoma as takeda. geometric distortion due to static field and local susceptibility effects has been a major concern in high - field mri (10). despite these technical issues, we found that the co - registered 7.0 t mri have only a little discrepancy in the positions of the physiological landmarks such as ac and pc as well as the location and shape of the brain tumor from 1.5 t mri. in conclusion, we report that 7t.0 mri can be safely utilized before and after surgery for the treatment of a patient with a small cell glioblastoma. we think this case report is an important landmark for the clinical application of ultra - high field mri in the field of neuro - oncology in near future. | a 45-yr - old female patient was admitted with one - month history of headache and progressive left hemiparesis. brain magnetic resonance imaging (mri) demonstrated a mass lesion in her right frontal lobe. her brain tumor was confirmed as a small cell glioblastoma. her follow - up brain mri, taken at 8 months after her initial surgery demonstrated tumor recurrence in the right frontal lobe. contrast - enhanced 7.0 t brain magnetic resonance imaging (mri) was safely performed before surgery and at the time of recurrence. compared with 1.5 t and 3.0 t brain mri, 7.0 t mri showed sharpened images of the brain tumor contexture with detailed anatomical information. the fused images of 7.0 t and 1.5 t brain mri taken at the time of recurrence demonstrated no significant discrepancy in the positions of the anterior and the posterior commissures. it is suggested that 7.0 t mri can be safely utilized for better images of the maligant gliomas before and after surgery.graphical abstract |
the treatment regimen includes multi - agent chemotherapy (three phases : induction therapy, intensification therapy, maintenance therapy) and central nervous system directed radiotherapy. immune suppression caused due to disease, and its therapy makes these children more prone to infections with severe stomatologic complications. the most common complications are mucositis, xerostomia, bleeding, dysgeusia and infections leading to pain, discomfort and interfere with the course and prognosis of the neoplasm. the study was planned to study the oral manifestations in pediatric population with acute leukemias during the induction phase only. to study the oral manifestation of acute leukemia during induction chemotherapy.to analyze the level of oral hygiene and find its correlation with induction outcome (short come outcomes in the form of increased febrile neutropenic episodes, induction deaths, number of admissions). to study the oral manifestation of acute leukemia during induction chemotherapy. to analyze the level of oral hygiene and find its correlation with induction outcome (short come outcomes in the form of increased febrile neutropenic episodes, induction deaths, number of admissions). to study the oral manifestation of acute leukemia during induction chemotherapy.to analyze the level of oral hygiene and find its correlation with induction outcome (short come outcomes in the form of increased febrile neutropenic episodes, induction deaths, number of admissions). to study the oral manifestation of acute leukemia during induction chemotherapy. to analyze the level of oral hygiene and find its correlation with induction outcome (short come outcomes in the form of increased febrile neutropenic episodes, induction deaths, number of admissions). this was a cross - sectional noninvasive study. thirty three patients both sexes age range (5 - 15 years) in pediatric oncology unit with diagnosis of acute lymphoblastic leukemia and acute myeloblastic leukemia were the subjects for the study. single clinician (dental surgeon) performed oral examination of children at the 1 week of induction and 4 week of induction of chemotherapy. patients who have completed first induction phase or those who were on maintenance therapy were excluded from the study. the parents / responsible guardians were informed and explained about the aim and character of the study. patients were evaluated for, oral hygiene by using oral hygiene index - simplified (ohi - s) [table 1a, b ], dental caries by using def index and decayed missing filled teeth (dmft) index, [table 2 ] and gingival findings by using the patients were also scrutinized for any other findings or lesions in the oral cavity, i.e., lesion including site and size of the lesion, candidiasis, any other infections as herpes simplex virus, varicella zoster virus, cytomegalovirus, acute necrotizing and ulcerative gingivitis. gingival index of loe and silness debris index : debris is defined as soft, foreign matter consisting of bacterial plaque and food debris. the criteria include : the same examination was again carried out in the 4 week of the induction phase. all the scores and observations were recorded in a diagnostic chart along with the brief patient 's history and complete blood count on the day of observation. these reports were collected and co - related with short term outcomes such as febrile / neutropenic episodes / morbidity / mortalities with the help of routine blood culture reports. the changes in the oral health status were being observed during first week and fourth week and were analyzed with wilkoxon 's signed rank test. the indices scores were converted in to percentage scale. for this pilot study, a cut - off criteria has been set as 50% for oral hygiene, gingival and def / dmft index. debris index : debris is defined as soft, foreign matter consisting of bacterial plaque and food debris. the criteria include : the same examination was again carried out in the 4 week of the induction phase. all the scores and observations were recorded in a diagnostic chart along with the brief patient 's history and complete blood count on the day of observation. these reports were collected and co - related with short term outcomes such as febrile / neutropenic episodes / morbidity / mortalities with the help of routine blood culture reports. the changes in the oral health status were being observed during first week and fourth week and were analyzed with wilkoxon 's signed rank test. the indices scores were converted in to percentage scale. for this pilot study, a cut - off criteria has been set as 50% for oral hygiene, gingival and def / dmft index. a total of 23 male and 10 female patients with acute leukemia were studied prospectively. we found that the dental status namely the gingival and periodontal tissues were affected in this period due to lack of good oral hygiene and overall poor health status following chemotherapy [table 1 ]. 15 out of 30 (50%) subjects showed increase in ohi - s, loe and silness gingival index. a significant change was observed in these indices (p = 0.002 ; 0.003, respectively). there were no extractions or dental filling done ; the decay factor was mainly noticed in this period. dental finding pre- and post - induction phase table 5 shows hematological finding pre- and post - induction phase. no significant difference was noted in hemoglobin, total leucocytic count, or absolute neutrophil count (p = 0.4 ; 0.11 ; 0.53 respectively), but highly significant difference was seen in platelet count p < 0.05. hematological finding pre- and post - induction phase in this study, no interruptions in induction chemotherapy was reported due to dental infections. three patients expired due to debilitating general health, but not due to induction therapy. palatal aphthous ulcer was observed in a child and it was 1.5 cm diameter in size. in another subject angular cheilitis was noted. all the remaining subjects presented no clinical signs of oral lesion. as a regular protocol the suspected foci of infection was cultured and it was found to be candida in angular cheilitis. in the second case of aphthous ulcer neutropenia was noticed as a cause. leukemia is a disease resulting from the malignant transformation of stem cells whose proliferation starts in the bone marrow. oral complaints precipitated by the side - effects of the antineoplastic agents are primarily those of discomfort, sensitivity of teeth and pain, ulceration, gingival hemorrhage, dryness and impaired taste sensation. maintenance of oral hygiene is the basic preventive aid from all kinds of oral infections. patients are advised against any vigorous mechanical dental cleansing aid (tooth brush / floss / interdental cleaning aids) to avoid triggering of any bleeding episodes. an alkaline saline rinse of warm water flavored with salt and sodium bicarbonate is recommended for oral cleansing. gingiva reacts differently to even slight amount of plaque if subject is on anti - neoplastic therapy for blood dyscrasias. in the present study, it was observed that the ohi - s count was increased in 50% of individuals. this change in ohi - s definitely affects other oral hard and soft - tissues. inflammation in the gingiva is most common and probably earliest presentation of changes in oral tissues due to plaque. the increased scores of gi in 50% of individuals clearly highlight the problems associated oral hygiene maintenance in these patients. the gingival inflammation, if not treated leads to periodontal problems and may eventually lead to loss of teeth in adult patients. dmft / def index was found to be increased in 10 patient 's, i.e., 33% of study population. since no active dental treatment is advocated (advisable) to any patient undergoing induction therapy, the difference in dmft was only in d, i.e., decay component of the dmft. although the induction therapy drugs are not directly responsible for the decay process, the change in oral environment due to excess plaque accumulation and/or change in quality and quantity of saliva due to drugs might have played important role in increased in caries process. in most of the patient 's erythematous oral mucosa, burning sensation of mouth was common, since mucositis was not primary objective of this study, the findings were not noted. one subject demonstrated an aphthous ulcer on palatal mucosa but it responded well to the symptomatic therapy. in another subject, angular cheilitis was seen. the angular cheilitis is generally a clinical sign of an inflammatory response to fungal infection. there was no statistical significant difference was observed in terms of short term outcomes of leukemia therapy due to dental problem. in this study, it was observed that the oral health status worsened following first induction chemotherapy in acute leukemic pediatric patients. there was a significant increase in level of ohi, gingival index and def / dmft. lesions like aphthous ulcer and angular cheilitis were noticed in two of the subjects while other subjects did not have any oral complications. these finding suggest that there is a need to institute optimum oral health, during and in between induction phases. further study is study with larger sample size is needed to conclude the role of oral hygiene in the induction outcome of pediatric patients receiving treatment for acute leukemia. | background : treatment of acute leukemia's- a common childhood malignancy, involves intensive and powerful multi - drug chemotherapeutic regime. oral lesions are a common complication in these patients affecting oral health status.aim:this study was conducted to evaluate and assess the oral health status of newly diagnosed leukemic pediatric patients during induction phase and its correlation to outcome of induction therapy.material methods : oral examinations was done in 33 children between the age group of 5 - 15 years with acute lymphoblastic leukemia (all) and acute myloblastic leukemia (aml), who were undergoing chemotherapy. oral hygiene index- simplified, (ohi - s) decayed missing filled teeth index (def / dmft), loe and sillness index for gingiva, and complete blood count at first and fourth week of induction phase were recorded for each patient. the changes in the oral health status were analyzed with wilcoxon signed rank test.results:during an induction phase it was observed that level of ohi - s (p = 0.002), loe and sillness index (p = 0.003), def / dmft index (p = 0.076), platelet count (p = 0.00) increased significantly and no significant difference was noted in hemoglobin (p = 0.4) and total leucocytes count (p = 0.11).conclusion : it was observed that, although oral health status had significantly worsened, the induction outcome was not affected. |
comprehensive women s healthcare includes basic reproductive health services, such as hormonal contraception, and specialized services, such as infertility evaluation / treatment and prenatal and obstetric care.1,2 as the number of women veterans enrolling in the veterans health administration (va) has increased, so has the demand for basic and specialized reproductive health services.36 while all va sites are required to provide basic women s health services on - site, specialized services may be delivered through referral to another va site or through non - va purchased (fee basis) or contract care from community (non - va) providers. specific specialized services, such as prenatal care, are almost exclusively provided through these mechanisms.2,79 on - site availability of reproductive services may be influenced by the practice context, including the site environment and organizational features such as practice structure, size, and resources.2,8,10 previous reports indicate that while the majority of sites serving a large number of women veterans are hospital - based and located in large urban areas,11 approximately 14 % of va sites serving at least 400 women are located in non - metropolitan areas.12 non - metropolitan areas have a documented shortage of reproductive service providers,13 and women veterans may face significant barriers to obtaining reproductive health services in these communities. va community - based outpatient clinics (cbocs) provide basic primary care for veterans.14 compared with hospital based clinics, cbocs frequently serve fewer women veterans and are often located in non - metropolitan areas.14,15 site location and type may jointly influence on - site availability of reproductive health services for women veterans,16 and lack of on - site availability of reproductive health care services may delay care for women veterans. currently, little is known regarding the on - site availability of reproductive health services for women veterans, particularly those served by cbocs or sites in non - metropolitan areas. therefore, the objectives of this study were to : 1) describe the overall on - site availability of hormonal contraceptives, intrauterine device (iud) placement, infertility evaluation / treatment, and prenatal care, by site location and type ; 2) describe the characteristics of sites providing these reproductive health services ; and 3) to examine, within this context, whether site location and type is associated with on - site availability of specialized reproductive services. we used cross - sectional survey data from the 2007 veterans health administration survey of women veterans health programs and practices (wvhp) merged with the area resource file and va administrative records. the protocol received institutional review board (irb) approval from va greater los angeles and an exemption from irb review by va puget sound health care system. the wvhp survey queried informants at the health care system (chief of staff, n = 123), and practice (senior clinician, n = 195) levels at sites serving at least 300 unique women veterans. chiefs of staff identified senior women s health clinicians most responsible for or knowledgeable regarding the women s health care delivery. response rates for chiefs of staff and senior clinicians were 93 % and 86 % respectively. this analysis included data from 193 sites and utilized the senior clinician portion of the survey, with the exception of the variable regarding a separate budget for the women s health program at a site, which used the chief of staff module when the senior clinician response was missing. primary outcomes were on - site availability of 1) hormonal contraception, 2) iud placement, 3) infertility evaluation / treatment, and 4) prenatal care. senior clinicians were asked to specify whether a given service was available at this va site, only available at another va site, available through contract or fee - basis on - site, available through contract or fee - basis off - site, not available, or available through some other arrangement. a service was considered to be available on - site if a response was either the primary exposure combined site location and type (metropolitan hospital - based clinic, non - metropolitan hospital - based clinic, metropolitan cboc). location was defined as metropolitan or non - metropolitan using data from the area resource file.17 non - metropolitan cbocs (n = 2) were excluded from the analysis. independent variables included elements of practice context and organization.8,10 variables related to practice context were the geographic region (northeast, midwest, south, and west) in which each va site was located, as designated by the us census bureau, and va regional networks known as veterans integrated service networks (visn) (n = 21). variables related to organization of the practice included : practice size, structure for providing care to women veterans, and resource availability. practice size was defined as the number of unique women veterans with at least one visit to the site in the year preceding the survey (2006) (< median, median). the structure for providing care to women veterans was characterized by the presence of specialized models for gender - specific care. specialized models for gender - specific care were not mutually exclusive and included presence of a separate women s clinic for primary care (yes, no) or gynecology clinic (yes, no). resource availability included informant ratings of the sufficiency of resources and personnel ; availability of formal training in women s health ; availability of a separate budget for women s health ; and change in resource availability over the preceding two years. informants rated the sufficiency of resources for the following items : overall clinical expertise in women s health, availability of same gender - providers, nursing staff, administrative and support staff, clinic space, examination rooms properly equipped for pelvic examinations and pap smears, female attendants to chaperone gender - sensitive examinations, and budget or funding for the women s health program. responses were dichotomized as always or usually sufficient and sometimes, rarely, or never sufficient. informants reported if formal training in women s health was available at their site (yes, no). combining the practice and chief of staff level responses, we determined whether there was a separate budget for the women s health program at each site. finally, informants reported whether resources for women veterans care had increased, decreased, or remained unchanged in the past 2 years. we compared characteristics of sites with and without onsite availability of hormonal contraception, iud placement, infertility evaluation / treatment, or prenatal care. continuous variables were compared via students t - test ; categorical variables were compared via the test or if cell sizes were < 5 using the fischer exact test. random effects models were used to examine association of site type and location with on - site availability of iud placement and infertility evaluation / treatment.18 two levels of independent variables were considered : visn was considered a level two variable since multiple sites are nested within a single visn, while all other variables were considered level one variables. adjustments for variables regarding structure for providing care to women veterans and resource availability were not made, as these characteristics were potentially consequences of site type and location.19 due to the extremely small number of facilities offering prenatal care (n = 11), we did not include availability of prenatal care as a separate outcome. we used cross - sectional survey data from the 2007 veterans health administration survey of women veterans health programs and practices (wvhp) merged with the area resource file and va administrative records. the protocol received institutional review board (irb) approval from va greater los angeles and an exemption from irb review by va puget sound health care system. the wvhp survey queried informants at the health care system (chief of staff, n = 123), and practice (senior clinician, n = 195) levels at sites serving at least 300 unique women veterans. chiefs of staff identified senior women s health clinicians most responsible for or knowledgeable regarding the women s health care delivery. response rates for chiefs of staff and senior clinicians were 93 % and 86 % respectively. this analysis included data from 193 sites and utilized the senior clinician portion of the survey, with the exception of the variable regarding a separate budget for the women s health program at a site, which used the chief of staff module when the senior clinician response was missing. primary outcomes were on - site availability of 1) hormonal contraception, 2) iud placement, 3) infertility evaluation / treatment, and 4) prenatal care. senior clinicians were asked to specify whether a given service was available at this va site, only available at another va site, available through contract or fee - basis on - site, available through contract or fee - basis off - site, not available, or available through some other arrangement. a service was considered to be available on - site if a response was either the primary exposure combined site location and type (metropolitan hospital - based clinic, non - metropolitan hospital - based clinic, metropolitan cboc). location was defined as metropolitan or non - metropolitan using data from the area resource file.17 non - metropolitan cbocs (n = 2) were excluded from the analysis. independent variables included elements of practice context and organization.8,10 variables related to practice context were the geographic region (northeast, midwest, south, and west) in which each va site was located, as designated by the us census bureau, and va regional networks known as veterans integrated service networks (visn) (n = 21). variables related to organization of the practice included : practice size, structure for providing care to women veterans, and resource availability. practice size was defined as the number of unique women veterans with at least one visit to the site in the year preceding the survey (2006) (< median, median). the structure for providing care to women veterans was characterized by the presence of specialized models for gender - specific care. specialized models for gender - specific care were not mutually exclusive and included presence of a separate women s clinic for primary care (yes, no) or gynecology clinic (yes, no). resource availability included informant ratings of the sufficiency of resources and personnel ; availability of formal training in women s health ; availability of a separate budget for women s health ; and change in resource availability over the preceding two years. informants rated the sufficiency of resources for the following items : overall clinical expertise in women s health, availability of same gender - providers, nursing staff, administrative and support staff, clinic space, examination rooms properly equipped for pelvic examinations and pap smears, female attendants to chaperone gender - sensitive examinations, and budget or funding for the women s health program. responses were dichotomized as always or usually sufficient and sometimes, rarely, or never sufficient. informants reported if formal training in women s health was available at their site (yes, no). combining the practice and chief of staff level responses, we determined whether there was a separate budget for the women s health program at each site. finally, informants reported whether resources for women veterans care had increased, decreased, or remained unchanged in the past 2 years. we compared characteristics of sites with and without onsite availability of hormonal contraception, iud placement, infertility evaluation / treatment, or prenatal care. continuous variables were compared via students t - test ; categorical variables were compared via the test or if cell sizes were < 5 using the fischer exact test. random effects models were used to examine association of site type and location with on - site availability of iud placement and infertility evaluation / treatment.18 two levels of independent variables were considered : visn was considered a level two variable since multiple sites are nested within a single visn, while all other variables were considered level one variables. adjustments for variables regarding structure for providing care to women veterans and resource availability were not made, as these characteristics were potentially consequences of site type and location.19 due to the extremely small number of facilities offering prenatal care (n = 11), we did not include availability of prenatal care as a separate outcome. table 1 shows the characteristics of the surveyed sites, including practice context and organization. of the 193 sites included in the analysis, 106 (55 %) were metropolitan hospital - based clinics, 23 were non - metropolitan hospital - based clinics (12 %), and 64 were metropolitan cbocs (33 %). the median practice size at geographically distinct sites was 1,209 (interquartile range : 6052,212). compared with metropolitan hospital - based clinics, non - metropolitan hospital - based clinics and metropolitan cbocs served fewer women veterans, were less likely to have a separate primary care women s health or gynecology clinic, and were less likely to have a separate budget for the women s health program.table 1characteristics of va sites serving 300 unique women veterans in 2006 by site location and type (n = 193)totalmetropolitan hospitalnon - metropolitan hospitalmetropolitan cbocn1931062364practice contextregion n (%) northeast30 (16)23 (22)2 (9)5 (8)midwest44 (23)25 (24)8 (35)11 (17)south73 (38)38 (36)7 (30)28 (43)west46 (24)20 (19)6 (26)20 (31)organization of the practice 1,209 women veterans in 200697 (51)77 (73)7 (30)13 (21)women s health clinic for primary care n (%) 130 (67)84 (79)16 (70)30 (47)gynecology clinic n (%) 85 (44)59 (56)8 (35)18 (28)women s health resources : always or usually sufficient n (%) clinical expertise160 (84)88 (85)20 (87)52 (83)same gender providers154 (82)90 (86)17 (77)47 (76)nursing staff142 (74)78 (74)17 (74)47 (75)administrative and support staff108 (57)60 (57)10 (44)38 (60)clinic space125 (66)68 (65)18 (78)39 (63)properly equipped examination rooms183 (96)101 (97)22 (96)60 (95)female attendants163 (86)91 (88)22 (96)50 (79)budget or funding73 (46)42 (47)8 (42)23 (46)formal women s health training n (%) 16 (8)12 (12)04 (6)separate budget or control point n (%) 32 (17)25 (24)3 (13)4 (7)change in women s health resources over the last 2 years n (%) increased62 (33)10 (39)7 (31)15 (24)unchanged102 (54)47 (45)12 (52)43 (68)decreased26 (13)17 (16)4 (17)5 (8)missing data : 1,209 women veterans in 2006, one missing ; clinical expertise, three missing ; same gender providers, four missing ; nursing staff, two missing ; administrative and support staff, two missing ; clinic space, four missing ; properly equipped examination rooms, three missing ; female attendants, three missing ; budget or funding, 35 missing ; formal women s health training, two missing ; separate budget or control point, five missing ; change in resources over the last 2 years, three missing characteristics of va sites serving 300 unique women veterans in 2006 by site location and type (n = 193) missing data : 1,209 women veterans in 2006, one missing ; clinical expertise, three missing ; same gender providers, four missing ; nursing staff, two missing ; administrative and support staff, two missing ; clinic space, four missing ; properly equipped examination rooms, three missing ; female attendants, three missing ; budget or funding, 35 missing ; formal women s health training, two missing ; separate budget or control point, five missing ; change in resources over the last 2 years, three missing table 2 indicates the on - site availability of the four reproductive health services. overall, 94 % of sites offered on - site hormonal contraception, 50 % offered on - site iud placement, 30 % offered on - site infertility evaluation / treatment, and 6 % offered on - site prenatal care. of the 11 sites not offering hormonal contraception on - site, five provided this service at another va site and six offered it through fee - basis or contract providers (data not shown). compared with metropolitan hospitals, a smaller percentage of non - metropolitan hospital - based clinics and metropolitan cbocs offered on - site iud placement. metropolitan cbocs were the least likely to offer infertility evaluation / treatment, but non - metropolitan hospital - based clinics were the most likely to offer on - site infertility evaluation only.table 2on - site availability of individual reproductive health care services and combinations of services by site location and typetotalmetropolitan hospitalnon - metropolitan hospitalmetropolitan cbocn1931062364individual services n (%) hormonal contraception182 (94)102 (96)22 (96)58 (91)iud placement97 (50)70 (66)9 (39)18 (28)infertility evaluation or treatment57 (30)39 (37)8 (35)10 (16)infertility evaluation only29 (15)19 (18)5 (22)5 (8)infertility treatment only6 (3)6 (6)00infertility evaluation or treatment22 (11)14 (13)3 (13)5 (8)prenatal care11 (6)7 (7)04 (6)number of services out of four n (%) none10 (5)3 (3)1 (4)6 (9)one74 (38)29 (27)11 (48)34 (53)two56 (29)35 (33)5 (22)16 (25)three51 (26)37 (35)6 (26)8 (13)four2 (1)2 (2)00combinations of servicesone service (n = 74)hormonal contraception73 (99)28 (97)11 (100)34 (100)iud placement1 (1)1 (3)00infertility evaluation or treatment0000prenatal care0000two services (n = 56)hormonal contraception and iud placement47 (84)31 (89)3 (60)13 (81)hormonal contraception and prenatal care1 (2)1 (3)00hormonal contraception and infertility evaluation or treatment8 (14)3 (8)2 (40)3 (19)iud placement and prenatal care0000iud placement and infertility evaluation or treatment0000prenatal care and infertility evaluation or treatment0000three services (n = 51)hormonal contraception, iud placement, and infertility evaluation or treatment43 (84)29 (88)5 (100)4 (50)hormonal contraception, iud placement, and prenatal care4 (8)3 (8)01 (12)hormonal contraception, prenatal care, and infertility evaluation or treatment4 (8)1 (3)03 (38)iud placement, infertility evaluation or treatment, and prenatal care0000 on - site availability of individual reproductive health care services and combinations of services by site location and type compared with sites not offering hormonal contraception, those offering this service were more likely to have a separate women s health clinic for primary care, and to report sufficient clinical expertise, availability of same gender providers, and properly equipped exam rooms (table 3). compared with sites not offering on - site iud placement, those offering this service were more likely to serve more women veterans, have a separate women s health clinic for primary care or gynecology clinic, report sufficient women s health clinical expertise, and have a separate women s health program budget (table 3). compared with sites not offering on - site infertility evaluation / treatment, those offering these services served more women veterans, were more likely to include a separate gynecology clinic, report sufficient women s health clinical expertise, and to have increased funding for women s health in the prior 2 years (table 4). compared with sites not offering prenatal care, those offering this service were more likely to have a formal women s health training program (table 4).table 3characteristics of sites by on - site availability of hormonal contraception and iud placement, (n = 193)hormonal contraceptioniud placementavailability : noyespnoyespn111829697practice contextregion n (%) northeast1 (9)29 (16)0.3116 (17)14 (14)0.65midwest1 (9)43 (24)25 (26)19 (20)south4 (36)69 (38)34 (35)39 (40)west5 (46)41 (23)21 (22)25 (26)organization of the practice 1,209 women veterans in 20065 (46)92 (51)0.7329 (30)68 (71) < 0.001women s health clinic for primary care n (%) 2 (18)128 (70)0.00152 (54)78 (80) < 0.001gynecology clinic n (%) 2 (18)83 (46)0.1219 (20)66 (68) < 0.001resources : always or usually sufficient n (%) clinical expertise6 (55)154 (86)0.0177 (81)83 (87)0.02same gender providers6 (55)145 (83)0.0276 (80)78 (83)0.60nursing staff6 (55)136 (76)0.1268 (72)74 (77)0.38administrative and support staff5 (46)103 (57)0.4556 (59)52 (54)0.51clinic space7 (64)118 (66)1.0066 (70)59 (62)0.24properly equipped examination rooms7 (78)176 (97)0.0489 (95)94 (97)0.72female attendants7 (70)156 (87)0.1677 (83)86 (87)0.25budget or funding3 (38)70 (47)0.7336 (49)37 (44)0.56formal women s health training n (%) 1 (9)15 (8)1.004 (4)12 (13)0.07separate budget or control point n (%) 1 (9)31 (18)0.6910 (11)22 (23)0.03change in resources over the last 2 years n (%) increased1 (9)61 (34)0.2026 (27)36 (38)0.35decreased2 (18)21 (13)14 (15)12 (13)unchanged8 (73)94 (53)54 (58)48 (50)table 4characteristics of sites by on - site availability of infertility evaluation / treatment and prenatal care, (n = 193)infertility evaluation / treatmentprenatal careavailability : noyespnoyespn1365718211practice contextregion n (%) northeast22 (16)8 (14)0.9326 (14)4 (36)n / amidwest32 (24)12 (21)44 (24)0south51 (38)22 (39)73 (40)0west31 (23)15 (26)39 (21)7 (64)organization of the practice1,209 women veterans in 200658 (43)39 (68)0.00188 (49)9 (82)0.06women s health clinic for primary care n (%) 86 (63)44 (77)0.06123 (68)7 (64)0.75gynecology clinic n (%) 51 (38)34 (60)0.0180 (44)5 (46)0.92resources : always or usually sufficient n (%) clinical expertise106 (80)54 (95)0.01151 (84)9 (82)0.69same gender providers104 (78)50 (89)0.08146 (82)8 (73)0.43nursing staff99 (74)43 (75)0.82137 (76)5 (46)0.03administrative and support staff83 (62)25 (44)0.02101 (56)7 (64)0.76clinic space86 (65)39 (68)0.66117 (66)8 (73)0.75properly equipped examination rooms130 (98)53 (93)0.20173 (97)10 (91)0.35female attendants116 (87)47 (83)0.39158 (88)5 (46)0.001budget or funding49 (45)24 (48)0.7669 (47)4 (36)0.55formal women s health training n (%) 12 (9)4 (7)0.7813 (7)3 (30)0.04separate budget or control point n (%) 18 (14)14 (25)0.0732 (18)0n / achange in resources over the last 2 years n (%) 38 (28)24 (44)0.01increased15 (11)11 (20)59 (33)3 (30)n / adecreased82 (61)20 (36)26 (14)0unchanged38 (28)24 (44)0.0195 (52)7 (70)n / a not applicable due to cell size of zero characteristics of sites by on - site availability of hormonal contraception and iud placement, (n = 193) characteristics of sites by on - site availability of infertility evaluation / treatment and prenatal care, (n = 193) n / a not applicable due to cell size of zero after adjusting for visn and practice size, compared with metropolitan hospital - based clinics, non - metropolitan hospital - based clinics had lower odds of offering on - site iud placement (or 0.57 ; 95 % ci 0.20, 1.62), but higher odds of offering on - site infertility evaluation / treatment (or 1.42 ; 95 % ci 0.48, 4.22), although these associations did not reach statistical significance. after adjustment, compared with metropolitan hospital - based clinics, metropolitan cbocs had 67 % lower odds of offering on - site iud placement (or 0.33 ; 95 % ci 0.14, 0.74). similar, although not statistically significant, associations were observed comparing metropolitan cbocs with metropolitan hospital - based clinics with respect to on - site infertility evaluation / treatment (or 0.43 ; 95 % ci 0.17, 1.11). table 1 shows the characteristics of the surveyed sites, including practice context and organization. of the 193 sites included in the analysis, 106 (55 %) were metropolitan hospital - based clinics, 23 were non - metropolitan hospital - based clinics (12 %), and 64 were metropolitan cbocs (33 %). the median practice size at geographically distinct sites was 1,209 (interquartile range : 6052,212). compared with metropolitan hospital - based clinics, non - metropolitan hospital - based clinics and metropolitan cbocs served fewer women veterans, were less likely to have a separate primary care women s health or gynecology clinic, and were less likely to have a separate budget for the women s health program.table 1characteristics of va sites serving 300 unique women veterans in 2006 by site location and type (n = 193)totalmetropolitan hospitalnon - metropolitan hospitalmetropolitan cbocn1931062364practice contextregion n (%) northeast30 (16)23 (22)2 (9)5 (8)midwest44 (23)25 (24)8 (35)11 (17)south73 (38)38 (36)7 (30)28 (43)west46 (24)20 (19)6 (26)20 (31)organization of the practice 1,209 women veterans in 200697 (51)77 (73)7 (30)13 (21)women s health clinic for primary care n (%) 130 (67)84 (79)16 (70)30 (47)gynecology clinic n (%) 85 (44)59 (56)8 (35)18 (28)women s health resources : always or usually sufficient n (%) clinical expertise160 (84)88 (85)20 (87)52 (83)same gender providers154 (82)90 (86)17 (77)47 (76)nursing staff142 (74)78 (74)17 (74)47 (75)administrative and support staff108 (57)60 (57)10 (44)38 (60)clinic space125 (66)68 (65)18 (78)39 (63)properly equipped examination rooms183 (96)101 (97)22 (96)60 (95)female attendants163 (86)91 (88)22 (96)50 (79)budget or funding73 (46)42 (47)8 (42)23 (46)formal women s health training n (%) 16 (8)12 (12)04 (6)separate budget or control point n (%) 32 (17)25 (24)3 (13)4 (7)change in women s health resources over the last 2 years n (%) increased62 (33)10 (39)7 (31)15 (24)unchanged102 (54)47 (45)12 (52)43 (68)decreased26 (13)17 (16)4 (17)5 (8)missing data : 1,209 women veterans in 2006, one missing ; clinical expertise, three missing ; same gender providers, four missing ; nursing staff, two missing ; administrative and support staff, two missing ; clinic space, four missing ; properly equipped examination rooms, three missing ; female attendants, three missing ; budget or funding, 35 missing ; formal women s health training, two missing ; separate budget or control point, five missing ; change in resources over the last 2 years, three missing characteristics of va sites serving 300 unique women veterans in 2006 by site location and type (n = 193) missing data : 1,209 women veterans in 2006, one missing ; clinical expertise, three missing ; same gender providers, four missing ; nursing staff, two missing ; administrative and support staff, two missing ; clinic space, four missing ; properly equipped examination rooms, three missing ; female attendants, three missing ; budget or funding, 35 missing ; formal women s health training, two missing ; separate budget or control point, five missing ; change in resources over the last 2 years, three missing table 2 indicates the on - site availability of the four reproductive health services. overall, 94 % of sites offered on - site hormonal contraception, 50 % offered on - site iud placement, 30 % offered on - site infertility evaluation / treatment, and 6 % offered on - site prenatal care. of the 11 sites not offering hormonal contraception on - site, five provided this service at another va site and six offered it through fee - basis or contract providers (data not shown). compared with metropolitan hospitals, a smaller percentage of non - metropolitan hospital - based clinics and metropolitan cbocs offered on - site iud placement. metropolitan cbocs were the least likely to offer infertility evaluation / treatment, but non - metropolitan hospital - based clinics were the most likely to offer on - site infertility evaluation only.table 2on - site availability of individual reproductive health care services and combinations of services by site location and typetotalmetropolitan hospitalnon - metropolitan hospitalmetropolitan cbocn1931062364individual services n (%) hormonal contraception182 (94)102 (96)22 (96)58 (91)iud placement97 (50)70 (66)9 (39)18 (28)infertility evaluation or treatment57 (30)39 (37)8 (35)10 (16)infertility evaluation only29 (15)19 (18)5 (22)5 (8)infertility treatment only6 (3)6 (6)00infertility evaluation or treatment22 (11)14 (13)3 (13)5 (8)prenatal care11 (6)7 (7)04 (6)number of services out of four n (%) none10 (5)3 (3)1 (4)6 (9)one74 (38)29 (27)11 (48)34 (53)two56 (29)35 (33)5 (22)16 (25)three51 (26)37 (35)6 (26)8 (13)four2 (1)2 (2)00combinations of servicesone service (n = 74)hormonal contraception73 (99)28 (97)11 (100)34 (100)iud placement1 (1)1 (3)00infertility evaluation or treatment0000prenatal care0000two services (n = 56)hormonal contraception and iud placement47 (84)31 (89)3 (60)13 (81)hormonal contraception and prenatal care1 (2)1 (3)00hormonal contraception and infertility evaluation or treatment8 (14)3 (8)2 (40)3 (19)iud placement and prenatal care0000iud placement and infertility evaluation or treatment0000prenatal care and infertility evaluation or treatment0000three services (n = 51)hormonal contraception, iud placement, and infertility evaluation or treatment43 (84)29 (88)5 (100)4 (50)hormonal contraception, iud placement, and prenatal care4 (8)3 (8)01 (12)hormonal contraception, prenatal care, and infertility evaluation or treatment4 (8)1 (3)03 (38)iud placement, infertility evaluation or treatment, and prenatal care0000 on - site availability of individual reproductive health care services and combinations of services by site location and type compared with sites not offering hormonal contraception, those offering this service were more likely to have a separate women s health clinic for primary care, and to report sufficient clinical expertise, availability of same gender providers, and properly equipped exam rooms (table 3). compared with sites not offering on - site iud placement, those offering this service were more likely to serve more women veterans, have a separate women s health clinic for primary care or gynecology clinic, report sufficient women s health clinical expertise, and have a separate women s health program budget (table 3). compared with sites not offering on - site infertility evaluation / treatment, those offering these services served more women veterans, were more likely to include a separate gynecology clinic, report sufficient women s health clinical expertise, and to have increased funding for women s health in the prior 2 years (table 4). compared with sites not offering prenatal care, those offering this service were more likely to have a formal women s health training program (table 4).table 3characteristics of sites by on - site availability of hormonal contraception and iud placement, (n = 193)hormonal contraceptioniud placementavailability : noyespnoyespn111829697practice contextregion n (%) northeast1 (9)29 (16)0.3116 (17)14 (14)0.65midwest1 (9)43 (24)25 (26)19 (20)south4 (36)69 (38)34 (35)39 (40)west5 (46)41 (23)21 (22)25 (26)organization of the practice 1,209 women veterans in 20065 (46)92 (51)0.7329 (30)68 (71) < 0.001women s health clinic for primary care n (%) 2 (18)128 (70)0.00152 (54)78 (80) < 0.001gynecology clinic n (%) 2 (18)83 (46)0.1219 (20)66 (68) < 0.001resources : always or usually sufficient n (%) clinical expertise6 (55)154 (86)0.0177 (81)83 (87)0.02same gender providers6 (55)145 (83)0.0276 (80)78 (83)0.60nursing staff6 (55)136 (76)0.1268 (72)74 (77)0.38administrative and support staff5 (46)103 (57)0.4556 (59)52 (54)0.51clinic space7 (64)118 (66)1.0066 (70)59 (62)0.24properly equipped examination rooms7 (78)176 (97)0.0489 (95)94 (97)0.72female attendants7 (70)156 (87)0.1677 (83)86 (87)0.25budget or funding3 (38)70 (47)0.7336 (49)37 (44)0.56formal women s health training n (%) 1 (9)15 (8)1.004 (4)12 (13)0.07separate budget or control point n (%) 1 (9)31 (18)0.6910 (11)22 (23)0.03change in resources over the last 2 years n (%) increased1 (9)61 (34)0.2026 (27)36 (38)0.35decreased2 (18)21 (13)14 (15)12 (13)unchanged8 (73)94 (53)54 (58)48 (50)table 4characteristics of sites by on - site availability of infertility evaluation / treatment and prenatal care, (n = 193)infertility evaluation / treatmentprenatal careavailability : noyespnoyespn1365718211practice contextregion n (%) northeast22 (16)8 (14)0.9326 (14)4 (36)n / amidwest32 (24)12 (21)44 (24)0south51 (38)22 (39)73 (40)0west31 (23)15 (26)39 (21)7 (64)organization of the practice1,209 women veterans in 200658 (43)39 (68)0.00188 (49)9 (82)0.06women s health clinic for primary care n (%) 86 (63)44 (77)0.06123 (68)7 (64)0.75gynecology clinic n (%) 51 (38)34 (60)0.0180 (44)5 (46)0.92resources : always or usually sufficient n (%) clinical expertise106 (80)54 (95)0.01151 (84)9 (82)0.69same gender providers104 (78)50 (89)0.08146 (82)8 (73)0.43nursing staff99 (74)43 (75)0.82137 (76)5 (46)0.03administrative and support staff83 (62)25 (44)0.02101 (56)7 (64)0.76clinic space86 (65)39 (68)0.66117 (66)8 (73)0.75properly equipped examination rooms130 (98)53 (93)0.20173 (97)10 (91)0.35female attendants116 (87)47 (83)0.39158 (88)5 (46)0.001budget or funding49 (45)24 (48)0.7669 (47)4 (36)0.55formal women s health training n (%) 12 (9)4 (7)0.7813 (7)3 (30)0.04separate budget or control point n (%) 18 (14)14 (25)0.0732 (18)0n / achange in resources over the last 2 years n (%) 38 (28)24 (44)0.01increased15 (11)11 (20)59 (33)3 (30)n / adecreased82 (61)20 (36)26 (14)0unchanged38 (28)24 (44)0.0195 (52)7 (70)n / a not applicable due to cell size of zero characteristics of sites by on - site availability of hormonal contraception and iud placement, (n = 193) characteristics of sites by on - site availability of infertility evaluation / treatment and prenatal care, (n = 193) n / a not applicable due to cell size of zero after adjusting for visn and practice size, compared with metropolitan hospital - based clinics, non - metropolitan hospital - based clinics had lower odds of offering on - site iud placement (or 0.57 ; 95 % ci 0.20, 1.62), but higher odds of offering on - site infertility evaluation / treatment (or 1.42 ; 95 % ci 0.48, 4.22), although these associations did not reach statistical significance. after adjustment, compared with metropolitan hospital - based clinics, metropolitan cbocs had 67 % lower odds of offering on - site iud placement (or 0.33 ; 95 % ci 0.14, 0.74). similar, although not statistically significant, associations were observed comparing metropolitan cbocs with metropolitan hospital - based clinics with respect to on - site infertility evaluation / treatment (or 0.43 ; 95 % ci 0.17, 1.11). while almost all surveyed va sites offered hormonal contraception on - site, the on - site availability of specialized reproductive health services, including iud placement, infertility evaluation / treatment, and prenatal care, varied considerably. however, 11 of the surveyed sites, including metropolitan and non - metropolitan hospitals and metropolitan cbocs, did not offer hormonal contraception on - site, which is a basic reproductive health service. lack of on - site provision of effective contraception may delay care and lead to increased risk of unplanned pregnancy.20 compared with metropolitan hospital - based clinics, metropolitan cbocs were less likely to offer specialized reproductive health services. in seeking to provide comprehensive reproductive health care for women veterans, va must consider the cost of providing these services on - site and the optimal means of ensuring the highest quality of care. although the number of women veterans in va is increasing, they remain a numerical minority in va, and, particularly at cbocs, it may not be feasible to offer all specialized reproductive health services on - site. metropolitan cbocs were least likely to have organizational features that facilitated on - site provision of specialized reproductive care, such as separate women s health clinics for primary care or women s gynecology clinics.12 women receiving care at cbocs may have to travel long distances to obtain reproductive health services if they are referred to another va site.14 while use of non - va (fee basis) or contract care may minimize travel distances, the efficiency and quality of these services is not well understood. introduction of innovative modalities, such as telegynecology or telematernity care, may improve access to certain reproductive health services. incorporating specialized models for gender - specific care within metropolitan cbocs may increase availability and access to specialized reproductive health services for women veterans. given the low volume of women patients, it may never be feasible to offer certain specialized reproductive health services (i.e. prenatal care) on - site. compared with metropolitan hospital - based clinics, non - metropolitan hospital - based clinics may be more likely to offer on - site infertility evaluation / treatment. non - metropolitan hospital - based clinics were almost as likely as their metropolitan counterparts to contain a separate women s clinic for primary care, but much less likely to have a gynecology clinic. infertility treatments are typically delivered by obstetrician gynecologist specialists ; however, infertility evaluation may have been facilitated at non - metropolitan hospitals by the presence of providers at women s clinics. alternatively, large metropolitan hospital - based clinics may opt to purchase such services in the community, while non - metropolitan hospital - based clinics may order laboratory tests for infertility evaluation on - site and only refer patients out once a need for infertility treatment is established. strengths of this study included the high response rate and large number of healthcare organizations. first, although rural setting was not an exclusion criterion, the survey was intentionally designed to query facilities with 300 or more women veterans, which likely differentially excluded rural sites. this is particularly important, given earlier findings indicating that facilities with smaller women veteran caseloads receive lower ratings for gender - related satisfaction and appropriateness compared with larger sites that incorporated tailored women s primary care models.11 second, it is possible that respondents may have been more likely to report availability of certain services due to social desirability bias or ambiguity of definitions of multi - faceted services, such as infertility evaluation or treatment. third, we were unable to determine distances between sites, which may impact on - site availability of specialized reproductive health services. metropolitan cbocs are less likely than their similarly located hospital counterparts to offer specialized reproductive services on - site. future studies should evaluate delivery of specialized reproductive health care services for women veterans in cbocs and clinics in non - metropolitan areas. | abstractintroductionwith the increasing number of women veterans enrolling in the veterans health administration (va), there is growing demand for reproductive health services. little is known regarding the on - site availability of reproductive health services at va and how this varies by site location and type.objectiveto describe the on - site availability of hormonal contraception, intrauterine device (iud) placement, infertility evaluation or treatment, and prenatal care by site location and type ; the characteristics of sites providing these services ; and to determine whether, within this context, site location and type is associated with on - site availability of these reproductive health services.methodswe used data from the 2007 veterans health administration survey of women veterans health programs and practices, a national census of va sites serving 300 or more women veterans assessing practice structure and provision of care for women. hierarchical models were used to test whether site location and type (metropolitan hospital - based clinic, non - metropolitan hospital - based clinic, metropolitan community - based outpatient clinic [cboc ]) were associated with availability of iud placement and infertility evaluation / treatment. non - metropolitan cbocs were excluded from this analysis (n = 2).resultsof 193 sites, 182 (94 %) offered on - site hormonal contraception, 97 (50 %) offered on - site iud placement, 57 (30 %) offered on - site infertility evaluation / treatment, and 11 (6 %) offered on - site prenatal care. after adjustment, compared with metropolitan hospital based - clinics, metropolitan cbocs were less likely to offer on - site iud placement (or 0.33 ; 95 % ci 0.14, 0.74).conclusioncompared with metropolitan hospital - based clinics, metropolitan cbocs offer fewer specialized reproductive health services on - site. additional research is needed regarding delivery of specialized reproductive health care services for women veterans in cbocs and clinics in non - metropolitan areas. |
although our review was protocol - driven, it allowed for evolution as we became more familiar with the literature. we undertook a systematic literature search for relevant empirical and theoretical sources, using electronic databases (pubmed, cinahl, scopus, psycinfo, sociological abstracts, dissertation abstracts) and google scholar, and also hand - searching relevant journals. we identified additional reports through our own collections and reference lists, using the context in which a reference was cited to assess its probable relevance. any item we thought might discuss social identity in health care was retrieved ; those discussing concepts other than social identity were sampled up to the point of theoretical saturation. two reviewers independently screened each abstract and subsequently assessed each report for inclusion ; disagreements were resolved by a tie - breaking vote for abstracts and by consensus for full reports. (further methodological details and a full list of included and excluded reports are available from the authors upon request.) to determine how the five sia dimensions were addressed, we undertook an inductive, iterative analysis of the literature, whereby we derived what we have termed conceptual currents. we did not rely on the authors espoused theories as a basis for categorization, as we discovered commonalities across theoretical labels and diversity within them ; moreover, many articles were atheoretical. instead, drawing on the approach of metanarrative synthesis (greenhalgh. we began with simple content analysis, coding sources for the presence of each of the five sia dimensions ; however, since merely documenting a concept 's presence provided no information about how it was addressed, we used this as a springboard for thematic analysis. this process involved constant comparison of the data against one another and an emerging list of thematic categories. one reviewer (sk) used the preliminary codes, along with qualitative notes recorded for each report, to sort articles according to the combination and interpretation of sia dimensions that they reflected ; the analysis was then opened to the other team members. we reviewed 348 reports representing 335 unique sources (166 qualitative, 77 quantitative, and 17 mixed - methods studies ; 71 essays, commentaries, or theoretical papers ; and 4 reviews). the literature revealed a strong focus on interprofessional and, secondarily, intraprofessional issues (n = 151 and 84, respectively), with some examination of management - staff relations (30), organizational change (29), and other organizational issues (34). as we confined our search to english - language reports, it is not surprising that most of the sources came from english - speaking countries such as the united states (90), united kingdom (88), australia (29), and canada (24). sit was referred to in 114 reports, representing 105 unique studies / essays (about a fifth of which addressed it only briefly or used it implicitly by discussing organizational identification). these did not constitute a unified literature ; pockets of articles cited one another, while many articles cited no others within the group. not all presented social identity as a group - related issue, instead treating it as an individual - difference variable or an input to interpersonal relationships (e.g., fuller. some authors defined key sit terms incorrectly (hallier and forbes 2005) or evinced awareness of only the earliest or simplest formulations of the theory, not its breadth and depth. for example, the efforts by mitchell and colleagues (2010) to integrate sit with an individualistic information - processing approach suggested a lack of familiarity with the extensive social identity literature on social cognition and social influence (e.g., abrams. furthermore, some of the papers that applied sit appropriately did not focus on health care, using it merely as a setting in which to test general hypotheses about intergroup or organizational behavior (bartels. 2010 ; oaker and brown 1986). thus, despite the apparently high number of sit citations, there was no evidence of a social identity literature on health care groups. content analysis suggested that sources featured an average of 2.5 of the 5 sia dimensions. whether or not a source cited sit was not a robust indicator of whether it addressed these dimensions in a manner consistent with the sia ; notably, we found several highly sia - congruent analyses among non - citers. for this reason, the theory - based synthesis does not privilege sit - citing sources, but discusses all sources together. in the next section, we group the conceptual currents (in italics) under the sia dimension that they most strongly reflect. to conserve space, we use citations for illustrative purposes, not exhaustively. the most basic insight of a group - based approach is that analyses of conflict or cooperation in health care must consider group, not merely interpersonal, dynamics. this insight was powerfully demonstrated in an expos of virulent intergroup conflict among hospital departments (hewett. drawing on sit, the study revealed that patient charts supposedly repositories of objective information were rife with examples of intergroup competition and in - group enhancement. the study also showed how interspecialty competition to own (or disown) patients could threaten safe care and may have led to a patient 's death. the authors argued that interpersonal - skills training would be an inadequate remedy for physicians dysfunctional communication, since the problem resulted not from a lack of skill but from the active expression of group identities. such recognition of the importance of groups is echoed in papers that variously invoke sit (shute 1997), role theory (booth and hewison 2002), evolutionary psychology (braithwaite, iedema, and jorm 2007), and other concepts (ferlie. 2005). however, this body of literature is primarily descriptive. to identify potential solutions, therefore, the health care landscape comprises groups of unequal power and status. without an understanding of this issue, naive calls for teamwork may actually reinforce professional divisions and hierarchies. for example, finn (2008) documented how various providers appealed to the concept of teamwork to advance their profession - specific norms and interests : to nurses, teamwork meant more equal and respectful relations ; to surgeons, that others efficiently followed their orders. many authors stress the gendered nature of the doctor - nurse dynamic, tracing its origins to the subordination of women within the sexual division of labor (campbell - heider and pollock 1987). others put power first and gender second, emphasizing how the specific unequal relationship between doctors and nurses produces certain patterns of interaction. for example, tellis - nayak and tellis - nayak (1984) elucidated how structural inequalities are reinforced by social rituals (concerning the differential use of space, time, language, and body language), arguing that any attempt to reduce the power imbalance must address both structural and symbolic factors. both gender- and power - related role expectations may constrain health care professionals to play the doctor - nurse game, in which the nurse must offer any suggestion indirectly so that it seems to be the doctor 's idea (stein 1967). many structural analyses also touched on identity content by noting how the values, discourses, sources of knowledge, and types of labor associated with high - status groups (men / physicians) are privileged over those of low - status groups (women / nurses) (wicks 1998). roberts (1983) contended that intergroup inequality also taints nurses intragroup relations, giving rise to oppressed group behavior in which nurses accept the physician - dominated structure as legitimate and inevitable. she explained horizontal violence within nursing in terms of nurses efforts to repudiate a devalued group identity and align themselves instead with the oppressing group. while offering a provocative examination of health care hierarchies, this literature has attracted three major criticisms : (1) its perspective tends to be static and deterministic, missing the context - dependent, strategic, and, above all, variable nature of doctor - nurse interactions, and sometimes denying nurses agency ; (2) its accounts are frequently partisan, reflecting the unquestioned assumption that increased power for a certain group is an absolute good ; and (3) its overwhelming focus on doctor - nurse relations obscures other power relations in health care. for example, a uk study (johns 1992) explored how the division of nurses into primary and associate roles created new power dynamics, including what might be described as a nurse - nurse game (i.e., associate nurses confining themselves to indirect suggestions to maintain a facade of harmony). from an sia perspective, all three limitations spring from the fact that unequal partners analyses reify observed phenomena as immutable social facts, not recognizing them as examples of generalized group processes. according to sit (tajfel and turner 1979), the behavior of unequal - status groups can be understood in terms of two basic responses : individual mobility (attempts to leave one group and enter the other) and social change (attempts to reduce, reverse, or increase the status difference between the groups). social change strategies include social creativity (creation of ideologies that affirm the in - group 's worth) and social competition (competition with the out - group for power or resources). sit does not treat these strategies as unique to oppressed groups or as dependent on a particular group psychology, but rather as a function of the in - group 's structural position relative to the out - group, the features of the structural relationship, and the strength of the member 's group identification. the sia 's generality and objectivity allow it to encompass both entrenched intergroup inequalities and scenarios in which power relations are more complex, unexpected, and context - dependent. (for a sit - informed account of doctor - nurse relations, see chattopadhyay, finn, and ashkanasy 2010.) the professional strategies tradition, grounded in the sociology of professions (freidson 1970), explores occupational groups struggles for territory and control. although medical dominance is a frequent focus, abbott (1988) underscored that the important phenomenon is not the empirical fact of a certain group 's dominance (which can change) but the ubiquity of intergroup competition which occurs both interprofessionally and among intraprofessional subgroups (currie, finn, and martin 2009). unlike the unequal partners literature, this stream tends to regard all groups as self - interested and their ideologies as self - serving. it also offers a less static view, illustrating how the intergroup landscape may change when a strategy succeeds or backfires. in a study of health care reorganization, daykin and clarke (2000) illuminated how nurses project of professionalization was impeded by contradictions between the strategies used to gain territory from medicine and to protect their own. to affirm their status vis - - vis medicine, nurses claimed a distinct body of knowledge grounded in caring, yet undermined this claim by devaluing the caring labor of health care assistants. the authors argued that, far from enhancing nurses professional status, such exclusionary tactics reduced their ability to resist the most pertinent threat : management 's fordist practices of routinization and de - skilling. the professional strategies literature often highlights the role of context ; for example, oncologists who worked in different settings used different means to promote their dominance over practitioners of complementary and alternative medicine (cam) (broom and tovey 2007). hospital physicians used the discourse of science to discredit cam, proclaiming the superiority of medicine 's scientific mind - set even in areas where no medical evidence existed. hospice physicians could not discredit cam, given its fit with their institution 's holistic philosophy ; instead, they found subtle ways to subsume cam within a biomedical paradigm, tightly controlling cam practice to avoid any challenge to (bio)medical dominance. with its rich description of both the material and rhetorical strategies that groups use to secure and enhance their professional status, the professional strategies literature depicts social competition and social creativity in action. echoing sit, it shows how members strategically compare their own group with others, choosing dimensions (may and fleming 1997), definitions (norris 2001), and comparison groups (fournier 2002) that maximize the in - group 's positive distinctiveness. it differs from sit, however, in focusing on groups instrumental motivation to gain power and autonomy, seldom recognizing the psychological motivation to maintain a positive social identity. the assumption that groups care only about tangible, not symbolic, benefits can lead to inaccurate predictions (e.g., that health care groups would soon abandon professional ideologies, competing instead over who could meet market needs most cost - effectively) (light 1988). skepticism about groups self - representations can be taken too far, to the point of dismissing all affirmations of group identity as strategic performances. the sia recognizes that such performances are not just strategic ; they often reflect highly valued identity content. group identities are not arbitrary but are defined by certain content : norms, values, and worldviews that are meaningful and important to members. a large literature frames this topic in terms of culture and cultural differences, delineating how professional groups differ in everything from values and attitudes (degeling, kennedy, and hill 2001) to myths and rituals (dombeck 1997). but bald descriptions of identity content do not elucidate group processes, and inspire few solutions apart from vague calls for cultural sensitivity. accounts of professional culture may also miss intraprofessional diversity ; even explorations of professional subcultures (leininger 1994) often stop short of asking what the different variants of identity mean. more sia - consistent studies may explain variability in terms of subgroups with different experiences or structural positions contesting (advancing their own versions of) identity content (e.g., pratt and rafaeli 1997). in contrast to the cultural differences discourse, which portrays identity content as static, the sia takes a dynamic view, examining how different representations of group identity are constructed and mobilized in order to achieve group goals. research on professional socialization considers, among other things, how educational and practice environments encourage or suppress the enactment of a patient - centered identity. patient - centeredness can be an integral part of physician identity, yet its expression may be actively discouraged through both the formal and informal curricula of medical school (apker and eggly 2004). in contrast, nurses are strongly socialized into patient - centeredness until they enter the working world, where demands for efficiency may imperil this core value, resulting in demoralization and disengagement (limoges 2007). odonohue and nelson (2007) stressed that professionals psychological contract with an organization depends not merely on individual rewards but on the organization 's respect for the values central to their collective identity. health care professionals are known to resist threats to their identity content (fiol and oconnor 2006) ; however, interventions perceived as identity threatening in one context may be perceived as benign, even identity affirming, in another. physicians may reject management - imposed system redesign as a threat to the doctor - patient relationship, yet actively support the same redesign when it is seen as contributing to the achievement of a patient - centered medical home (kreindler 2008). when the threat involves alternative practitioners encroachment on medical territory, medicine is a science (broom and tovey 2007)yet, when it involves managerial pressure to comply with clinical practice guidelines, medicine is an art (mcdonald, waring, and harrison 2006). at first glance, the finding that such varied, even contradictory, discourses are used to resist change seems to imply that change is impossible. the flexible (re)construction of identity, however, may itself be a key to change. in one study, gps who became change leaders constructed a hybrid identity that affirmed the primacy of their physician identity while incorporating management skills (redefined as a trivial subset of skills that most physicians possess) (hotho 2008). they found this new identity more attractive than that of management (still seen as a low - status out - group with objectionable values)or of rank - and - file gps (seen as routine oriented and averse to change). organizational change efforts have been observed to founder when administrators either ignore social identity (seeking to shape employees behavior through individual rewards and sanctions alone) or assume that staff can be rallied behind an imposed organizational identity (charles - jones, latimer, and may 2003 ; mcdonald 2004). success is more likely when leaders guide change as it grows from the real values of existing groups (brooks 1996 ; obrien. the fullest elaboration of this strategy occurs within the sia literature, with the aspire (actualizing social and personal identity resources) model of organizational development ; the aspire process engages employees in building a mosaic identity that recognizes both common goals and the distinct contributions of valued subgroups (haslam, eggins, and reynolds 2003). (bevan 2008), organic development of a network community (bate 2000), and other concepts. one hospital used the norms of physician culture (concrete, expert - led, decision - oriented discussions) to encourage physician engagement in restructuring (ohare and kudrle 2007). other studies of positive physician - manager relationships have emphasized the development of a shared identity, facilitated by shared decision making grounded in common values (in particular, service and excellence ; see graham and steele 2001 ; kirkpatrick., they also can be mobilized to cope with change, or to achieve it. as the social identity literature has revealed, mobilization is not simply a matter of invoking a ready - made image of group identity, but involves crafting a sense of us (haslam, reicher, and platow, 2011) that supports a desired change. goodrick and reay (2010) showed how nursing textbooks, using the idea of continuity with the past to foster the reconstruction of nursing identity, highlighted only those aspects of the past consistent with the new vision. attempts to promote identification with a certain group, to restructure groups, or to mobilize identities must reckon with members existing patterns of group identification. one stream of literature is concerned primarily with health care employees qua employees, examining organizational identification, its predictors (e.g., good communication up and down the hierarchy, value congruence between organization and employee, the organization 's prestige, and the employee 's sense of being respected), and its consequences (e.g., cooperation, organizational citizenship) (dukerich, golden, and shortell 2002). nearly all these studies cite sit ; problematically, though, many are characterized by a management - centric assumption that organizations should, or even can, manipulate employees social identities (e.g., han and harms 2010). yet even within this literature, it becomes clear that employee commitment is not so easily manipulable. for example, while participative decision making is among the strongest predictors of organizational identification, opportunities to participate are unlikely to foster organizational identification unless employees see them as genuine and relevant (joensson 2008 ; tangirala and ramanujam 2008). a second stream of literature recognizes that most health care providers are strongly identified with their profession. strong professional and organizational identification are not incompatible ; indeed, the two are often positively correlated (bartels. however, most professionals identify more strongly with their profession than with their organization, which typically offers a less distinctive identity and a shorter period of socialization (callan. 2007). moreover, the combination of high professional and low organizational identification is linked to behavior that, from the organization 's perspective, is undesirable. physicians exhibiting this combination of attitudes have been found to resist influence from management, repay perceived organizational support with reduced compliance, and retaliate against perceived violation of their psychological contract with the organization (hekman. tradition is that most of the studies, concentrating as they do on drawing general conclusions about organizational psychology, are not firmly grounded in the health care context. for instance, some studies have drawn inferences about high - status staff from analyses that excluded physicians or combined them with senior nurses (callan. 2007 ; obrien. several qualitative studies have amplified the point that group membership does not equate with group identification. multidisciplinary team. however, direct care nurses and health care assistants, who continued to be excluded from decision making, did not share in this team identity ; rather, they felt alienated and unwilling to cooperate with the team 's directives. physicians are unlikely to identify with an organization whose management they perceive as a devalued out - group in particular, one that threatens their autonomy in the service of values abhorrent to their profession (fiol, pratt, and oconnor 2009 ; hekman. moreover, hoff (1999) found that creating physician - managers failed to bridge the divide between these two groups ; on the contrary, the physician - manager population itself became divided. those who saw themselves primarily as managers embraced their new identity, complete with management jargon, and even defended organizational policies that negatively affected physicians. those who continued to see themselves primarily as physicians asserted this identity by resisting and sabotaging the activities of the first group, whom they viewed as traitors. these findings invite the application of such sia concepts as categorization threat (the threat of one 's being viewed as a member of a devalued group ; see ellemers, spears, and doosje 2002) and the black sheep effect (in which an in - group member who flouts group norms attracts more censure than does an out - group member ; see marques, yzerbyt, and leyens 1988). as we have seen, physician - managers can develop hybrid identities (hotho 2008), but this is not inevitable. doolin (2002) found that physicians revised their identities to incorporate a new management role only when they perceived the latter as congruent with their physician identity. context refers to the external conditions such as organizational structures, working practices, and physical features of the work environment that support a particular system of group relations. the sia holds that changing the context can change the way people view and relate to one another, ultimately altering existing patterns of group interaction. a handful of articles described successful organizational - development initiatives that put intergroup issues front and center (van de vliert 1995). staff have been invited to participate in collective reflection on group dynamics, and the insights derived from this process used to develop or implement new structures and working practices (context changes) that support more positive group relations (bate 2000 ; bleakley. although the literature abounds with warnings that the mere establishment of a team does not guarantee true teamwork, some studies with a longer follow - up have reached more optimistic conclusions. a british study (hudson 2002) found that effective teamwork between health and social care providers had begun to develop in practices where providers were co - located and informal working patterns were shifting. a longitudinal study of an interdisciplinary health team traced members gradual progression from defensiveness and stereotyping to a shared team culture in which roles were less differentiated (farrell, schmitt, and heinemann 2001). such findings suggest that what are seen as intractable problems with multidisciplinary teams may actually just be growing pains. there is some evidence, however, that role revision and teamwork are more likely to become realities when social identity is taken into account. a study of an unsuccessful attempt to introduce an enhanced nursing role suggested that the change process failed to engage with the entrenched social identity dynamics associated with a rigid professional hierarchy (currie, finn, and martin 2010). despite its superficial adoption, the new role was assimilated into the existing social structure and produced little real change. furthermore, a single organizational intervention may not suffice ; context change may entail addressing broader environmental factors that impede collaboration. for example, such contextual factors as organizational integration, resource availability, and various facilitators of long - term working relationships strongly influenced whether the introduction of teams reproduced or transformed professional hierarchies (finn, currie, and martin 2010). interprofessional education (ipe) is the only area in which sia - influenced interventions have been tested through controlled experiments. however, the interventions studied have not mined the depth of the social identity approach. this may be because their foundation is not actually the sia but the more interpersonally oriented contact hypothesis, which prescribes intergroup contact as a remedy for prejudice (allport 1954). many ipe articles noted that health care professionals propensity to categorize themselves in terms of a uniprofessional identity can impede interprofessional collaboration (lidskog, lofmark, and ahlstrom 2008). the literature also reflects an awareness of the various options for reshaping social categorizations (hean and dickinson 2005). two possible approaches are decategorization (encouraging people to see themselves and each other as individuals, not group members) and recategorization (emphasizing a common in - group identity, not subgroup identities). however, both these approaches can provoke identity threat and backlash from highly identified health care professionals. most of the contemporary sia - influenced literature advocates a third option : acknowledging and valuing both a common superordinate identity and distinct subgroup identities. in contrast to the bottom - up approach of the aspire model (haslam, eggins, and reynolds 2003), the strategies reflected in the ipe literature are top - down : they involve controlling participants experiences in order to influence their attitudes in specific, planned ways. whereas the bottom - up approach makes identity content the driver of change, the top - down approach tends to treat it as incidental. proponents of contact - based interventions have maintained that structural issues are beyond their sphere of influence (carpenter and hewstone 1996). unfortunately, the enterprise of creating favorable interprofessional contexts within an inegalitarian social structure is fraught with challenges and contradictions. first, it is not always possible to create equal - status contact between unequal - status groups as illustrated by a shared education program in which the location and curriculum had been arranged to meet the needs of medical students, thereby marginalizing dental students (ajjawi. second, even if an equal - status bubble can be created, attitudes developed under such artificial conditions may evaporate when participants return to the real world. this difficulty has led contact theorists to develop increasingly elaborate techniques to encourage the generalization of newly acquired attitudes. this individualistic approach, which locates the problem in personal attitudes and stereotypes, is incompatible with a group - based perspective. the latter, because it views stereotypes as a symptom of a system of group relations that entrenches intergroup conflict, sees context as the necessary target of intervention. contact theorists preoccupation with decontextualized stereotypes has created ironic situations ; for example, researchers downplayed an ipe program 's successes with team function or client outcomes, lamenting instead that pencil - and - paper tests detected no change in stereotypes (barnes, carpenter, and dickinson 2000 ; carpenter. the contexts for contact approach 's attentiveness to social categorization represents an advance over approaches that ignore groups or treat them as a nuisance. but without a substantial focus on identity content or social structure, this approach misses a lot of what is social about social identity, and falls short of harnessing the power of social identities to stimulate change. fiol, pratt, and oconnor (2009) have advanced a sequential, sia - based model for resolving intractable identity(-based) conflict (iic) (e.g., between physicians and hospital administrators). in such cases, each in - group draws part of its identity from negating the out - group, making collaboration impossible. the iic model holds that de - escalation of such conflict must progress through the following stages : (1) readiness (to come to the table), (2) mindfulness (openness to other ways of conceptualizing the situation), (3) positive in - group distinctiveness (ensuring a secure in - group identity without the need to negate the out - group), (4) simultaneous intergroup differentiation and unity (cooperation around specific objectives while maintaining separate, distinct groups), and (5) integrative goals and structures. organizations may take steps to help a fractious relationship progress from one stage to the next. however, attempts to achieve a higher stage before lower ones have been completed (e.g., making appeals to unity while each group still views the other as a threat) are likely to backfire. the sequence of stages 1 through 4 was borne out in a canadian study of the relationship between physicians and regional health authorities ; the parties might have ultimately progressed to stage 5, but the government dissolved the regional system (reay and hinings 2009). in an australian study, allied health professionals appeared to traverse stages 3 through 5, progressing from unidisciplinary identities to targeted collaboration and finally dual (disciplinary and allied health) identity (boyce 2006). in its bottom - up approach to the development of a superordinate identity, the most basic insight of a group - based approach is that analyses of conflict or cooperation in health care must consider group, not merely interpersonal, dynamics. this insight was powerfully demonstrated in an expos of virulent intergroup conflict among hospital departments (hewett. drawing on sit, the study revealed that patient charts supposedly repositories of objective information were rife with examples of intergroup competition and in - group enhancement. the study also showed how interspecialty competition to own (or disown) patients could threaten safe care and may have led to a patient 's death. the authors argued that interpersonal - skills training would be an inadequate remedy for physicians dysfunctional communication, since the problem resulted not from a lack of skill but from the active expression of group identities. such recognition of the importance of groups is echoed in papers that variously invoke sit (shute 1997), role theory (booth and hewison 2002), evolutionary psychology (braithwaite, iedema, and jorm 2007), and other concepts (ferlie. 2005). however, this body of literature is primarily descriptive. to identify potential solutions, therefore the health care landscape comprises groups of unequal power and status. without an understanding of this issue, naive calls for teamwork may actually reinforce professional divisions and hierarchies. for example, finn (2008) documented how various providers appealed to the concept of teamwork to advance their profession - specific norms and interests : to nurses, teamwork meant more equal and respectful relations ; to surgeons, that others efficiently followed their orders. many authors stress the gendered nature of the doctor - nurse dynamic, tracing its origins to the subordination of women within the sexual division of labor (campbell - heider and pollock 1987). others put power first and gender second, emphasizing how the specific unequal relationship between doctors and nurses produces certain patterns of interaction. for example, tellis - nayak and tellis - nayak (1984) elucidated how structural inequalities are reinforced by social rituals (concerning the differential use of space, time, language, and body language), arguing that any attempt to reduce the power imbalance must address both structural and symbolic factors. both gender- and power - related role expectations may constrain health care professionals to play the doctor - nurse game, in which the nurse must offer any suggestion indirectly so that it seems to be the doctor 's idea (stein 1967). many structural analyses also touched on identity content by noting how the values, discourses, sources of knowledge, and types of labor associated with high - status groups (men / physicians) are privileged over those of low - status groups (women / nurses) (wicks 1998). roberts (1983) contended that intergroup inequality also taints nurses intragroup relations, giving rise to oppressed group behavior in which nurses accept the physician - dominated structure as legitimate and inevitable. within nursing in terms of nurses efforts to repudiate a devalued group identity and align themselves instead with the oppressing group. while offering a provocative examination of health care hierarchies, this literature has attracted three major criticisms : (1) its perspective tends to be static and deterministic, missing the context - dependent, strategic, and, above all, variable nature of doctor - nurse interactions, and sometimes denying nurses agency ; (2) its accounts are frequently partisan, reflecting the unquestioned assumption that increased power for a certain group is an absolute good ; and (3) its overwhelming focus on doctor - nurse relations obscures other power relations in health care. for example, a uk study (johns 1992) explored how the division of nurses into primary and associate roles created new power dynamics, including what might be described as a nurse - nurse game (i.e., associate nurses confining themselves to indirect suggestions to maintain a facade of harmony). from an sia perspective, all three limitations spring from the fact that unequal partners analyses reify observed phenomena as immutable social facts, not recognizing them as examples of generalized group processes. according to sit (tajfel and turner 1979), the behavior of unequal - status groups can be understood in terms of two basic responses : individual mobility (attempts to leave one group and enter the other) and social change (attempts to reduce, reverse, or increase the status difference between the groups). social change strategies include social creativity (creation of ideologies that affirm the in - group 's worth) and social competition (competition with the out - group for power or resources). sit does not treat these strategies as unique to oppressed groups or as dependent on a particular group psychology, but rather as a function of the in - group 's structural position relative to the out - group, the features of the structural relationship, and the strength of the member 's group identification. the sia 's generality and objectivity allow it to encompass both entrenched intergroup inequalities and scenarios in which power relations are more complex, unexpected, and context - dependent. (for a sit - informed account of doctor - nurse relations, see chattopadhyay, finn, and ashkanasy 2010.) the professional strategies tradition, grounded in the sociology of professions (freidson 1970), explores occupational groups struggles for territory and control. although medical dominance is a frequent focus, abbott (1988) underscored that the important phenomenon is not the empirical fact of a certain group 's dominance (which can change) but the ubiquity of intergroup competition which occurs both interprofessionally and among intraprofessional subgroups (currie, finn, and martin 2009). unlike the unequal partners literature, this stream tends to regard all groups as self - interested and their ideologies as self - serving. it also offers a less static view, illustrating how the intergroup landscape may change when a strategy succeeds or backfires. in a study of health care reorganization, daykin and clarke (2000) illuminated how nurses project of professionalization was impeded by contradictions between the strategies used to gain territory from medicine and to protect their own. to affirm their status vis - - vis medicine, nurses claimed a distinct body of knowledge grounded in caring, yet undermined this claim by devaluing the caring labor of health care assistants. the authors argued that, far from enhancing nurses professional status, such exclusionary tactics reduced their ability to resist the most pertinent threat : management 's fordist practices of routinization and de - skilling. the professional strategies literature often highlights the role of context ; for example, oncologists who worked in different settings used different means to promote their dominance over practitioners of complementary and alternative medicine (cam) (broom and tovey 2007). hospital physicians used the discourse of science to discredit cam, proclaiming the superiority of medicine 's scientific mind - set even in areas where no medical evidence existed. hospice physicians could not discredit cam, given its fit with their institution 's holistic philosophy ; instead, they found subtle ways to subsume cam within a biomedical paradigm, tightly controlling cam practice to avoid any challenge to (bio)medical dominance. with its rich description of both the material and rhetorical strategies that groups use to secure and enhance their professional status, the professional strategies literature depicts social competition and social creativity in action. echoing sit, it shows how members strategically compare their own group with others, choosing dimensions (may and fleming 1997), definitions (norris 2001), and comparison groups (fournier 2002) that maximize the in - group 's positive distinctiveness. it differs from sit, however, in focusing on groups instrumental motivation to gain power and autonomy, seldom recognizing the psychological motivation to maintain a positive social identity. the assumption that groups care only about tangible, not symbolic, benefits can lead to inaccurate predictions (e.g., that health care groups would soon abandon professional ideologies, competing instead over who could meet market needs most cost - effectively) (light 1988). skepticism about groups self - representations can be taken too far, to the point of dismissing all affirmations of group identity as strategic performances. the sia recognizes that such performances are not just strategic ; they often reflect highly valued identity content. many authors stress the gendered nature of the doctor - nurse dynamic, tracing its origins to the subordination of women within the sexual division of labor (campbell - heider and pollock 1987). others put power first and gender second, emphasizing how the specific unequal relationship between doctors and nurses produces certain patterns of interaction. for example, tellis - nayak and tellis - nayak (1984) elucidated how structural inequalities are reinforced by social rituals (concerning the differential use of space, time, language, and body language), arguing that any attempt to reduce the power imbalance must address both structural and symbolic factors. both gender- and power - related role expectations may constrain health care professionals to play the doctor - nurse game, in which the nurse must offer any suggestion indirectly so that it seems to be the doctor 's idea (stein 1967). many structural analyses also touched on identity content by noting how the values, discourses, sources of knowledge, and types of labor associated with high - status groups (men / physicians) are privileged over those of low - status groups (women / nurses) (wicks 1998). roberts (1983) contended that intergroup inequality also taints nurses intragroup relations, giving rise to oppressed group behavior in which nurses accept the physician - dominated structure as legitimate and inevitable. she explained horizontal violence within nursing in terms of nurses efforts to repudiate a devalued group identity and align themselves instead with the oppressing group. while offering a provocative examination of health care hierarchies, this literature has attracted three major criticisms : (1) its perspective tends to be static and deterministic, missing the context - dependent, strategic, and, above all, variable nature of doctor - nurse interactions, and sometimes denying nurses agency ; (2) its accounts are frequently partisan, reflecting the unquestioned assumption that increased power for a certain group is an absolute good ; and (3) its overwhelming focus on doctor - nurse relations obscures other power relations in health care. for example, a uk study (johns 1992) explored how the division of nurses into primary and associate roles created new power dynamics, including what might be described as a nurse - nurse game (i.e., associate nurses confining themselves to indirect suggestions to maintain a facade of harmony). from an sia perspective, all three limitations spring from the fact that unequal partners analyses reify observed phenomena as immutable social facts, not recognizing them as examples of generalized group processes. according to sit (tajfel and turner 1979), the behavior of unequal - status groups can be understood in terms of two basic responses : individual mobility (attempts to leave one group and enter the other) and social change (attempts to reduce, reverse, or increase the status difference between the groups). social change strategies include social creativity (creation of ideologies that affirm the in - group 's worth) and social competition (competition with the out - group for power or resources). sit does not treat these strategies as unique to oppressed groups or as dependent on a particular group psychology, but rather as a function of the in - group 's structural position relative to the out - group, the features of the structural relationship, and the strength of the member 's group identification. the sia 's generality and objectivity allow it to encompass both entrenched intergroup inequalities and scenarios in which power relations are more complex, unexpected, and context - dependent. (for a sit - informed account of doctor - nurse relations, see chattopadhyay, finn, and ashkanasy 2010.) the professional strategies tradition, grounded in the sociology of professions (freidson 1970), explores occupational groups struggles for territory and control. although medical dominance is a frequent focus, abbott (1988) underscored that the important phenomenon is not the empirical fact of a certain group 's dominance (which can change) but the ubiquity of intergroup competition which occurs both interprofessionally and among intraprofessional subgroups (currie, finn, and martin 2009). unlike the unequal partners literature, this stream tends to regard all groups as self - interested and their ideologies as self - serving. it also offers a less static view, illustrating how the intergroup landscape may change when a strategy succeeds or backfires. in a study of health care reorganization, daykin and clarke (2000) illuminated how nurses project of professionalization was impeded by contradictions between the strategies used to gain territory from medicine and to protect their own. to affirm their status vis - - vis medicine, nurses claimed a distinct body of knowledge grounded in caring, yet undermined this claim by devaluing the caring labor of health care assistants. the authors argued that, far from enhancing nurses professional status, such exclusionary tactics reduced their ability to resist the most pertinent threat : management 's fordist practices of routinization and de - skilling. the professional strategies literature often highlights the role of context ; for example, oncologists who worked in different settings used different means to promote their dominance over practitioners of complementary and alternative medicine (cam) (broom and tovey 2007). hospital physicians used the discourse of science to discredit cam, proclaiming the superiority of medicine 's scientific mind - set even in areas where no medical evidence existed. hospice physicians could not discredit cam, given its fit with their institution 's holistic philosophy ; instead, they found subtle ways to subsume cam within a biomedical paradigm, tightly controlling cam practice to avoid any challenge to (bio)medical dominance. with its rich description of both the material and rhetorical strategies that groups use to secure and enhance their professional status, the professional strategies literature depicts social competition and social creativity in action. echoing sit, it shows how members strategically compare their own group with others, choosing dimensions (may and fleming 1997), definitions (norris 2001), and comparison groups (fournier 2002) that maximize the in - group 's positive distinctiveness. it differs from sit, however, in focusing on groups instrumental motivation to gain power and autonomy, seldom recognizing the psychological motivation to maintain a positive social identity. the assumption that groups care only about tangible, not symbolic, benefits can lead to inaccurate predictions (e.g., that health care groups would soon abandon professional ideologies, competing instead over who could meet market needs most cost - effectively) (light 1988). skepticism about groups self - representations can be taken too far, to the point of dismissing all affirmations of group identity as strategic performances. the sia recognizes that such performances are not just strategic ; they often reflect highly valued identity content. group identities are not arbitrary but are defined by certain content : norms, values, and worldviews that are meaningful and important to members. a large literature frames this topic in terms of culture and cultural differences, delineating how professional groups differ in everything from values and attitudes (degeling, kennedy, and hill 2001) to myths and rituals (dombeck 1997). but bald descriptions of identity content do not elucidate group processes, and inspire few solutions apart from vague calls for cultural sensitivity. accounts of professional culture may also miss intraprofessional diversity ; even explorations of professional subcultures (leininger 1994) often stop short of asking what the different variants of identity mean. more sia - consistent studies may explain variability in terms of subgroups with different experiences or structural positions contesting (advancing their own versions of) identity content (e.g., pratt and rafaeli 1997). in contrast to the cultural differences discourse, which portrays identity content as static, the sia takes a dynamic view, examining how different representations of group identity are constructed and mobilized in order to achieve group goals. research on professional socialization considers, among other things, how educational and practice environments encourage or suppress the enactment of a patient - centered identity. patient - centeredness can be an integral part of physician identity, yet its expression may be actively discouraged through both the formal and informal curricula of medical school (apker and eggly 2004). in contrast, nurses are strongly socialized into patient - centeredness until they enter the working world, where demands for efficiency may imperil this core value, resulting in demoralization and disengagement (limoges 2007). odonohue and nelson (2007) stressed that professionals psychological contract with an organization depends not merely on individual rewards but on the organization 's respect for the values central to their collective identity. health care professionals are known to resist threats to their identity content (fiol and oconnor 2006) ; however, interventions perceived as identity threatening in one context may be perceived as benign, even identity affirming, in another. physicians may reject management - imposed system redesign as a threat to the doctor - patient relationship, yet actively support the same redesign when it is seen as contributing to the achievement of a patient - centered medical home (kreindler 2008). when the threat involves alternative practitioners encroachment on medical territory, medicine is a science (broom and tovey 2007)yet, when it involves managerial pressure to comply with clinical practice guidelines, medicine is an art (mcdonald, waring, and harrison 2006). at first glance, the finding that such varied, even contradictory, discourses are used to resist change seems to imply that change is impossible. the flexible (re)construction of identity, however, may itself be a key to change. in one study, gps who became change leaders constructed a hybrid identity that affirmed the primacy of their physician identity while incorporating management skills (redefined as a trivial subset of skills that most physicians possess) (hotho 2008). they found this new identity more attractive than that of management (still seen as a low - status out - group with objectionable values)or of rank - and - file gps (seen as routine oriented and averse to change). organizational change efforts have been observed to founder when administrators either ignore social identity (seeking to shape employees behavior through individual rewards and sanctions alone) or assume that staff can be rallied behind an imposed organizational identity (charles - jones, latimer, and may 2003 ; mcdonald 2004). success is more likely when leaders guide change as it grows from the real values of existing groups (brooks 1996 ; obrien. 2004). the fullest elaboration of this strategy occurs within the sia literature, with the aspire (actualizing social and personal identity resources) model of organizational development ; the aspire process engages employees in building a mosaic identity that recognizes both common goals and the distinct contributions of valued subgroups (haslam, eggins, and reynolds 2003). (bevan 2008), organic development of a network community (bate 2000), and other concepts. one hospital used the norms of physician culture (concrete, expert - led, decision - oriented discussions) to encourage physician engagement in restructuring (ohare and kudrle 2007). other studies of positive physician - manager relationships have emphasized the development of a shared identity, facilitated by shared decision making grounded in common values (in particular, service and excellence ; see graham and steele 2001 ; kirkpatrick. just as social identities can be mobilized to resist change, they also can be mobilized to cope with change, or to achieve it. as the social identity literature has revealed, mobilization is not simply a matter of invoking a ready - made image of group identity, but involves crafting a sense of us (haslam, reicher, and platow, 2011) that supports a desired change. goodrick and reay (2010) showed how nursing textbooks, using the idea of continuity with the past to foster the reconstruction of nursing identity, highlighted only those aspects of the past consistent with the new vision. attempts to promote identification with a certain group, to restructure groups, or to mobilize identities must reckon with members existing patterns of group identification. one stream of literature is concerned primarily with health care employees qua employees, examining organizational identification, its predictors (e.g., good communication up and down the hierarchy, value congruence between organization and employee, the organization 's prestige, and the employee 's sense of being respected), and its consequences (e.g., cooperation, organizational citizenship) (dukerich, golden, and shortell 2002). nearly all these studies cite sit ; problematically, though, many are characterized by a management - centric assumption that organizations should, or even can, manipulate employees social identities (e.g., han and harms 2010). yet even within this literature, it becomes clear that employee commitment is not so easily manipulable. for example, while participative decision making is among the strongest predictors of organizational identification, opportunities to participate are unlikely to foster organizational identification unless employees see them as genuine and relevant (joensson 2008 ; tangirala and ramanujam 2008). a second stream of literature recognizes that most health care providers are strongly identified with their profession. strong professional and organizational identification are not incompatible ; indeed, the two are often positively correlated (bartels.. however, most professionals identify more strongly with their profession than with their organization, which typically offers a less distinctive identity and a shorter period of socialization (callan. 2007). moreover, the combination of high professional and low organizational identification is linked to behavior that, from the organization 's perspective, is undesirable. physicians exhibiting this combination of attitudes have been found to resist influence from management, repay perceived organizational support with reduced compliance, and retaliate against perceived violation of their psychological contract with the organization (hekman. tradition is that most of the studies, concentrating as they do on drawing general conclusions about organizational psychology, are not firmly grounded in the health care context. for instance, some studies have drawn inferences about high - status staff from analyses that excluded physicians or combined them with senior nurses (callan. 2007 ; obrien. several qualitative studies have amplified the point that group membership does not equate with group identification. multidisciplinary team. however, direct care nurses and health care assistants, who continued to be excluded from decision making, did not share in this team identity ; rather, they felt alienated and unwilling to cooperate with the team 's directives. physicians are unlikely to identify with an organization whose management they perceive as a devalued out - group in particular, one that threatens their autonomy in the service of values abhorrent to their profession (fiol, pratt, and oconnor 2009 ; hekman. moreover, hoff (1999) found that creating physician - managers failed to bridge the divide between these two groups ; on the contrary, the physician - manager population itself became divided. those who saw themselves primarily as managers embraced their new identity, complete with management jargon, and even defended organizational policies that negatively affected physicians. those who continued to see themselves primarily as physicians asserted this identity by resisting and sabotaging the activities of the first group, whom they viewed as traitors. these findings invite the application of such sia concepts as categorization threat (the threat of one 's being viewed as a member of a devalued group ; see ellemers, spears, and doosje 2002) and the black sheep effect (in which an in - group member who flouts group norms attracts more censure than does an out - group member ; see marques, yzerbyt, and leyens 1988). as we have seen, physician - managers can develop hybrid identities (hotho 2008), but this is not inevitable. doolin (2002) found that physicians revised their identities to incorporate a new management role only when they perceived the latter as congruent with their physician identity. one stream of literature is concerned primarily with health care employees qua employees, examining organizational identification, its predictors (e.g., good communication up and down the hierarchy, value congruence between organization and employee, the organization 's prestige, and the employee 's sense of being respected), and its consequences (e.g., cooperation, organizational citizenship) (dukerich, golden, and shortell 2002). nearly all these studies cite sit ; problematically, though, many are characterized by a management - centric assumption that organizations should, or even can, manipulate employees social identities (e.g., han and harms 2010). yet even within this literature, it becomes clear that employee commitment is not so easily manipulable. for example, while participative decision making is among the strongest predictors of organizational identification, opportunities to participate are unlikely to foster organizational identification unless employees see them as genuine and relevant (joensson 2008 ; tangirala and ramanujam 2008). a second stream of literature recognizes that most health care providers are strongly identified with their profession. strong professional and organizational identification are not incompatible ; indeed, the two are often positively correlated (bartels. however, most professionals identify more strongly with their profession than with their organization, which typically offers a less distinctive identity and a shorter period of socialization (callan. 2007). moreover, the combination of high professional and low organizational identification is linked to behavior that, from the organization 's perspective, is undesirable. physicians exhibiting this combination of attitudes have been found to resist influence from management, repay perceived organizational support with reduced compliance, and retaliate against perceived violation of their psychological contract with the organization (hekman. tradition is that most of the studies, concentrating as they do on drawing general conclusions about organizational psychology, are not firmly grounded in the health care context. staff from analyses that excluded physicians or combined them with senior nurses (callan. several qualitative studies have amplified the point that group membership does not equate with group identification.. however, direct care nurses and health care assistants, who continued to be excluded from decision making, did not share in this team identity ; rather, they felt alienated and unwilling to cooperate with the team 's directives. physicians are unlikely to identify with an organization whose management they perceive as a devalued out - group in particular, one that threatens their autonomy in the service of values abhorrent to their profession (fiol, pratt, and oconnor 2009 ; hekman. moreover, hoff (1999) found that creating physician - managers failed to bridge the divide between these two groups ; on the contrary, the physician - manager population itself became divided. those who saw themselves primarily as managers embraced their new identity, complete with management jargon, and even defended organizational policies that negatively affected physicians. those who continued to see themselves primarily as physicians asserted this identity by resisting and sabotaging the activities of the first group, whom they viewed as traitors. these findings invite the application of such sia concepts as categorization threat (the threat of one 's being viewed as a member of a devalued group ; see ellemers, spears, and doosje 2002) and the black sheep effect (in which an in - group member who flouts group norms attracts more censure than does an out - group member ; see marques, yzerbyt, and leyens 1988). as we have seen, physician - managers can develop hybrid identities (hotho 2008), but this is not inevitable. doolin (2002) found that physicians revised their identities to incorporate a new management role only when they perceived the latter as congruent with their physician identity. context refers to the external conditions such as organizational structures, working practices, and physical features of the work environment that support a particular system of group relations. the sia holds that changing the context can change the way people view and relate to one another, ultimately altering existing patterns of group interaction. a handful of articles described successful organizational - development initiatives that put intergroup issues front and center (van de vliert 1995). staff have been invited to participate in collective reflection on group dynamics, and the insights derived from this process used to develop or implement new structures and working practices (context changes) that support more positive group relations (bate 2000 ; bleakley. although the literature abounds with warnings that the mere establishment of a team does not guarantee true teamwork, some studies with a longer follow - up have reached more optimistic conclusions. a british study (hudson 2002) found that effective teamwork between health and social care providers had begun to develop in practices where providers were co - located and informal working patterns were shifting. a longitudinal study of an interdisciplinary health team traced members gradual progression from defensiveness and stereotyping to a shared team culture in which roles were less differentiated (farrell, schmitt, and heinemann 2001). such findings suggest that what are seen as intractable problems with multidisciplinary teams may actually just be growing pains. there is some evidence, however, that role revision and teamwork are more likely to become realities when social identity is taken into account. a study of an unsuccessful attempt to introduce an enhanced nursing role suggested that the change process failed to engage with the entrenched social identity dynamics associated with a rigid professional hierarchy (currie, finn, and martin 2010). despite its superficial adoption, the new role was assimilated into the existing social structure and produced little real change. furthermore, a single organizational intervention may not suffice ; context change may entail addressing broader environmental factors that impede collaboration. for example, such contextual factors as organizational integration, resource availability, and various facilitators of long - term working relationships strongly influenced whether the introduction of teams reproduced or transformed professional hierarchies (finn, currie, and martin 2010). interprofessional education (ipe) is the only area in which sia - influenced interventions have been tested through controlled experiments. however, the interventions studied have not mined the depth of the social identity approach. this may be because their foundation is not actually the sia but the more interpersonally oriented contact hypothesis, which prescribes intergroup contact as a remedy for prejudice (allport 1954). many ipe articles noted that health care professionals propensity to categorize themselves in terms of a uniprofessional identity can impede interprofessional collaboration (lidskog, lofmark, and ahlstrom 2008). the literature also reflects an awareness of the various options for reshaping social categorizations (hean and dickinson 2005). two possible approaches are decategorization (encouraging people to see themselves and each other as individuals, not group members) and recategorization (emphasizing a common in - group identity, not subgroup identities). however, both these approaches can provoke identity threat and backlash from highly identified health care professionals. most of the contemporary sia - influenced literature advocates a third option : acknowledging and valuing both a common superordinate identity and distinct subgroup identities. in contrast to the bottom - up approach of the aspire model (haslam, eggins, and reynolds 2003), the strategies reflected in the ipe literature are top - down : they involve controlling participants experiences in order to influence their attitudes in specific, planned ways. whereas the bottom - up approach makes identity content the driver of change, the top - down approach tends to treat it as incidental. proponents of contact - based interventions have maintained that structural issues are beyond their sphere of influence (carpenter and hewstone 1996). unfortunately, the enterprise of creating favorable interprofessional contexts within an inegalitarian social structure is fraught with challenges and contradictions. first, it is not always possible to create equal - status contact between unequal - status groups as illustrated by a shared education program in which the location and curriculum had been arranged to meet the needs of medical students, thereby marginalizing dental students (ajjawi. bubble can be created, attitudes developed under such artificial conditions may evaporate when participants return to the real world. this difficulty has led contact theorists to develop increasingly elaborate techniques to encourage the generalization of newly acquired attitudes. this individualistic approach, which locates the problem in personal attitudes and stereotypes, is incompatible with a group - based perspective. the latter, because it views stereotypes as a symptom of a system of group relations that entrenches intergroup conflict, sees context as the necessary target of intervention. contact theorists preoccupation with decontextualized stereotypes has created ironic situations ; for example, researchers downplayed an ipe program 's successes with team function or client outcomes, lamenting instead that pencil - and - paper tests detected no change in stereotypes (barnes, carpenter, and dickinson 2000 ; carpenter. the contexts for contact approach 's attentiveness to social categorization represents an advance over approaches that ignore groups or treat them as a nuisance. but without a substantial focus on identity content or social structure, this approach misses a lot of what is social about social identity, and falls short of harnessing the power of social identities to stimulate change. fiol, pratt, and oconnor (2009) have advanced a sequential, sia - based model for resolving intractable identity(-based) conflict (iic) (e.g., between physicians and hospital administrators). in such cases, each in - group draws part of its identity from negating the out - group, making collaboration impossible. the iic model holds that de - escalation of such conflict must progress through the following stages : (1) readiness (to come to the table), (2) mindfulness (openness to other ways of conceptualizing the situation), (3) positive in - group distinctiveness (ensuring a secure in - group identity without the need to negate the out - group), (4) simultaneous intergroup differentiation and unity (cooperation around specific objectives while maintaining separate, distinct groups), and (5) integrative goals and structures. organizations may take steps to help a fractious relationship progress from one stage to the next. however, attempts to achieve a higher stage before lower ones have been completed (e.g., making appeals to unity while each group still views the other as a threat) are likely to backfire. the sequence of stages 1 through 4 was borne out in a canadian study of the relationship between physicians and regional health authorities ; the parties might have ultimately progressed to stage 5, but the government dissolved the regional system (reay and hinings 2009). in an australian study, allied health professionals appeared to traverse stages 3 through 5, progressing from unidisciplinary identities to targeted collaboration and finally dual (disciplinary and allied health) identity (boyce 2006). in its bottom - up approach to the development of a superordinate identity, a handful of articles described successful organizational - development initiatives that put intergroup issues front and center (van de vliert 1995). staff have been invited to participate in collective reflection on group dynamics, and the insights derived from this process used to develop or implement new structures and working practices (context changes) that support more positive group relations (bate 2000 ; bleakley. although the literature abounds with warnings that the mere establishment of a team does not guarantee true teamwork, some studies with a longer follow - up have reached more optimistic conclusions. a british study (hudson 2002) found that effective teamwork between health and social care providers had begun to develop in practices where providers were co - located and informal working patterns were shifting. a longitudinal study of an interdisciplinary health team traced members gradual progression from defensiveness and stereotyping to a shared team culture in which roles were less differentiated (farrell, schmitt, and heinemann 2001). such findings suggest that what are seen as intractable problems with multidisciplinary teams may actually just be growing pains. there is some evidence, however, that role revision and teamwork are more likely to become realities when social identity is taken into account. a study of an unsuccessful attempt to introduce an enhanced nursing role suggested that the change process failed to engage with the entrenched social identity dynamics associated with a rigid professional hierarchy (currie, finn, and martin 2010). despite its superficial adoption, the new role was assimilated into the existing social structure and produced little real change. furthermore, a single organizational intervention may not suffice ; context change may entail addressing broader environmental factors that impede collaboration. for example, such contextual factors as organizational integration, resource availability, and various facilitators of long - term working relationships strongly influenced whether the introduction of teams reproduced or transformed professional hierarchies (finn, currie, and martin 2010). interprofessional education (ipe) is the only area in which sia - influenced interventions have been tested through controlled experiments. however, the interventions studied have not mined the depth of the social identity approach. this may be because their foundation is not actually the sia but the more interpersonally oriented contact hypothesis, which prescribes intergroup contact as a remedy for prejudice (allport 1954). many ipe articles noted that health care professionals propensity to categorize themselves in terms of a uniprofessional identity can impede interprofessional collaboration (lidskog, lofmark, and ahlstrom 2008). the literature also reflects an awareness of the various options for reshaping social categorizations (hean and dickinson 2005). two possible approaches are decategorization (encouraging people to see themselves and each other as individuals, not group members) and recategorization (emphasizing a common in - group identity, not subgroup identities). however, both these approaches can provoke identity threat and backlash from highly identified health care professionals. most of the contemporary sia - influenced literature advocates a third option : acknowledging and valuing both a common superordinate identity and distinct subgroup identities. in contrast to the bottom - up approach of the aspire model (haslam, eggins, and reynolds 2003), the strategies reflected in the ipe literature are top - down : they involve controlling participants experiences in order to influence their attitudes in specific, planned ways. whereas the bottom - up approach makes identity content the driver of change, the top - down approach tends to treat it as incidental. proponents of contact - based interventions have maintained that structural issues are beyond their sphere of influence (carpenter and hewstone 1996). unfortunately, the enterprise of creating favorable interprofessional contexts within an inegalitarian social structure is fraught with challenges and contradictions. first, it is not always possible to create equal - status contact between unequal - status groups as illustrated by a shared education program in which the location and curriculum had been arranged to meet the needs of medical students, thereby marginalizing dental students (ajjawi. second, even if an equal - status bubble can be created, attitudes developed under such artificial conditions may evaporate when participants return to the real world. this difficulty has led contact theorists to develop increasingly elaborate techniques to encourage the generalization of newly acquired attitudes. this individualistic approach, which locates the problem in personal attitudes and stereotypes, is incompatible with a group - based perspective. the latter, because it views stereotypes as a symptom of a system of group relations that entrenches intergroup conflict, sees context as the necessary target of intervention. contact theorists preoccupation with decontextualized stereotypes has created ironic situations ; for example, researchers downplayed an ipe program 's successes with team function or client outcomes, lamenting instead that pencil - and - paper tests detected no change in stereotypes (barnes, carpenter, and dickinson 2000 ; carpenter. the contexts for contact approach 's attentiveness to social categorization represents an advance over approaches that ignore groups or treat them as a nuisance. but without a substantial focus on identity content or social structure, this approach misses a lot of what is social about social identity, and falls short of harnessing the power of social identities to stimulate change. fiol, pratt, and oconnor (2009) have advanced a sequential, sia - based model for resolving intractable identity(-based) conflict (iic) (e.g., between physicians and hospital administrators). in such cases, each in - group draws part of its identity from negating the out - group, making collaboration impossible. the iic model holds that de - escalation of such conflict must progress through the following stages : (1) readiness (to come to the table), (2) mindfulness (openness to other ways of conceptualizing the situation), (3) positive in - group distinctiveness (ensuring a secure in - group identity without the need to negate the out - group), (4) simultaneous intergroup differentiation and unity (cooperation around specific objectives while maintaining separate, distinct groups), and (5) integrative goals and structures. organizations may take steps to help a fractious relationship progress from one stage to the next. however, attempts to achieve a higher stage before lower ones have been completed (e.g., making appeals to unity while each group still views the other as a threat) are likely to backfire. the sequence of stages 1 through 4 was borne out in a canadian study of the relationship between physicians and regional health authorities ; the parties might have ultimately progressed to stage 5, but the government dissolved the regional system (reay and hinings 2009). in an australian study, allied health professionals appeared to traverse stages 3 through 5, progressing from unidisciplinary identities to targeted collaboration and finally dual (disciplinary and allied health) identity (boyce 2006). in its bottom - up approach to the development of a superordinate identity, prior research has provided a rich description of how power structures, group norms and values, strength of identification, and contextual factors interact with various social identifications to produce different patterns of group behavior. the social identity approach has the potential to serve as a coherent framework for synthesizing this diverse information and identifying the most promising mechanisms for change. in order to realize this potential, it is important to take advantage of the full depth and complexity of the approach, rather than stop at the basic insight that people engage in social categorization. further research should also focus on deepening our understanding of the currently neglected group level, rather than assume that a potpourri of group - based and individualistic approaches can offer a better or more complete analysis. it also is essential that research be sensitive to the unique features of the health care context not because transferability is unimportant, but because overlooking social identifications, structures, or elements of identity content that are highly salient in a particular context can result in shallow or misleading analysis. finally, since much of the literature has concentrated on the micro level of interprofessional silos and clinical teams, future research might emphasize the macro level of interorganizational silos and system integration. while the same basic mechanisms may apply in both contexts, two directions for future research are (1) in - depth exploration of social identity dynamics during system - integration efforts (e.g., development of accountable care organizations in the united states), with a focus on identity mobilization and context change ; and (2) further testing of the aspire and iic models in health care systems. a recurring theme in literature from disparate traditions is the importance of identity mobilization and/or context change in driving system transformation. change seems most likely to occur when both mechanisms are present : without mobilization of valued identities, attempts to impose context change may provoke identity threat and invite implementation failure ; without changes to the real conditions under which people work, identity mobilization may amount to just another staff development workshop. the two processes may also reinforce each other cyclically : mobilization of shared identities can facilitate the adoption of concrete changes (graham and steele 2001 ; kerfoot 2007), while changes in working arrangements can stimulate the reshaping and reinterpretation of social identities (farrell, schmitt, and heinemann 2001 ; hotho 2008). the aspire model) ; however, context change can be the impetus for identity reconstruction. in a study of primary care reform, changes at the institutional and organizational levels (capitation, multidisciplinary teams, co - location, etc.) created a context for physicians to reframe teamwork, preventive medicine, and guideline adherence as identity congruent (chreim, williams, and hinings 2007). whereas managerial attempts to colonize staff identities are likely to be resisted or subverted, context changes that can be meaningfully incorporated into existing identities may stimulate constructive engagement (levay and waks 2009). in the course of such engagement, providers not only reconstruct their identities to fit the intervention but often reconstruct the intervention to fit their identities. although such reconstruction may sometimes serve narrow professional interests (mcdonald, harrison, and checkland 2008), in other cases the result advances the interests of professionals, managers, and, most important, patients (waring and currie 2009). in advancing a social identity perspective on health care silos, we do not mean to imply that silos are wholly a function of social identity dynamics. the sia recognizes that individual and interpersonal factors remain important, particularly when group identification or social - identity salience is low. moreover, although practical and operational problems often have a social - identity component, solutions can not be found by addressing social identity alone. the contribution of the sia is to articulate when and how various factors will be relevant and to prevent misconstrual of a group - level issue as an interpersonal or purely operational one. we suggest the sia as a framework, not a replacement, for other group - level theories ; for example, while the sia highlights the ubiquity of politics in organizations, it is not a theory of politics, and other approaches delve more fully into structural relations or power enactment. in short, our claim is not that everything is a social identity problem, but that every problem involving interactions within or among health care groups probably has a social identity dimension, and that understanding this dimension will enable more effective responses. because this is not an evidence review but we do offer the following guiding questions for health care leaders confronting the problem of silos : who are the relevant groups, and what are their relationships ? how might social identity be playing a role in current organizational problems or conflicts ? which groups need to be around the table to develop new ways of working ? can they come together immediately, or does intergroup tension necessitate that they first work separately ? what change messages (and messengers) will fit the values and attributes cherished by each group ? how might day - to - day factors that reinforce silos and conflict be replaced by others that promote more cooperative and equal interactions ? as change proceeds, how can identity threat (to valued groups existence, status, distinctiveness, values, etc.) be minimized ? as the more than 300 reports in our review reveal, social identity is a powerful reality in the functioning of the health care system. rather than attempt to ignore, expunge, or manipulate social identities, we can embrace the opportunity to work with and through them to unite providers around the values that all health care professionals share. social identity thinking can unlock new options for overcoming silos and bringing about a harmoniously functioning, well - coordinated health care system. | contextone of health care 's foremost challenges is the achievement of integration and collaboration among the groups providing care. yet this fundamentally group - related issue is typically discussed in terms of interpersonal relations or operational issues, not group processes.methodswe conducted a systematic search for literature offering a group - based analysis and examined it through the lens of the social identity approach (sia). founded in the insight that group memberships form an important part of the self - concept, the sia encompasses five dimensions : social identity, social structure, identity content, strength of identification, and context.findingsour search yielded 348 reports, 114 of which cited social identity. however, sia - citing reports varied in both compatibility with the sia 's metatheoretical paradigm and applied relevance to health care ; conversely, some non - sia - citers offered sia - congruent analyses. we analyzed the various combinations and interpretations of the five sia dimensions, identifying ten major conceptual currents. examining these in the light of the sia yielded a cohesive, multifaceted picture of (inter)group relations in health care.conclusionsthe sia offers a coherent framework for integrating a diverse, far - flung literature on health care groups. further research should take advantage of the full depth and complexity of the approach, remain sensitive to the unique features of the health care context, and devote particular attention to identity mobilization and context change as key drivers of system transformation. our article concludes with a set of guiding questions to help health care leaders recognize the group dimension of organizational problems, identify mechanisms for change, and move forward by working with and through social identities, not against them. |
maximal ball release velocity is a crucial variable for successful performance in many sport games such as team handball, baseball, soccer and water polo (van den tillaar and ettema, 2003). the velocity of the ball in an overarm throw depends on optimal throwing mechanics and body segments characteristics. the overarm throw is determined by a proximal - to - distal principle (calabrese, 2013 ; putnam, 1993 ; wagner., 2012 ; weber., 2014) which describes progressive contribution of body segments to the momentum of the throwing object, beginning from the base of support and progressing through to the hand. this progression can be observed by monitoring peak angular velocities of the involved segments or by monitoring the activation of the muscles moving these segments (escamilla and andrews, 2009 ; hancock and hawkins, 1996 ; hirashima., 2002 ; in addition, it is evident that the delay of the activation of the distal muscles with respect to the proximal ones should be optimal not too short and not too long ; if the delay is shorter than optimal, there is less time available for the contraction of the proximal muscles which then do less work and vice versa (alexander, 1991 ; chowdhary and challist, 1999). the stretch - shortening cycle (ssc) is another mechanism which can contribute to the final throw velocity (grezios., 2006), as it enhances concentric muscle action due to recovery of elastic energy stored during preceding eccentric contraction and increased agonist muscle innervation as a result of the stretch reflex (bosco., 1981). there are indeed many factors contributing to the final velocity of the ball at release. to evaluate these factors different demanding and time consuming acquisition and analysis methods are required including kinematic and electromyography assessments. however, successful implementation of all complex mechanisms discussed would generally result in a high final velocity of the ball, conversely low velocities would imply that throwing mechanics were not optimal. due to its ballistic nature, an overarm throw is performed in a short space of time and is controlled based on an open - loop system, which is a feed forward process and has no feedback (magill, 2011). due to a time limitation, the motor program controlling the involved effectors (muscles) containing all the information needed to carry out the throw is generated in the brain prior to the throw ; there is no time to continually register, evaluate and implement the information to control the movement while it is in the process. the natural part of it is sensory - perceptual information referred to as task - intrinsic feedback, while the velocity added on presents augmented feedback. augmented feedback of the velocity of a ball can be provided by a radar gun after an overarm throw. the information given by the radar gun falls to the subcategory of the extrinsic feedback known as knowledge of results (kr), while the category where the information concerning the movement characteristics is given is known as knowledge of performance (kp). kp is commonly provided verbally by the teaching or coaching staff during regular training sessions or by video recordings of the performance being carried out and shown to athletes. kr provides information that a subject is unable to detect using his / her own sensory system about performing a throw and can therefore add it to intrinsic sensory feedback (magill, 2011). there has been a lack of research evaluating the effectiveness of improvement of the throwing velocity due to immediate kr. knowing the velocity of the ball enables the thrower to consolidate the right sensory perceptual information and increases the chance that the thrower will qualitatively repeat the performance. feedback also involves the motivational component, encouraging the subject to continue performing a skill at the highest possible level. kermode and carlton (1992) studied differences in the maximum throwing distances between the groups who received either kp about their throwing technique or kr about the throwing distance and they found that the kp group demonstrated better results. the aim of this study was to determine whether training with instantly provided quantitative feedback information of the velocity of the ball after every throw (in addition to the usual knowledge of performance feedback provided by the teaching staff) would enhance the gain of velocity with respect to the same training intervention where no kr information was provided. in addition, the secondary aim was to examine how such training (throwing a normal ball) would affect the velocities of the heavy ball throws which present different sensory perceptual information for the thrower. indeed, sensory perceptual information might change due to different external conditions (van den tillaar and ettema, 2011), such as different weight of the ball (the force and time conditions vary with respect to the normal ball). in order to verify differences in the throwing velocity increase during a six week training period, 50 female and 73 male students were randomly assigned to two groups including the experimental group that received knowledge of their results (kr) and a control group that received no knowledge of the results (nokr). all subjects performed 2 series of 10 set shots twice per week for six weeks. the kr group received feedback information about throwing velocity measured by a radar gun and displayed immediately after every shot, while the nokr group did not receive any feedback. throwing velocity measurements of a normal (nb) and heavy ball (hb) were performed pre- and post- training to determine the increase in velocity. dependant variables included final velocity of the normal and heavy ball with respect to their initial velocities as well as their relative change. independent variables consisted of the different training regimens hypothesized to impact differently the dependent variables, training with and without frequently provided throwing velocity value after every shot. according to the results of this study fifty female (age 21.1 2.1 years, body height 165.4 6.2 cm, body mass 59.1 7.4 kg) and seventy - three male (age 21.4 2 years, body height 180.1 5.4 cm, body mass 77.8 7 kg) students participated in the intervention, divided into groups of 15 to 20 students. each subject was considered healthy and injury - free at the time of the study. the experiment was performed with the university of ljubljana (faculty of sport) ethics committee s approval ; each subject was provided with a full explanation of the protocols and signed informed consent was received before the study commenced. the experiment was conducted within regular practical education classes on theory and methodology of handball at the faculty of sport following a well - established program. practical classes (groups) of students were divided by gender. before the commencement of the experiment, basic information regarding handball in general and the overarm throw called the three step set shot was provided to all students during the first four week learning sessions (two sessions per week). after the introduction period, the participants were randomly assigned to the experimental (kr) or control (nokr) group ; the groups were also divided by gender. during the next twelve sessions (twice per week for six weeks), all participants first performed a standardised warm up and then executed two series of ten handball three step set shots with maximal effort. the experimental group (kr) was provided with the quantitative feedback on the highest ball velocity measured with the radar gun after every shot which was shown immediately on the display board, while the participants of the control group (nokr) were not. all subjects were provided with verbal encouragement during their throws to perform them with maximal effort. the initial measurements were carried out in the last session of the introduction period and final measurements after six weeks of training. every participant performed two series of three handball three step set shots. due to possible muscle potentiation effect, series were not randomly assigned ; first series of three shots were performed with the dominant arm using a normal size handball (nb : the volume 0.54 m, weight 375 grams, size 2) and the second series with the dominant arm using a heavy ball (hb ; weight 800 grams). the shots were executed with maximal effort in the direction of the radar gun (stalker ats professional sports, applied concepts, inc., usa), which was placed behind an ordinary handball port, seven metres from the shooter at the height of 1.5 m. a rest period of 15 s was allowed between the shots. during pre- and post - training measurements the highest ball velocity of every shot was measured and the highest of the three velocities was used for further analysis. the initial (pre) measurement velocities were labelled nbi and hbi for the throws with normal and heavy ball, respectively, and final (post) measurement velocities were labelled nbf and hbf. analysis of covariance (ancova) was used to verify the differences between the velocities of the ball measured after the intervention (training) between the feedback and no - feedback groups, setting the pre - training velocity as the covariate and the post - training velocity as the dependent variable. pre- to post - test velocity change was also expressed in percentages and the differences in the changes were calculated executing the independent t - test. the alpha level for significance fifty female (age 21.1 2.1 years, body height 165.4 6.2 cm, body mass 59.1 7.4 kg) and seventy - three male (age 21.4 2 years, body height 180.1 5.4 cm, body mass 77.8 7 kg) students participated in the intervention, divided into groups of 15 to 20 students. each subject was considered healthy and injury - free at the time of the study. the experiment was performed with the university of ljubljana (faculty of sport) ethics committee s approval ; each subject was provided with a full explanation of the protocols and signed informed consent was received before the study commenced. the experiment was conducted within regular practical education classes on theory and methodology of handball at the faculty of sport following a well - established program. practical classes (groups) of students were divided by gender. before the commencement of the experiment, basic information regarding handball in general and the overarm throw called the three step set shot was provided to all students during the first four week learning sessions (two sessions per week). after the introduction period, the participants were randomly assigned to the experimental (kr) or control (nokr) group ; the groups were also divided by gender. during the next twelve sessions (twice per week for six weeks), all participants first performed a standardised warm up and then executed two series of ten handball three step set shots with maximal effort. the experimental group (kr) was provided with the quantitative feedback on the highest ball velocity measured with the radar gun after every shot which was shown immediately on the display board, while the participants of the control group (nokr) were not. all subjects were provided with verbal encouragement during their throws to perform them with maximal effort. the initial measurements were carried out in the last session of the introduction period and final measurements after six weeks of training. every participant performed two series of three handball three step set shots. due to possible muscle potentiation effect, series were not randomly assigned ; first series of three shots were performed with the dominant arm using a normal size handball (nb : the volume 0.54 m, weight 375 grams, size 2) and the second series with the dominant arm using a heavy ball (hb ; weight 800 grams). the shots were executed with maximal effort in the direction of the radar gun (stalker ats professional sports, applied concepts, inc., usa), which was placed behind an ordinary handball port, seven metres from the shooter at the height of 1.5 m. a rest period of 15 s was allowed between the shots. during pre- and post - training measurements, the highest ball velocity of every shot was measured and the highest of the three velocities was used for further analysis. the initial (pre) measurement velocities were labelled nbi and hbi for the throws with normal and heavy ball, respectively, and final (post) measurement velocities were labelled nbf and hbf. analysis of covariance (ancova) was used to verify the differences between the velocities of the ball measured after the intervention (training) between the feedback and no - feedback groups, setting the pre - training velocity as the covariate and the post - training velocity as the dependent variable. pre- to post - test velocity change was also expressed in percentages and the differences in the changes were calculated executing the independent t - test. the alpha level for significance absolute values of the velocities of the ball measured pre- and post - training are shown in table 1. velocities of the ball (km / h) for kr and nokr groups measured pre and post training for men and women nb normal ball, hb heavy ball, i initial (pre - test), f final (post - test). ancova results are presented in table 2 and showed a significant effect of the intervention group (kr / nokr) on post - test velocities after controlling the initial throwing velocity for both normal and heavy ball shots. all effects irrespective of the gender and the ball used for testing (normal and heavy ball) were found to be significant (p < 0.001 and p < 0.05 for final velocities of normal and heavy ball, respectively). ancova results for the effect of the intervention group (kr / nokr) on post - test velocities after controlling for the initial throwing velocity for both normal and heavy ball shots relative changes of the velocity of the ball measured pre- and post - intervention with the significance of the t - test are shown in figure 1. all changes were found significant for men and women separately as well as for all participants. relative velocity changes for throws performed with a normal ball (nb) and a heavy ball (hb) for kr (full columns) and nokr (empty columns) groups. left all participants, middle women, right men. p < 0.001, p < 0.01, p < 0.05. no significant effect for gender in relative changes for the kr group was found, (normal ball, t(60) = 0.657, p = 0.514 ; heavy ball t(60) = 1.317, p = 0.193), as well as for the nokr group for heavy ball throws (t(59) = 0.45, p = 0.965). however, for the nokr group gender showed a significant effect for a normal ball relative to changes of velocity (t(56,4) = 2.060, p = 0.044) ; the relative changes of velocity were 3.3 and 1.9% for women and men, respectively. the aim of the study was to examine if the information on the velocity of the ball at release, provided quantitatively immediately after each throw, could help the thrower to gain more velocity following a training period of six weeks as compared to the subjects who were not provided with such feedback. the results showed that 1) final velocity measured after six week training increased significantly in all groups ; however, providing kr resulted in a greater relative increase of velocity of the ball with respect to the same intervention when kr was not provided ; 2) the relative increase of velocity was larger when using a normal handball compared to the heavy ball. in the present study, these results are consistent with previous research that have shown that precise quantitative kr is generally more effective for learning than qualitative kr (bennett and simmons, 1984 ; magill and wood, 1986 ; reeve., 1990 ; salmoni., 1983). however, some researchers suggest that giving augmented feedback after every performance is neither practical nor optimal for learning because it may overload attention capacity or may make the learner dependent on kr (winstein and schmidt, 1990). therefore, to their conviction some type of relative or reduced frequency feedback may be more appropriate. (1998) studied the influence of the kr frequency on learning the complex skill of skiing slalom and they observed that the group with 100% of kr achieved higher performance than the group provided with 50% kr. moran. (2012) also indicated that tennis players could not accurately judge service speed without augmented feedback. keller. (2014) reported that the greatest long - term drop jump height increase was achieved when participants were provided with 100% of augmented feedback, compared to 50% and 0%. they also found a significant within - session effect of augmented feedback, meaning that providing augmented feedback increased drop jump height immediately as well as long - term. results of our study, therefore, confirm that kr could present a powerful tool for detecting the best trials of several similar performances, which can help the subject to direct the following actions and, consequently, gradually optimise his / hers throwing mechanics. another aspect of the discussion regarding kr concerns the time that lapses before feedback is given. (1990) formulated a hypothesis that there should be a minimum amount of time delay before kr was given ; however, they found that providing kr too soon could interfere with task intrinsic feedback and, therefore, it should be delayed. considering that in the present study, the use of a radar gun precluded the delayed display, the kr was provided to the subjects immediately after the shot and the options of providing the kr with a delay were not considered. a normal ball was used during the training period, the differences in the velocity increase were also found when the heavy ball was thrown ; however, the increase was much smaller compared to the normal ball. according to schmidt and wrisberg (2008), the programming process must include specifications such as particular muscles needed to produce the action, their precise order and level of activation, the relative timing and sequencing of the contractions and the duration of the respective contraction. although these variables were not measured, previous research had shown that some of these specifications changed when throwing a heavy ball, especially the level of muscle activation and kinematics of the major contributors to overarm throwing : elbow extension and internal rotation of the shoulder (van den tillaar and ettema, 2011). conversely, the muscles involved, sequencing of their activation and relative timing might be similar ; hence, the reason why training with a normal ball had some effect on the velocity of throwing the heavy ball, although this effect was noticeably smaller compared to the increase in velocity of a normal ball throw. perhaps this finding can support the generalised motor programme theory of motor learning, which states that a pattern of movement rather than specific movement is programmed and can, therefore, be flexible to meet some altered environmental demands (schmidt and wrisberg, 2008). the influence of feedback depends on the skill and the performer (magill, 2011). perhaps this is the cue for understanding the differences obtained in the amount of the velocity increase between males and females. nevertheless, all the subjects that volunteered to participate in the research were given the same treatment and amount of encouragement. it could be possible that the males showed more enthusiasm or / and competitiveness during the training period and, therefore, the increase in velocity was higher. this was observed in the nokr group, where differences between genders were found to be significant. it had been evident previously that males strived more for success in sport (findlay and bowker, 2009 ; gill, 1988) and that they participated in sports substantially more often than girls and women in general (deaner., 2012). however, it appeared that in the kr group, the motivation arising from the feedback (velocity displayed) stimulated women in the same way as men. the experiment was carried out within the regular faculty programme and schedule and, as a consequence, the participants were not available for a retention test to evaluate the longterm effects of the intervention. different times of the delay of kr were not investigated ; moreover, we did not monitor or evaluate precisely the kp feedback. physical education students constituted rather a diverse population, therefore comparisons to elite athletes should be made with some caution. additionally, in our study a smaller ball weighing 100 grams less than the official size 3 ball used in handball by men and male youth over the age of 16 was utilised. shooting with a lighter ball must have influenced shooting biomechanics resulting in different muscular activation and coordination of the muscles involved. the same balls were used to test the male and female participants ; therefore, some precautions should be made regarding our findings since they could be compromised by different anthropometric characteristics of the hand and arm, which might also have influenced throwing performance. on the other hand, the size of the balls used should not have influenced our findings regarding the main aim of the study which was to investigate the effects of providing or not providing feedback on the ball velocity gain. limitations of the study listed above also present some issues to be considered in future studies. the results clearly showed that providing immediate 100% frequent kr for six weeks resulted in a three to four times greater increase of the velocity of the ball at release compared to the same intervention when kr was not provided. this information should encourage athletes and coaches to use kr frequently in sports where the velocity of a throwing projectile is of paramount importance. nowadays, devices for measuring velocities of projectiles are readily available and easy to use. a shooting session (or one of the circuit conditioning stations) could be used without the necessity of presence of a goalkeeper ; such an approach would be a valuable variation from typical sessions possibly augmenting velocity gain, engagement and motivation of the players. | abstractin the present study, the effect of frequent, immediate, augmented feedback on the increase of throwing velocity was investigated. an increase of throwing velocity of a handball set shot when knowledge of results was provided or not provided during training was compared. fifty female and seventy - three male physical education students were assigned randomly to the experimental or control group. all participants performed two series of ten set shots with maximal effort twice a week for six weeks. the experimental group received information regarding throwing velocity measured by a radar gun immediately after every shot, whereas the control group did not receive any feedback. measurements of maximal throwing velocity of an ordinary handball and a heavy ball were performed, before and after the training period and compared. participants who received feedback on results attained almost a four times greater relative increase of the velocity of the normal ball (size 2) as compared to the same intervention when feedback was not provided (8.1 3.6 vs. 2.7 2.9%). the velocity increases were smaller, but still significant between the groups for throws using the heavy ball (5.1 4.2 and 2.5 5.8 for the experimental and control group, respectively). apart from the experimental group throwing the normal ball, no differences in velocity change for gender were obtained. the results confirmed that training oriented towards an increase in throwing velocity became significantly more effective when frequent knowledge of results was provided. |
cellular mediated immune responses to brucellosis drive a broad range of manifestations of the disease that vary from subclinical infection (more common with brucella abortus) to undulant fever, to focal pyogenic infection, to chronic fatigue syndrome - like illness (yingst and hoover, 2003 ; yang., 2005). however, the molecular mechanisms that determine the variable manifestations of brucella infection remain to be elucidated. information on acquired immunity to human brucellosis has been accumulated through observational studies of naturally infected hosts (cattle, goats), experimental models (mice), and observations of human disease. three predominant brucella species are seen frequently in human infections : b. melitensis, b. abortus, and b. suis. of these three species, b. melitensis infections are most commonly seen in humans and seem to be the most pathogenic (pappas., 2005). in the united states, domesticated cattle, which are potential reservoirs for the organism, are vaccinated against b. abortus (rb51 or s19) ; elsewhere (in the middle east and latin america), goats and sheep may be vaccinated with rev-1, an attenuated strain of b. melitensis. currently available veterinary vaccines are comprised of live - attenuated organisms but are unacceptable in humans because they cause clinical disease (kinikli., 2005 ; durward., brucellosis can occur in several forms : acute / subacute (associated with positive blood cultures, high titer agglutination serologies), focal (blood culture negative, serologically variable, and positive local site culture), and chronic (blood culture negative, serologically variable but often negative, sometimes bone marrow culture positive, often diagnosed clinically in response to therapy ; jimenez de bagues., 2005). however, it is currently unknown what causes some individuals to have the acute form and some to progress and develop chronic disease. as hypothesized in this review and elsewhere, it seems probable that immunogenetics of cell - mediated immune responses to brucella protein antigens determines clinical manifestations and outcome. sometimes, despite treatment for brucellosis, there are still some bacterial foci that may persist despite antibiotics or brucella dnaemia may persist, presumably because of deficient t cell activation of infected macrophage / dendritic cells (dcs ; vrioni., 2008). additional mechanisms may also include altered innate immune responses determined by the pathogenetic properties of the bacteria themselves. there have been studies demonstrating that brucella epitopes can include those recognized by peptide - specific cd8 + t cells associated with protective responses at least in a mouse model (durward., while there has been an experimental interferon - gamma release assay developed for bovine brucellosis, there has not been one developed for human infection to differentiate immune responses associated with different forms of brucellosis, to definitely diagnose previous exposure, or identify targets of protective immunity. understanding the precise molecular targets (protein, peptidic epitope) of t cell - mediated immune responses has the promise to translate to further investigations into new vaccine and diagnostic brucella t cell epitopes, and their role in specific t cell - mediated responses. infections may occur after ingestion or inhalation of brucellae that penetrate mucosal surface such as the upper respiratory or gastrointestinal mucosa via lymphoid cells. once the bacteria are phagocytosed by macrophages, dcs (billard., 2007), and other antigen presenting cells (apcs), approximately 4050% of the bacteria resist digestion within these cells. b. abortus and b. melitensis that have smooth lps (with intact o - antigen chain) are able to survive better intracellularly than b. canis that has rough lps (lacks o - antigen side chain ; vrioni., 2008). lps is also composed of longer carbon chains (c28) as apposed to the usual 1216 carbons in the lps from enterobacteriaceae. in addition, brucella spp. produce proteins (e.g., vi antigen), which create a capsule around the lps, therefore limiting it to have contact with tlr4 receptors (lapaque. in addition, the domain for the flagellin protein in brucella spp. does not stimulate tlr5 receptors, and is another way for the bacteria to evade the immune system during early infection (tsolis., 2008). to others in the same family to evade detection by the immune system during the infection and possibly allowing the bacteria to persist in the reticuloendothelial system (tsolis., 2008 ; barquero - calvo., produce interleukin-1 (il-1), interleukin-6 (il-6), tumor necrosis factor alpha (tnf-), and gamma - interferon (ifn-) initiating innate immune responses (including natural killer cells) that may limit the initial spread of organisms. infected apc in which organisms residing within unactivated phagolysosomes are likely to present some subset of peptidic brucella antigens (hitherto unknown) to cd4 + and cd8 + cells, and thus inducing a th1 response associated with ifn- release. the functional consequences of antigen - specific ifn- release is unclear but does not lead to elimination of organisms during active, symptomatic infection and likely results in clinical symptomatology (i.e., fever, sweating, weight loss). clonal t cell expansion is initiated with production of interleukin-2 (il-2) and interleukin-12 (il-12), which initiates a cd8 + cytotoxic response on brucella - infected cells. infected macrophages produce il-12 and ifn- which regulate antigen presentation and may contribute to the limitation of intracellular bacterial replication through unknown mechanisms (akbulut., 2005). also modify the initial immune response once phagocytosed into apcs. to survive within apcs, brucella use certain gene products to subvert certain phagocyte intracellular processes, particularly phagosome lysosome fusion which would be associated with bacterial killing. one way that the organism survives within apcs is through brucella - containing vacuoles (bcvs), which the organism secretes sar1, a critical protein which allows the organism to replicate within these vacuoles (celli., 2005). cd64, also known as fcri, is a macrophage - expressed gene whose expression is down - regulated in b. melitensis infections (lapaque., 2005), reflecting the reduction of antigen processing in brucella - infected apcs and possibly inhibiting the killing of infected cells initiated by cd4 + and cd8 + t cells. this mechanism may be important for decreasing superoxide and reactive nitrogen compounds as another pathogen - associated localized immunosuppression. other gene products such as cystatin c, serpina3c, and gas2 (natural peptidase inhibitors) appear to alter macrophage chemotaxis, cell migration, and proliferation, which may further allow brucella to avoid immune surveillance and lead to enhanced multiplication (lapaque., 2005). decreased transcription of cyp4a10 is thought to be associated with the reduction of oxidative stress that creates an environment conducive to bacterial proliferation. prkca is another macrophage - associated gene potentially modulated during brucella infection, who functions includes regulating phagosome prkca expression is dramatically reduced in brucella - infected macrophages, and interestingly, has been shown to be regulated by other intracellular pathogens such as salmonella, leishmania, and legionella. all these mechanisms contribute to allowing brucellae to survive within the intracellular environment, and evade not only the innate immunity, but cd4 + - and cd8 + -mediated host cell killing (covert., alteration of t cell function may be key to explaining the clinico - pathological manifestations of chronic or relapsing brucellosis. specifically, a decreased th1 cytokine response by apcs (with decreased activation of cytotoxic t cells via ifn-, il-12, and possibly il-17 (pasquevich., 2010), and toward a th2 response (which decreases phagocyte function and reduced cytotoxic response via il-4, il-5, and il-10 ; jimenez de bagues., 2005 these infected cells fail to produce ifn-, and decrease clonal expansion of brucella - specific cd4 + t cells. subsequently, brucella - specific cd8 + t cells would not initiate destruction of infected cells by perforin and granzyme injection (cell lysis) or through stimulation of fas ligand (cellular apoptosis ; yingst and hoover, 2003 ; skendros., 2008). based on these considerations, the unusual and diverse manifestations of chronic and relapsing brucellosis could be related to several potential immunopathogenic mechanisms : an ineffective cd4 + effector response, a down - regulated cd8 + t cell response or a continued, established th2 response, each of which could result in an incomplete resolution of the infection (giambartolomei., 2004). in comparing cytokine responses in patients with acute and chronic brucellosis, before and after treatment, (2005) found that in chronic brucellosis, both the absolute number of cd4 + cells and the quantitative secretion of ifn- were reduced. (2006) confirmed these results, and further demonstrated that il-13 is increased in chronic brucellosis, further demonstrating the initial association of th1 cytokine responses with acute brucellosis illness, which gradually becomes th2 cytokine dominant. these observations suggest that once acute brucellosis has resolved, that both the number of cd4 + cells and cd4 + functional response is reduced. whether antigen - specific memory cd4 + cells are produced during acute or chronic brucellosis remains to be determined (moreno - lafont. recently elfaki and al - hokail observed that mice deficient in 2-microglobulin produced an impaired cd8 + response associated with increased brucella bacterial load and decreased clearance (moreno - lafont., 2002 ; elfaki and al - hokail, 2009). extrapolating such results to human disease, chronic or relapsing brucellosis might be explained in that cd69 expression on both cd4 + t cells and cd8 + t cells are significantly decreased in this patient subset. further experimentation indicates that there is an unknown individual effect on the immune system after an episode of acute brucellosis, and therefore one develop chronic and relapsing brucellosis. epitopes are the molecular subset of any macromolecule recognized by antibodies, b cells, t cells, and nk cells. t cells recognize peptides generally of 820 amino acids bound to mhc molecules (hla class i associated with cd8 + t cells ; hla class ii associated with cd4 + t cells) presented on the surface of a b cell, macrophage or dendritic cell (yang., 2005). non - classical mhc molecules also present non - peptidic epitopes such as glycolipids to non - restricted lymphocytes, particularly nk cells. there are only a few studies in the literature that have demonstrated either a humoral or cellular response to brucella epitopes. the epitopes of greatest interest to date include bp26 (a periplasmic protein), trigger factor (a chaperone protein ; yang., 2005), and the outer membrane lipoproteins such as omp 10, 16, and 19 (tibor., 1999 interestingly, these antigens, especially the outer membrane lipoproteins, appear to be potent in inducing cytokine responses from memory t cells. in contrast, brucella lps and brucella dna do not elicit intense immune responses (giambartolomei. previous vaccine studies in mice have used some of these outer membrane epitopes (pasquevich., 2010) as well as certain enzymes such as cu yet, these were not demonstrated to offer protection for humans or t cell cytokine release assays to prove their efficacy. these antigens have been identified in both b. melitensis and b. abortus. bp26 and trigger factor have been shown to be recognized by the immune system (yang., 2005), yet with a reduced activation in patients with chronic / relapsing brucellosis. (2010, 2011) reported comprehensive systems biology analyses of human antibody responses in acute b. melitensis brucellosis in peru. a collection of sera isolated from individuals from one of the following groups was used to probe large scale b. melitensis protein microarrays including a 1400 proteins array and 3300 proteins array representing nearly the entire encoded proteome : brucella blood culture positive, blood culture negative with positive rose bengal, blood culture negative with negative rose bengal, and two nave groups (from both american and peruvian individuals.) sets of proteins that differentiated acutely infected from uninfected patient groups were identified that were recognized by patient igg responses. these brucella protein epitopes were then further separated by the patient groups which recognized them, as some were only recognized by culture positive or culture negative patients / rose bengal positive patients compared to nave patients (table 1), others cross react in both the culture positive patients and the nave patients (table 2) and some antigens produced a positive response in the culture positive group but not the culture negative group / rose bengal positive group (table 3). this broad spectrum of antibody responses demonstrates the differences between in the manner that these groups respond to brucella protein epitopes. interestingly, some of the epitopes mentioned previously (i.e., bp 26) produced a strong antibody response by peruvian brucellosis patients, who were culture positive or culture negative patients / rose bengal positive patients. these antibody responses could reflect the substantial differences, which these protein epitopes have on the immune system activation, and a large number of antigens recognized by brucellosis patients were identified which have yet to be studied or have limited understanding of their function. cross - reactive antigens for culture+ or culture-/rose bengal+ vs. peruvian nave (negative controls).. there could be a potential difference in the antibody responses compared to cytokine release assays for these specific epitopes, which produced antibody responses. based on unpublished data, bp26, which gave a strong antibody response, does not give a strong th1 response ; virb8 does generate a strong th1 response, yet does not give a particularly strong antibody response. the use of an epitope database has previously been used for mycobacteria tuberculosis, and other infections by estimating the immune responses to epitopes associated with a certain organism (blythe., 2007). based on the fact that antibody production is not protective in chronic brucellosis, we must conclude that t cell assays would be a more appropriate method to pursue with regards to not only diagnostic purposes, but for development of a recombinant protein vaccine as well. brucella spp. are important intracellular human and animal pathogens associated with fascinating mechanisms of immune modulation and subversion of apcs as an intrinsic mechanism of the diseases that they cause. new systems biology analyses of antigens recognized by human immune responses in brucellosis have identified large numbers of protein antigens with potential for understanding mechanisms of pathogenesis and immune evasion and may point the way toward novel vaccine and diagnostic approaches. these approaches have generalized applicability to the analysis of t cell responses associated chronic bacterial, fungal and parasitic infections other than brucella. 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. | brucella spp., are gram negative bacteria that cause disease by growing within monocyte / macrophage lineage cells. clinical manifestations of brucellosis are immune mediated, not due to bacterial virulence factors. acquired immunity to brucellosis has been studied through observations of naturally infected hosts (cattle, goats), mouse models (mice), and human infection. even though brucella spp. are known for producing mechanisms that evade the immune system, cell - mediated immune responses drive the clinical manifestations of human disease after exposure to brucella species, as high antibody responses are not associated with protective immunity. the precise mechanisms by which cell - mediated immune responses confer protection or lead to disease manifestations remain undefined. descriptive studies of immune responses in human brucellosis show that th1 (interferon--producing t cells) are associated with dominant immune responses, findings consistent with animal studies. whether these t cell responses are protective, or determine the different clinical responses associated with brucellosis is unknown, especially with regard to undulant fever manifestations, relapsing disease, or are associated with responses to distinct sets of brucella spp. antigens are unknown. few data regarding t cell responses in terms of specific recognition of brucella spp. protein antigens and peptidic epitopes, either by cd4 + or cd8 + t cells, have been identified in human brucellosis patients. additionally because current attenuated brucella vaccines used in animals cause human disease, there is a true need for a recombinant protein subunit vaccine for human brucellosis, as well as for improved diagnostics in terms of prognosis and identification of unusual forms of brucellosis. this review will focus on current understandings of antigen - specific immune responses induced brucella peptidic epitopes that has promise for yielding new insights into vaccine and diagnostics development, and for understanding pathogenetic mechanisms of human brucellosis. |
the agency for toxic substances and disease registry (atsdr) is a public health agency with responsibility for assessing the public health implications associated with uncontrolled releases of hazardous substances into the environment. the biological effects of low - level exposures are a primary concern in these assessments. one of the tools used by the agency for this purpose is the risk assessment paradigm originally outlined and described by the national academy of science in 1983. because of its design and inherent concepts, risk assessment has been variously employed by a number of environmental and public health agencies and programs as a means to organize information, as a decision support tool, and as a working hypothesis for biologically based inference and extrapolation. risk assessment has also been the subject of significant critical review. the atsdr recognizes the utility of both the qualitative and quantitative conclusions provided by traditional risk assessment, but the agency uses such estimates only in the broader context of professional judgment, internal and external peer review, and extensive public review and comment. this multifaceted approach is consistent with the council on environmental quality 's description and use of risk analysis as an organizing construct based on sound biomedical and other scientific judgment in concert with risk assessment to define plausible exposure ranges of concern rather than a single numerical estimate that may convey an artificial sense of precision. in this approach biomedical opinion, host factors, mechanistic interpretation, molecular epidemiology, and actual exposure conditions are all critically important in evaluating the significance of environmental exposure to hazardous substances. as such, the atsdr risk analysis approach is a multidimensional endeavor encompassing not only the components of risk assessment but also the principles of biomedical judgment, risk management, and risk communication. within this framework of risk analysis, the atsdr may rely on one or more of a number of interrelated principles and approaches to screen, organize information, set priorities, make decisions, and define future research needs and directions.imagesfigure 1 |
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several studies demonstrate that rats (rattus novergicus) infected with protozoan parasite toxoplasma gondii exhibit lesser fear to cat odors. this is thought to increase transmission of the parasite to its definitive hosts, i.e. cats. this is an example of extended phenotype where a gene of an organism allegedly creates a phenotype in another organism. we examined a possible proximate mechanism for this phenotype, describing an epigenetic change in arginine vasopressin gene in medial amygdala of male rats. exogenously mimicking medial amygdala dna hypomethylation resulted in reduction of fear to cat odors in uninfected animals, thus suggesting sufficiency. systemic blockade of infection - induced dna hypomethylation countermanded infection - induced behavioral change, thus suggesting necessity. this leads us to propose an epigenetic basis for this extended phenotype. |
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retinitis pigmentosa (rp) comprises a heterogeneous group of inherited retinal disorders that primarily affects the rod and cone photoreceptors and provokes a progressive loss of them beginning in the periphery and progressing toward the central retina.13 histopathologic studies on patients with rp have shown earlier anatomic changes, such as shortening and distortion of the outer segments of rods and cones.4,5 currently there is no treatment that can recover lost vision or halt disease progression. however, novel treatments including gene therapy,6,7 tissue transplantation,8,9 or retinal prosthesis1013 offer optimism on the subject. this makes better evaluation of the functional and structural changes of the macula, which is better preserved until the late stages of the disease. optical coherence tomography (oct) is a noninvasive technique that provides information about the morphology of the retina, and especially of the macular area in vivo. some studies support the idea that oct determines structural changes in the macula that are correlated with subjective visual function, including visual acuity (va) and visual threshold in patients with rp. additionally, multifocal electroretinogram (mferg) evaluation can be useful in monitoring macular function in rp, and mferg responses have been shown to be associated with the subjective visual field size.14,15 however, to date, there are relatively few reports concerning the combined use of oct and mferg for the investigation of the correlation between retinal morphology and function of the macula in patients with rp.16,17 the purpose of our study was to investigate the usefulness of mferg in the detection of early changes of macular function, and to determine whether a significant correlation exists between the amplitude of mferg and the retinal thickness in the central area of the macula in patients with rp. the study was based on 66 eyes of 33 patients (17 males and 16 females) with rp, examined in the first department of ophthalmology, university of athens, greece. also, a group of 20 age- and sex - matched volunteers who were ophthalmologically normal, without ocular or systemic diseases, served as control subjects. diagnosis was based on family history, fundus examination, and international society for clinical electrophysiology of vision standard full - field electroretinograms (ergs). the exclusion criteria were atypical rp - like central rp, sector rp, or unilateral rp, cystoid macular edema, cataract, or glaucoma, which may affect mferg and oct recordings. the inheritance pattern in 17 cases was autosomal dominant, and in seven cases was autosomal recessive, while in nine cases, rp was characterized as simplex, as it was difficult to determine its inheritance. interestingly, two patients presented with usher syndrome ii, which is characterized by congenital bilateral sensorineural hearing loss that is mild to moderate in the low frequencies and severe to profound in the higher frequencies, intact vestibular responses, and rp.18 a complete ophthalmic examination, including va measurement by means of snellen charts, oct recordings, and mferg recordings were performed. the patients had functional visual fields of at least 10 using the humphrey field analyzer 24 - 2 threshold test (carl zeiss meditec ag, jena, germany). informed consent was obtained from each patient after they were provided with an explanation of the nature of the study. oct examination was performed with the oct model 3000 (stratus oct ; carl zeiss meditec ag). the retinal mapping software was used, calculating the average retinal thickness of the central ring. all eyes were scanned in a radial spoke pattern centered on the foveola with a scan length of 6 mm. the subjects were asked to gaze at the fixation light within the machine, and the foveolar fixation was confirmed by observing the retina through the infrared monitoring camera. the retinal thickness was calculated as the distance between the two boundaries along each a - scan using the attached automatic boundary detection software. for the recording of the mferg, the veris iii (visual evoked response imaging system ; tomey corporation, nagoya, japan) was used. the stimulus matrix consisted of 61 pattern scaled segments displayed on a cathode ray tube color monitor (sony corporation, tokyo, japan) driven at a frame of 72 hz. the luminance of the stimulus element in the light state was 100 cd / m in the lighted state and < room lights should be on and produce illumination close to that of the stimulus screen. each hexagon was independently alternated between black and white at a rate of 72 hz. the pattern seems to flicker randomly, but each element follows a fixed predetermined sequence (presently an the bandwidth of the amplifier was 10300 hz and the amplification was 10.000. for signal acquisition, a bipolar contact lens was used in which the active and reference electrodes were incorporated in the contact lens. the fellow eye was closed, and the duration of the data acquisition was 8 minutes divided into eight sessions of 60 seconds. the recording procedure was repeated if there were spurious potentials from eye blinks or if ocular movements were recorded. the response density (amplitude per unit retinal area, nv / deg) of each local response was estimated as the dot product between the normalized response template and each local response. the normal ranges for these amplitudes were defined by calculation of the median and the 95% confidence intervals (cis) in both eyes of 20 normal volunteers (group b). the mferg stimuli location and anatomic areas corresponded roughly as follows : ring 1 to the fovea (02), ring 2 to the parafovea (27), ring 3 to the perifovea (713), ring 4 to the near periphery (322), and ring 5 to the central part of the middle periphery (2230.5). the amplitude of each group was scaled to reflect the angular size of the stimulus hexagon, which produces the response. these averages give a more accurate view of the relative response densities of each group. the retinal response density (rrd) decreases with eccentricity, although there is no further decrease from ring 4 to ring 5. the protocol followed the recommended guidelines of the international society of electrophysiology of vision for basic mferg.19 pearson s coefficient was used to evaluate correlation. several univariate linear regression models were performed to explore the association of va with study parameters ; va was regarded as the outcome variable, whereas exploratory variables were defined as follows : foveal retinal thickness (model 1) ; mferg amplitude in ring 1 (model 2) ; mferg amplitude in ring 2 (model 3) ; latency in ring 1 (model 4) ; and latency in ring 2 (model 5). further on, multivariate linear regression analysis was used in order to study the simultaneous insertion of foveal retinal thickness, mferg amplitude, and latency. significance was accepted at the p < 0.05 level. the sas statistical package (version 9.1 ; sas institute inc, cary, nc, usa) oct examination was performed with the oct model 3000 (stratus oct ; carl zeiss meditec ag). the retinal mapping software was used, calculating the average retinal thickness of the central ring. all eyes were scanned in a radial spoke pattern centered on the foveola with a scan length of 6 mm. the subjects were asked to gaze at the fixation light within the machine, and the foveolar fixation was confirmed by observing the retina through the infrared monitoring camera. the retinal thickness was calculated as the distance between the two boundaries along each a - scan using the attached automatic boundary detection software. for the recording of the mferg, the veris iii (visual evoked response imaging system ; tomey corporation, nagoya, japan) was used. the stimulus matrix consisted of 61 pattern scaled segments displayed on a cathode ray tube color monitor (sony corporation, tokyo, japan) driven at a frame of 72 hz. the luminance of the stimulus element in the light state was 100 cd / m in the lighted state and < room lights should be on and produce illumination close to that of the stimulus screen. each hexagon was independently alternated between black and white at a rate of 72 hz. the pattern seems to flicker randomly, but each element follows a fixed predetermined sequence (presently an the bandwidth of the amplifier was 10300 hz and the amplification was 10.000. for signal acquisition, a bipolar contact lens was used in which the active and reference electrodes were incorporated in the contact lens. the fellow eye was closed, and the duration of the data acquisition was 8 minutes divided into eight sessions of 60 seconds. the recording procedure was repeated if there were spurious potentials from eye blinks or if ocular movements were recorded. the response density (amplitude per unit retinal area, nv / deg) of each local response was estimated as the dot product between the normalized response template and each local response. the normal ranges for these amplitudes were defined by calculation of the median and the 95% confidence intervals (cis) in both eyes of 20 normal volunteers (group b). the mferg stimuli location and anatomic areas corresponded roughly as follows : ring 1 to the fovea (02), ring 2 to the parafovea (27), ring 3 to the perifovea (713), ring 4 to the near periphery (322), and ring 5 to the central part of the middle periphery (2230.5). the amplitude of each group was scaled to reflect the angular size of the stimulus hexagon, which produces the response. these averages give a more accurate view of the relative response densities of each group. the retinal response density (rrd) decreases with eccentricity, although there is no further decrease from ring 4 to ring 5. the protocol followed the recommended guidelines of the international society of electrophysiology of vision for basic mferg.19 several univariate linear regression models were performed to explore the association of va with study parameters ; va was regarded as the outcome variable, whereas exploratory variables were defined as follows : foveal retinal thickness (model 1) ; mferg amplitude in ring 1 (model 2) ; mferg amplitude in ring 2 (model 3) ; latency in ring 1 (model 4) ; and latency in ring 2 (model 5). further on, multivariate linear regression analysis was used in order to study the simultaneous insertion of foveal retinal thickness, mferg amplitude, and latency. significance was accepted at the p < 0.05 level. the sas statistical package (version 9.1 ; sas institute inc, cary, nc, usa) the mean age of our study sample was 45.94 11.68 years, ranging from 25 years to 69 years. table 1 shows the distribution of va, foveal retinal thickness, mferg amplitude, and latency values. the va of 22 out of 66 eyes (33.3%) expressed in a decimal scale number was 1.0. the mean rrd of mferg was calculated at 91.52 49.82 nv / deg and at 33.67 25.95 nv / deg in ring 1 and ring 2, respectively. the mean latency in ring 1 was 44.77 7.55 ms and in ring 2 was 44.05 8.49 ms. correlation analysis was performed between va, foveal retinal thickness, mferg amplitude, and latency measurements (table 2). foveal retinal thickness, mferg amplitude in ring 1, and latency in ring 1 and ring 2 were independently and positively associated with va (p = 0.002 ; p < 0.0001 ; p = 0.029 ; and p = 0.002, respectively), whereas there was no evidence for a correlation between va and mferg amplitude in ring 2. the evidence of a correlation between foveal retinal thickness and mferg amplitude in ring 1 was of borderline significance (p = 0.047). subsequently, the data were modeled through linear regression analyses using va as the dependent variable (table 3). according to model 1, a 10 m increase in foveal retinal thickness increases the mean va by 0.039 (coefficient b = 0.039 ; 95% ci : 0.016, 0.062 ; p = 0.002), whereas according to model 2, a 10 nv / deg increase in mferg amplitude increases the mean va by 0.042 (b = 0.042 ; 95% ci : 0.026, 0.059 ; p when foveal retinal thickness and mferg amplitude in ring 1 were inserted simultaneously into the same model, both were found to be strongly associated with va. regarding mferg amplitude in ring 2 (model 3) there was no evidence for an association with va (p = 0.484). there was evidence for an inverse association of va with latency in ring 1 (model 4) and ring 2 (model 5). specifically, a 10 ms increase of latency in ring 1 decreases the mean va by 0.138 (b = 0.138 ; 95% ci : 0.261, 0.015 ; p = 0.029), and a 10 ms increase of latency (ring 2) decreases the mean va by 0.168 (b = 0.168 ; 95% ci : 0.274, 0.063 ; p = 0.002). in accordance with the univariate findings, when foveal retinal thickness, mferg amplitude, and latency in ring 1 were inserted simultaneously into the same model, all the three variables remained significantly associated with va (p = 0.016 ; p < 0.0001 ; and p = 0.031, respectively). further investigation into the spread of individual values revealed that most of the foveal retinal thickness and mferg amplitude in ring 1 measurements of the study participants confirmed that both techniques are associated with the level of va. specifically, 15 eyes showed an increased retinal thickness value, whereas the respective va was relatively low. conversely, in seven eyes, despite the decreased foveal retinal thickness, the respective va was high. regarding mferg amplitude in ring 1, five eyes showed an increased mferg value when the respective va was relatively low. conversely, in ten eyes, despite the decreased mferg amplitude, the respective va was high. graphically, the relationships between va and foveal retinal thickness, and va and mferg amplitude in ring 1 are depicted in figures 1 and 2, respectively. our results demonstrated that the rrd of mferg in ring 1 and foveal thickness were associated with va, whereas there was no evidence of a correlation between va and mferg in ring 2. furthermore, there was evidence for an inverse association of va with latency in ring 1. indeed, a 10 ms increase of latency in ring 1 decreases the mean va by 0.138, and a 10 ms decrease of latency in ring 2 increases the mean va by 0.168. this is in contrast with previous investigations, which have shown that patients with rp can have preserved mferg timing in the central retina despite the decrease of mferg amplitudes or the reduced temporal contrast sensitivity function.20,21 another interesting finding of our study is that there were patients lacking a central mferg response despite good va, in line with gerth,22 who also found that some patients miss a central mferg response despite a va of 0.4 (decimal scale) or better and a normal humphrey visual field foveal threshold. seiple showed a similar example of a patient with rp, a va of 20/25, and preserved mferg responses in the peripheral area without a central response.23 a possible explanation in these cases could be that the number of intact photoreceptors may be sufficient to resolve a small visual angle required for good va.22 concerning the oct findings, our results showed that foveal retinal thickness generally is positively associated with va, and the evidence of a correlation between mferg and oct was of borderline significance. specifically, 15 eyes showed a normal retinal thickness of the fovea, whereas the respective va was relatively low. in these eyes, conversely in seven eyes, despite the decreased foveal thickness, the respective va was normal or quasi - normal. in three of these eyes, these findings raise some questions about the correlation between the foveal retinal thickness and va at least in some cases. it must be mentioned that sugita postulated that there are some patients with rp whose macular oct images are relatively well preserved, but their electrophysiological functions are severely reduced. the real reason for this discrepancy was not determined. according to the authors, there are two possibilities. first, these patients may have very subtle structural changes which third - generation stratus oct (carl zeiss meditec ag) can not determine, or the functional abnormality may precede structural changes in some patients with rp. similar findings were described in some patients with leber congenital amaurosis and very low va, where the cone photoreceptors and inner retinal architecture in the central retina was retained.24,25 if this second possibility is correct, only the combined assessment of macular structure by oct and macular function by psychophysics or erg can provide important information on the macula of patients with rp.15 on the contrary, the use of only oct or mferg for the evaluation of the macula may lead to unreliable results and erroneous decisions for the feasibility of upcoming treatments in the future treatment of rp.26 a potential limitation of our study pertains to the fact that the duration of the disease was not included in the analysis. in addition, spectral domain oct, which is more accurate, was not able to be performed. our study suggested that rrd of mferg in ring 1 was associated with va, while no association was remarked in ring 2. additionally, there was a significant association between the foveal thickness in oct and va. therefore, the combined use of oct with mferg appears to be more appropriate for the estimation of macular function. nevertheless, further studies are important for improving the combination of structural imaging and electrophysiological investigations of the macula for a better estimation of its activity. | introductionour purpose was to study the correlation between the macular morphology and function in eyes with retinitis pigmentosa (rp).methodssixty - six eyes from 33 patients with rp and with different visual acuity (va) were studied using optical coherence tomography (oct) and multifocal electroretinogram (mferg). correlation analysis was performed between va, macular thickness, mferg amplitude, and latency.resultsretinal thickness, retinal response density, and latency of the mferg in the foveal area were independently and positively associated with va (p = 0.002 ; p < 0.0001 ; p = 0.029 ; and p = 0.002, respectively), whereas there was no evidence for a correlation between va and the amplitude of mferg in the parafoveal area. evidence of a correlation between the oct and the mferg evaluation was of borderline significance (p = 0.047). also, there was evidence for an inverse association of va with latency in ring 1 and ring 2 (b = 0.138 ; 95% confidence interval : 0.261, 0.015 ; p = 0.029). in accordance with the univariate findings, when foveal retinal thickness, mferg amplitude, and latency in ring 1 were inserted simultaneously in the same model, all the three variables remained significantly associated with va (p = 0.016 ; p < 0.0001 ; and p = 0.031, respectively). nevertheless, some individual values deviated from the expected range. more specifically, 15 eyes showed a normal retinal thickness, whereas the respective va was relatively low and the mferg values were abnormal. conversely, in seven eyes, despite the low retinal thickness, the respective va was high.conclusionthe combined use of oct with mferg appears to be more appropriate for the estimation of macular function. |
empowerment as a primary outcome has been considered in most interventions related to women s health, and focusing on other issues has caused the clinical importance of the incensement of empowerment and its subsequent health benefits to be neglected. empowerment of women was designed to develop skills and resources needed to effectively cope with future stresses and traumas. given the fact that stress and anxiety during pregnancy cause mental health problems for pregnant women, factors affecting their psychological state should be recognized so that mothers are provided with the means to achieve health and psychological empowerment. prenatal empowerment improves people s ability to help themselves, especially in times of difficulty making it a useful quality, especially during parenting. research has shown that more capable mothers experienced better conditions during prenatal care, and coped better during delivery and in caring for and bringing up their children ; ultimately their empowerment strengthens their families foundations. the international conference on population & development (icpd) identifies the empowerment of women as an essential element in achieving results in reproductive health in the program of action conference ; empowerment is credited with improving the health status of women and is deemed essential for achieving sustainable social development. the concept of empowerment is used in a wide range of contexts and levels. therefore, empowerment is a psychological state that allows an individual to engage in effective social communication and subsequently leads them to take responsibility for their own health. ultimately, increased empowerment can play an important role in the improvement of prenatal care and maternal health. however, the main factors affecting psychological empowerment of mothers are a matter of debate. several studies have been conducted on women s empowerment and its contributing factors. in such a study in ethiopia, mother s employment was the predictor of women s empowerment and a study in uganda showed that the maternal health status of mothers is affected by violence against women and early marriage as well as the mother s income. however, there is dearth of research on psychological empowerment of pregnant women (pepw) in iranian context. due to the fact that empowerment is a context based issue therefore, given the importance of maternal empowerment, the current study was conducted with the aim of studying direct and indirect components of pepw with the use of path analysis. in this model - testing study, weused spritzer s psychological empowerment scale based on the assumption framework of conger and kanungo and thomas and velthouse. this scale consists of 12 items in seven - point likertscale (1=quite disagree to 7=quite agree) with four sub - scales (meaning, impact, self determination and competence) and total score is between 12 to 84. in golparvar s study, the spritzer s questionnaire was translated to persian and exploratory factor analysis was performed, with varimax rotation method. kaiser normalization was equal to.86, and bartlett s test of sphericity was equal to 2286/18 (p1 and factor analysis obtained total variance explained equal to 77.85% and cronbach s alpha for four subscales was 0.79 - 0.90. in this study, we calculated cronbach s alpha 90.9% for this scale. although spritzer s scale is not specific to pepw, many researchers have used this questionnaire. this tool is the most reliable scale for psychological empowerment, and thus it was used in the present study. marital relation satisfaction was measured based on fundamental items of locke - wallace marital adjustment. the mothers were asked about the level of satisfaction in marriage, the degree of expressing love to their partner, and mutual understanding in the marriage on a four - point scale ranging from 1 to 4 (4=excellent ; 3=very good ; 2=good ; 1=bad) with total score 3 - 12. violence was classified in four ways (physical, verbal, behavioral and sexual) and each was rated between 0and4 (0=never ; 1=seldom ; 2=occasionally ; 3=often, 4=constant) and sum of this score was considered as the score of violence with a minimum of 0 and a maximum of 16. in this model - testing study, we did not have any model for pepw.as such, it was necessary to go through five steps of modeling process. for investigating markers of pepw, these markers were first calculated by linear regression and the two following parts were done : part 1 : investigating markers of pepw model in this study, multiple regressions have been used to predict empowerment, so that with the use of indicator variables, the empowerment of mothers could be modeled. for regression, ensuring normal distribution of variables is important and therefore it is necessary to examine bivariate relationships in order to assure their linearity. however, if the relationship is linear and the dependent variable for each independent variable is normally distributed, then distribution of residuals should be almost normal ; then it can be examined through a regression - standardized residual histogram ; meanwhile, the plotted scores in the normal probability plot are placed close to a line. also, for examining the independent residuals, durbin - watson (dw) statistics have been used : these should be from 1.5 to 2.5. moreover, to check whether the residuals are linear uncorrelated, collinerality test was used. if statistics were vif0.1, those conditions are established. before doing linear regression, the correlation between predictor variables and psychological empowerment was measured using a correlation coefficient matrix. then, variables including mother s age, marriage age, length of marital relationship, employment status of mothers, participation in prenatal education classes, marital satisfaction score, violence score, ownership score, high literacy, financial independence, living status and spiritual support score, as determining factors in empowerment, are imported to the linear regression in backward model. the results of regression in table 1 showed that psychological empowerment levels of mothers are predictable, taking into account individual factors such as age of marriage and employment status as well as psychological factors such as the experience of violence by a spouse and marital satisfaction (r square=0.609). demographic characteristics of the survey sample part 2 : specification of conceptual model what is certain is that employment is an important factor affecting empowerment, as confirmed by many researchers such gholipour. therefore, in this conceptual model, employment is considered as an exogenous variable affecting other variables in the model. women with high economic empowerment are more noticed and better understood in the family and experienced less violence at the hands of their spouses. therefore, in this conceptual model, we bring violence under the influence of the mother s employment. on the other hand, domestic violence, as a measure of women s empowerment has a negative correlation ; the higher the level of empowerment, the less violence is experienced by the woman. age of marriage is also considered an important element of women s empowerment, one that is affected by the mother s employment ; and with the increase of women s employment rate, the age of marriage rises. it is also affected by domestic violence ; those who are married at a younger age experienced more violence from their spouses. as can be seen in figure 1 the conceptual model of pepw is formed as a recursive model, because there is a one - way causal flow and all routes between variables are one way, as assumed in path analysis.. conceptual model of pepw based on the nunally and bernstein method, path model uses at least 30 persons per independent variable ; in the model above, there is a dependent variable (spitzer s psychological empowerment) and four independent variables (violence score, mothers employment, marriage age and marital satisfaction score) ; therefore, a sample of at least 120 is required. pregnant women were selected from 10 urban medical centers and clinics as primary centers via two stage random clustering method with design effect equal to 1.5. thus we needed 180 participants (120 1.5=180). based on the importance of the maximum variation in the samples, we used private and public institutions. after determining the exact number of existing centers and bases, two centers from the east, south, north, west and center of gorgan - for a total of 10 centers - were randomly selected. from each center and based on the number of low - risk nulliparous pregnant, visiting women who were literate, and eligible were randomly selected. based on the letters list, 180 nulliparous pregnant mothers were selected. after the exclusion of 20 participants (11.11 %) due to lack of interest in participation, response rate was in normal distribution clusters. eventually, 160 pregnant women participated in 25, march to 25, june 2015. all nulliparous pregnant women who were urban residents and were able to read and write were entered into the research ; the high - risk pregnant women and mothers who were not willing to participate were excluded from the study. in the next phase, amos 18 software (ibm, armonk, ny, usa) was used for identification of the model. in this model the independent variable is not affected by other variables, and the dependent psychological empowerment variable is an endogenous variable. also, marital satisfaction, violence and age of marriage are endogenous variables because they are influenced by the other variables in the model. in the next phase, path analysis was performed by amos 18 software to indicate the relationship between psychological empowerment variables, which will be discussed further. path analysis is basically a regression model ; it is a causal modeling technique that theorizes the relationships between variables and is used to answer study questions relating to the impact of independent variables on the dependent variable in a model. for evaluating the suitability of the model the best and most famous indicator for determining the fitness of structural equation modeling and path analysis, and the optimum conditions for these indicators is expressed by hu and bentler. permission for this study was issued after it was approved by a research committee and a regional ethics committee on 25.01.2014 with 921488 code. all ethical considerations such as voluntary participation with complete awareness and obtaining permission were done. mothers were reassured that the information they provided would remain confidential and that only researchers had access to collected data. personal information (name and surname) were entered as code numbers into software, so that all data would be securely protected. in this model - testing study, we did not have any model for pepw.as such, it was necessary to go through five steps of modeling process. for investigating markers of pepw, these markers were first calculated by linear regression and the two following parts were done : part 1 : investigating markers of pepw model in this study, multiple regressions have been used to predict empowerment, so that with the use of indicator variables, the empowerment of mothers could be modeled. for regression, ensuring normal distribution of variables is important and therefore it is necessary to examine bivariate relationships in order to assure their linearity. however, if the relationship is linear and the dependent variable for each independent variable is normally distributed, then distribution of residuals should be almost normal ; then it can be examined through a regression - standardized residual histogram ; meanwhile, the plotted scores in the normal probability plot are placed close to a line. also, for examining the independent residuals, durbin - watson (dw) statistics have been used : these should be from 1.5 to 2.5. moreover, to check whether the residuals are linear uncorrelated, collinerality test was used. if statistics were vif0.1, those conditions are established. before doing linear regression, the correlation between predictor variables and psychological empowerment was measured using a correlation coefficient matrix. then, variables including mother s age, marriage age, length of marital relationship, employment status of mothers, participation in prenatal education classes, marital satisfaction score, violence score, ownership score, high literacy, financial independence, living status and spiritual support score, as determining factors in empowerment, are imported to the linear regression in backward model. the results of regression in table 1 showed that psychological empowerment levels of mothers are predictable, taking into account individual factors such as age of marriage and employment status as well as psychological factors such as the experience of violence by a spouse and marital satisfaction (r square=0.609). demographic characteristics of the survey sample part 2 : specification of conceptual model what is certain is that employment is an important factor affecting empowerment, as confirmed by many researchers such gholipour. therefore, in this conceptual model, employment is considered as an exogenous variable affecting other variables in the model. women with high economic empowerment are more noticed and better understood in the family and experienced less violence at the hands of their spouses. therefore, in this conceptual model, we bring violence under the influence of the mother s employment. on the other hand, domestic violence, as a measure of women s empowerment has a negative correlation ; the higher the level of empowerment, the less violence is experienced by the woman. age of marriage is also considered an important element of women s empowerment, one that is affected by the mother s employment ; and with the increase of women s employment rate, the age of marriage rises. it is also affected by domestic violence ; those who are married at a younger age experienced more violence from their spouses. as can be seen in figure 1 the conceptual model of pepw is formed as a recursive model, because there is a one - way causal flow and all routes between variables are one way, as assumed in path analysis.. based on the nunally and bernstein method, path model uses at least 30 persons per independent variable ; in the model above, there is a dependent variable (spitzer s psychological empowerment) and four independent variables (violence score, mothers employment, marriage age and marital satisfaction score) ; therefore, a sample of at least 120 is required. pregnant women were selected from 10 urban medical centers and clinics as primary centers via two stage random clustering method with design effect equal to 1.5. based on the importance of the maximum variation in the samples, we used private and public institutions. after determining the exact number of existing centers and bases, two centers from the east, south, north, west and center of gorgan - for a total of 10 centers - were randomly selected. from each center and based on the number of low - risk nulliparous pregnant, visiting women who were literate, and eligible were randomly selected. based on the letters list, 180 nulliparous pregnant mothers were selected. after the exclusion of 20 participants (11.11 %) due to lack of interest in participation, response rate was in normal distribution clusters. eventually, 160 pregnant women participated in 25, march to 25, june 2015. all nulliparous pregnant women who were urban residents and were able to read and write were entered into the research ; the high - risk pregnant women and mothers who were not willing to participate were excluded from the study. in the next phase, amos 18 software (ibm, armonk, ny, usa) was used for identification of the model. in this model the independent variable is not affected by other variables, and the dependent psychological empowerment variable is an endogenous variable. also, marital satisfaction, violence and age of marriage are endogenous variables because they are influenced by the other variables in the model. in the next phase, path analysis was performed by amos 18 software to indicate the relationship between psychological empowerment variables, which will be discussed further. path analysis is basically a regression model ; it is a causal modeling technique that theorizes the relationships between variables and is used to answer study questions relating to the impact of independent variables on the dependent variable in a model. for evaluating the suitability of the model the best and most famous indicator for determining the fitness of structural equation modeling and path analysis, and the optimum conditions for these indicators is expressed by hu and bentler. permission for this study was issued after it was approved by a research committee and a regional ethics committee on 25.01.2014 with 921488 code. all ethical considerations such as voluntary participation with complete awareness and obtaining permission were done. mothers were reassured that the information they provided would remain confidential and that only researchers had access to collected data. personal information (name and surname) were entered as code numbers into software, so that all data would be securely protected. descriptive information on the demographic characteristics of mothers who participated in the study is shown in table 1. meanwhile, the average psychological empowerment in this study was calculated as 60.0569.105 using spitzer s scale with a minimum of 32 and maximum of 84. the psychological empowerment indicators using linear regression analysis were : mother s age of marriage, mother s employment ; marital satisfaction ; and experience of violence. increases in levels of mothers age of marriage, mothers employment, and marital satisfaction of the mother during pregnancy saw the empowerment of women increase. increased violence against pregnant mothers reduced their psychological empowerment (table 2). results of linear regression model for predictive factors of empowerment durbin - watson (dw)=1.600 ; r square=0.609 according to the conceptual model of the study, the following path analysis of the model was provided by amos software 18 (figure 2). path diagram of psychological empowerment in pregnant women indicators of goodness of fit of the model were good and demonstrated the fitness of the model (table 3). on the other hand, the low index of 2/df shows little difference between the conceptual model and observed data, while rmsea value indicated the goodness of fit. other indicators such as cmin=0.957, cmin / df=0.957, p - close=0.418, 2=0.957 and probability level=0.328 the fact that the model is suitable. as a result, the model has the necessary fitness and is approved in its entirety, showing that the set relationships of variables on the theoretical framework are reasonable. goodness of fit indexes of the model in comparison with the acceptable and the ideal range chi - square testing should not be significant because in the null hypothesis an inappropriate model by reducing the goodness of fit would be accepted. if the chi - square is not significant, the model will fit the data, and a normal fit index (nfi) indicator calculates the simple differences between the two models by dividing chi - square and a number equal to or higher than 0.90 (it is better to be higher than 0.95) approves the goodness of fit of this model. another index is comparative fit index (cfi), which is the best index even for small samples and it stands between zero and one. numbers higher than 0.95 (or 0.9 and above) show the goodness of fit of the model. cmin / df is an index that shows how the fitness of data decreases in the model. if we lose one or two tracks, and if the index is above 2 or 3, we have lost a lot of routes. finally, the rmsea indicator (root mean square error of approximation) estimates the lack of proportion in comparison to the saturated model ; a number of 0.05 or lower means goodness of fit and a number of 0.08 or less means sufficient fitness. path coefficients show the severity of the impact of a variable on other variables in the model. using path coefficients allows us to recognize which independent variable had the most direct impact on the dependent variable. determining direct and indirect impacts of dependent variables is one of the advantages of path analysis, and with its use, the general impact of a dependent variable can be recognized. moreover, in this way, it can be decided which dependent variable is used in the intervention as target. we know that if the relationship between variables is significant, there is a causal relationship. otherwise, the relationship would be non - causal. in the model above, the only relationship that is not significant is the relationship between marital satisfaction and experience of violence : the rest are significant relationships. therefore, the mother s employment does not have a causal relationship with marital satisfaction and experience of violence, but it has direct and indirect impacts on their levels. mother s employment with high coefficient has a causal relationship with the age of marriage. if the age of marriage is higher, the rate of employment is also higher. in addition, there is a negative causality relationship between the age of marriage and violence, with the reduction of the age in marriage, women s score of violence increases. on the other hand, there is a negative causality relationship between marital satisfaction and score of violence ; with the increase of marital satisfaction, the score of violence decreases (table 4). maximum likelihood estimates of variables in model on each other as is seen in tables 4 and 5, the mothers employment is the independent variable that has the greatest impact on the psychological empowerment of pregnant women. the greatest path coefficient is related to the influence of the mothers employment on the age of marriage. finally, all the effects of independent variables in the model (age of marriage, employment, marital satisfaction and experience of violence) on pepw are shown to boost confidence intervals (table 5). direct, indirect and total standardized effect of variables in model on psychological empowerment ci : confidence interval ; bci : bias - corrected confidence interval ; bc : bootstrap confidence in this study, because of the ideal range of goodness of fit indexes of model, we did nt make any modification for better fitness. according to the conceptual model of the study, the following path analysis of the model was provided by amos software 18 (figure 2). path diagram of psychological empowerment in pregnant women indicators of goodness of fit of the model were good and demonstrated the fitness of the model (table 3). on the other hand, the low index of 2/df shows little difference between the conceptual model and observed data, while rmsea value indicated the goodness of fit. other indicators such as cmin=0.957, cmin / df=0.957, p - close=0.418, 2=0.957 and probability level=0.328 the fact that the model is suitable. as a result, the model has the necessary fitness and is approved in its entirety, showing that the set relationships of variables on the theoretical framework are reasonable. goodness of fit indexes of the model in comparison with the acceptable and the ideal range chi - square testing should not be significant because in the null hypothesis an inappropriate model by reducing the goodness of fit would be accepted. if the chi - square is not significant, the model will fit the data, and a normal fit index (nfi) indicator calculates the simple differences between the two models by dividing chi - square and a number equal to or higher than 0.90 (it is better to be higher than 0.95) approves the goodness of fit of this model. when the number is closer to 1 another index is comparative fit index (cfi), which is the best index even for small samples and it stands between zero and one. numbers higher than 0.95 (or 0.9 and above) show the goodness of fit of the model. cmin / df is an index that shows how the fitness of data decreases in the model. if we lose one or two tracks, and if the index is above 2 or 3, we have lost a lot of routes. finally, the rmsea indicator (root mean square error of approximation) estimates the lack of proportion in comparison to the saturated model ; a number of 0.05 or lower means goodness of fit and a number of 0.08 or less means sufficient fitness. path coefficients show the severity of the impact of a variable on other variables in the model. using path coefficients allows us to recognize which independent variable had the most direct impact on the dependent variable. determining direct and indirect impacts of dependent variables is one of the advantages of path analysis, and with its use, the general impact of a dependent variable can be recognized. moreover, in this way, it can be decided which dependent variable is used in the intervention as target. we know that if the relationship between variables is significant, there is a causal relationship. otherwise, the relationship would be non - causal. in the model above, the only relationship that is not significant is the relationship between marital satisfaction and experience of violence : the rest are significant relationships. therefore, the mother s employment does not have a causal relationship with marital satisfaction and experience of violence, but it has direct and indirect impacts on their levels. mother s employment with high coefficient has a causal relationship with the age of marriage. if the age of marriage is higher, the rate of employment is also higher. in addition, there is a negative causality relationship between the age of marriage and violence, with the reduction of the age in marriage, women s score of violence increases. on the other hand, there is a negative causality relationship between marital satisfaction and score of violence ; with the increase of marital satisfaction, the score of violence decreases (table 4). maximum likelihood estimates of variables in model on each other as is seen in tables 4 and 5, the mothers employment is the independent variable that has the greatest impact on the psychological empowerment of pregnant women. the greatest path coefficient is related to the influence of the mothers employment on the age of marriage. finally, all the effects of independent variables in the model (age of marriage, employment, marital satisfaction and experience of violence) on pepw are shown to boost confidence intervals (table 5). direct, indirect and total standardized effect of variables in model on psychological empowerment ci : confidence interval ; bci : bias - corrected confidence interval ; bc : bootstrap confidence in this study, because of the ideal range of goodness of fit indexes of model, we did nt make any modification for better fitness. the most important advantage of using path analysis is that in the regression analysis, in addition to direct effects, indirect effects of each independent variable on the dependent variable could be identified. in this study, mothers employment is the indigenous independent variable in the model that has the greatest impact on women s psychological empowerment. of course, we know many factors affecting women s empowerment. of these in fact, employment and skill acquisition in women give them more ownership of their lives and have a more important role in the economic empowerment of women. in addition to helping to improve their economic conditions, women s employment can also influence social, political and psychological dimensions of empowerment. therefore, it can be said that access to resources (employment and income) is an important factor in women s empowerment. in a study in ethiopia, employment status and economic factors such as owning property were associated with the empowerment of mothers. also, mothersage and wealth were important factors during prenatal care in the presence of the spouse. there is a strong positive relationship between socioeconomic status and the health of pregnant women. we can alsoconsider control of household income as a criterion in the empowerment of women. the employment of the mother had direct and indirect non - eminent effectson the experience of violence and marital satisfaction of mothers ; it means that working mothers experienced less violence and more marital satisfaction. therefore, the mothers employment not only has an economic dimension, but also has drastic effects on the lives of pregnant mothers in its psychological dimensions. it has been suggested that women s economic situation scan influence their risk of violence, and that financial independence is protective or associated with increased risk, but many studies such as this study have shown that the mother s employment empowered women as a protective personal resource against domestic violence. in programs for women who have experienced domestic violence, accessing financial resources and being employed are executive solutions. in a study of empowerment in ghana, domestic violence the researchers in this study suggest that while strong links between the different aspects of empowerment such as freedom of movement and control over finance have been observed, abuse by a partnerwhether emotional, physical or sexualis a neglected empowerment indicator. domestic violence is associated with the health care of women and to increase women s mental health, physical and sexual violence by husbands should be prevented, as this violence determines the mental health of women. on the other hand, domestic violence as an empowerment criterion for women has a negative correlation with marital satisfaction ; with an increase in marital satisfaction, less violence is experienced by women. in this study, it was also found that the violence experienced by pregnant women at the hands of the husband is one of the great eminent factors in the empowerment of women ; this is highly influenced by the marital satisfaction variable. at the same time, in addition to its direct influence on psychological empowerment, marital satisfaction has an indirect impact on domestic violence. moreover, many studies have also shown that people s health is associated with their marriage quality and that marital satisfaction causes people to be healthier, while couples who experience marital distress have fewer skills in terms of emotional disclosure. therefore, limited emotional expression may cause damage to people s physical and psychological health, for the reason that marital distress is an important risk factor for mental health ; and it can be noted that socio - familial empowerment in contexts of marital conflict or negotiation, and domestic violence can determine women s empowerment. although the age of marriage in this study does nt have a high coefficient in the path analysis its elimination causes the model to lose its balance, and therefore having the lowest coefficient plays an important role in mother s empowerment. experience of violence has a causal relationship with the age of marriage ; the lower the mother s age, the more violence they experience. it is also believed that age, age of marriage and age at first pregnancy as underlying factors that affect empowerment. in fact, in the present study, the age of marriage was a causal factor in mother s employment ; mothers who married later had higher rates of employment. in support of this study, in a study in 36 countries in africa and southwest asia, it was found that each year of delay in marriage is associated with a half - year increase in a girl s education (one semester). and therefore with a reduction in the age of marriage, women s employment has also decreased, affecting the level of violence experienced by them and ultimately their pepw. in support of this, ahmad believes that to improve mothers maternal health in developing countries, their economic and social status and education should be increased. other researchers found an association between education and socioeconomic status with decision - making power of individuals to seek medical care. it was found that the acceptance of women as equal partners for achieving development is growing every day in all communities. barriers to women s health, along with socio - economic obstacles, can cause a community s efforts to achieve sustainable development to fail. therefore, consideration should be given to women s education and their access to economic resourcesespecially in terms of their fertilityas the main factors in empowering more women. similar to this article, a study in uganda showed that the maternal health status of mothers is affected by key determinants of health, such as violence against women and early marriage, as well as the mother s income. one of the limitations of this study was the exclusion of multiparous and high - risk pregnant women. the cross - sectional measures did not capture the dynamicity of empowerment. despite these limitations, the study s strengths are the use of diverse ethnicity, randomized sampling, path analysis and compatibility with existing tools. one of the limitations of this study was the exclusion of multiparous and high - risk pregnant women. the cross - sectional measures did not capture the dynamicity of empowerment. despite these limitations, the study s strengths are the use of diverse ethnicity, randomized sampling, path analysis and compatibility with existing tools. it can be concluded that if the mother has married at an early age and become pregnant immediately the chances are high that she wo nt be sufficiently psychologically empowered to cope with pregnancy. but this fact should not lead to the assumption that later marriage will solve the problem of mothers psychological empowerment ; perhaps with empowerment strategies, this issue could be resolved. however, increasing the possibility of mothers employment should be made a priority in planning and policymaking and certainly will not be possible in the short term. strategies to increase marital satisfaction should be used to reduce domestic violence and increase the mothers psychological empowerment. in another study, it would be better to examine this study in multiparous with a larger sample size, so that the findings can be used in the empowerment programs during pregnancy. | abstractbackground : women s empowerment programs during pregnancy focus primarily on increasing women s health goals and psychological empowerment has been considered important in most issues related to pregnant mothers mental health. using path analysis, this study aims to examine the direct and indirect components of psychological empowerment of pregnant mothers. methods : this model - testing study was conducted in gorgan, northwest of iran during three months in spring of 2015. through random cluster sampling, a total number of 160 pregnant women were selected from 10 urban medical centers and clinics as primary centers. we used spritzer s psychological empowerment scale. suitable sampling based on nunally and bernstein was followed in the model. the relationships between the dependent variables were then examined by means of path analysis using amos 18. results : the psychological empowerment of pregnant mothers (pepw) model is impacted by individual factors, such as marriage age and employment, including some subjectively rated factors such as marital satisfaction and experience of violence. the pepw model was deemed appropriate as optimum conditions indicators of goodness of fit ; low index of 2/df shows little difference between the conceptual model and observed data, while rmsea value indicated the goodness of fit. other indicators such as cmin=0.957, cmin / df=0.957, p - close=0.418, 2=0.957 and probability level=0.328 the fact that the model is ideal. the mothers employment had the highest coefficient in the pepw path model.731 (0.443, 0.965) bootstrap confidence intervals by 95%, and with a p - value of less than 0.05. conclusions : the mothers employment is the most important factor in psychological empowerment, but it can not be addressed quickly. programming to increase marital satisfaction followed by a decrease in family violence and prevention of early marriage are necessary for promotion of psychological empowerment during pregnancy. |
this study included 189 patients who were diagnosed with mm between 2001 and 2011 at the asan medical center, seoul, korea. disease was considered to be plasmablastic when > 30% of myeloma cells in the bm biopsy exhibited plasmablastic morphology. all patients met the following criteria : bm involvement ; no previous treatment ; no previous history of other malignancies, transplantation or immunosuppression ; no anti - human immunodeficiency virus antibodies ; and availability of laboratory and radiologic data and follow - up information. amc and alc were obtained from routine complete blood count (cbc) with a four - part differential (lymphocytes, monocytes, eosinophils, and neutrophils) using a sysmex automated hematology analyzer (model e-4000, se-9000 or xe-2100, sysmex co., kobe, japan), which was performed at the time of the diagnosis.19 overall survival (os) was defined as the time between the date of diagnosis and the date of death from any cause. for living patients, os was defined as the time between diagnosis and the last follow - up date. os was analyzed using the kaplan - meier method and log - rank testing was used to compare groups. median follow - up with a 95% confidence interval was calculated using the reverse kaplan - meier method.20 multivariate analyses of demographic and clinical characteristics prognostic for os were performed using the cox proportional hazards regression model. the maximal chi - square method was adapted to evaluate the cutoff points in the dataset that best segregated patients into poor and good prognosis subgroups (based on the likelihood of surviving), with the log - rank test as statistic used to measure the strength of the grouping. (r development core team, vienna, austria ; http://www.r-project.org), was used to identify the optimal cut - off points for alc, amc and the alc / amc ratio.21,22 correlations between alc, amc, and the alc / amc ratio with categorical variables were analyzed using pearson 's chi - square test or fisher 's exact test, and the mann - whitney u test was used to evaluate associations with continuous variables. all statistical analyses were performed using the spss ver. 18.0 (spss inc.,. a p - value of.05 was considered to be statistically significant. the institutional review board (irb) of asan medical center (seoul, korea) approved this study protocol and provided all necessary ethical permissions. this study included 189 patients who were diagnosed with mm between 2001 and 2011 at the asan medical center, seoul, korea. disease was considered to be plasmablastic when > 30% of myeloma cells in the bm biopsy exhibited plasmablastic morphology. all patients met the following criteria : bm involvement ; no previous treatment ; no previous history of other malignancies, transplantation or immunosuppression ; no anti - human immunodeficiency virus antibodies ; and availability of laboratory and radiologic data and follow - up information. amc and alc were obtained from routine complete blood count (cbc) with a four - part differential (lymphocytes, monocytes, eosinophils, and neutrophils) using a sysmex automated hematology analyzer (model e-4000, se-9000 or xe-2100, sysmex co., kobe, japan), which was performed at the time of the diagnosis.19 overall survival (os) was defined as the time between the date of diagnosis and the date of death from any cause. for living patients, os was defined as the time between diagnosis and the last follow - up date. os was analyzed using the kaplan - meier method and log - rank testing was used to compare groups. median follow - up with a 95% confidence interval was calculated using the reverse kaplan - meier method.20 multivariate analyses of demographic and clinical characteristics prognostic for os were performed using the cox proportional hazards regression model. the maximal chi - square method was adapted to evaluate the cutoff points in the dataset that best segregated patients into poor and good prognosis subgroups (based on the likelihood of surviving), with the log - rank test as statistic used to measure the strength of the grouping. (r development core team, vienna, austria ; http://www.r-project.org), was used to identify the optimal cut - off points for alc, amc and the alc / amc ratio.21,22 correlations between alc, amc, and the alc / amc ratio with categorical variables were analyzed using pearson 's chi - square test or fisher 's exact test, and the mann - whitney u test was used to evaluate associations with continuous variables. the institutional review board (irb) of asan medical center (seoul, korea) approved this study protocol and provided all necessary ethical permissions. the mean age of the 189 patients with mm in the study was 60 years (range, 29 to 84 years), and the male to female ratio was 1.077:1. paraprotein type was determined by immunoelectrophoresis ; igg was the most common (n=95, 50.3%), followed by light chain (n=48, 25.4%), iga (n=34, 18%), igd (n=7, 3.7%), and igm (n=5, 2.6%). of the 189 patients, 57 (30.2%) exhibited plasmablastic disease morphology. punched - out, osteolytic bone lesions (> 3 lesions) were identified in 96 patients (50.8%). following diagnosis, patients were treated with chemotherapy (47.1%), radiotherapy (6.3%), chemotherapy followed by asct (35.4%), or conservative treatment (11.1%). at the time of analysis, 146 patients (77.2%) had died of mm, and the estimated 5-year os was 26%. at the time of diagnosis, the median amc was 372 cells/l (range, 41 to 2,040 cells/l), the median alc was 1,581 cells/l (range, 319 to 5,742 cells/l) and the median alc / amc ratio was 4 (range, 0.83 to 33). the patients were divided into two groups according to alc, amc, and the alc / amc ratio at diagnosis. cut - off points for the division of groups, chosen according to the results of maximal chi - square analysis to best segregate patients, were as follows : alc 1,400 cells/l, amc 490 cells/l, and an alc / amc ratio of 2.9. one hundred and fifteen patients (60.8%) belonged to the high alc (1,400 cells/l) group, 57 patients (30.2%) to the high amc (490 cells/l) group and 135 patients (71.4%) to the high alc / amc ratio (2.9) group. the low alc, high amc, and low alc / amc ratio groups were associated with poor prognostic factors such as high international staging system (iss) stage, plasmablastic morphology, hypoalbuminemia and high 2-microglobulin (2 m). patients with a low alc at diagnosis were more likely to present with a high iss stage (p=.004), plasmablastic morphology (p=.027), hypoalbuminemia (p=.004), anemia (p=.005), low platelets (p=.001), and leukopenia (p=.001). patients with a high amc at diagnosis were more likely to be male (p 3 lesions) were identified in 96 patients (50.8%). following diagnosis, patients were treated with chemotherapy (47.1%), radiotherapy (6.3%), chemotherapy followed by asct (35.4%), or conservative treatment (11.1%). at the time of analysis, 146 patients (77.2%) had died of mm, and the estimated 5-year os was 26%. at the time of diagnosis, the median amc was 372 cells/l (range, 41 to 2,040 cells/l), the median alc was 1,581 cells/l (range, 319 to 5,742 cells/l) and the median alc / amc ratio was 4 (range, 0.83 to 33). the patients were divided into two groups according to alc, amc, and the alc / amc ratio at diagnosis. cut - off points for the division of groups, chosen according to the results of maximal chi - square analysis to best segregate patients, were as follows : alc 1,400 cells/l, amc 490 cells/l, and an alc / amc ratio of 2.9. one hundred and fifteen patients (60.8%) belonged to the high alc (1,400 cells/l) group, 57 patients (30.2%) to the high amc (490 cells/l) group and 135 patients (71.4%) to the high alc / amc ratio (2.9) group. the low alc, high amc, and low alc / amc ratio groups were associated with poor prognostic factors such as high international staging system (iss) stage, plasmablastic morphology, hypoalbuminemia and high 2-microglobulin (2 m). patients with a low alc at diagnosis were more likely to present with a high iss stage (p=.004), plasmablastic morphology (p=.027), hypoalbuminemia (p=.004), anemia (p=.005), low platelets (p=.001), and leukopenia (p=.001). patients with a high amc at diagnosis were more likely to be male (p<.0001) and present with leukocytosis (p<.0001) and high 2 m (p=.015), serum creatinine (p=.014) and platelets (p=.028). patients with a low alc / amc ratio at diagnosis were more likely to present with plasmablastic morphology (p=.008) and leukocytosis (p=.003), and be male (p=.011) (table 1). the median follow - up period was 938 days (range, 2 to 5,011 days). univariate analysis showed that low alc, high amc, and low alc / amc ratio were correlated with poor os (p=.002, p=.038, and p=.001, respectively) (fig. the 5-year os rate was also shorter in the low alc (17% vs 33% in the high alc group), high amc (18% vs 30% in the low amc group), and low alc / amc ratio groups (13% vs 31% in the high alc / amc ratio group). univariate analyses also showed that other factors related to poor os included plasmablastic morphology (p=.016), older age (65 years ; p=.001), high iss stage disease (p<.0001), high durie - salmon stage (p<.0001), high 2 m (p<.0001), hypoalbuminemia (p=.009) and treatment without asct (p<.0001) (table 2). multivariate analysis including alc, amc, morphology, age, iss stage, durie - salmon stage, and treatment type showed that only treatment type (asct vs no asct) was an independent prognostic factor for os (p=.001) ; alc and amc were not statistically significant (p=.106 and p=.107, respectively). multivariate analysis including the alc / amc ratio, morphology, age, iss stage, durie - salmon stage, and treatment type showed that the alc / amc ratio and treatment type were both independent prognostic markers (p=.047 and p=.001, respectively) (table 3). we also assessed alc, amc and the alc / amc ratio in patients subdivided for factors that can affect prognosis and treatment selection (such as asct vs no asct, age, sex, and iss) to determine whether they could predict os. among patients who did not undergo asct, those with low alc, high amc, and low alc / amc ratio had significantly shorter os (p=.029, p=.025, and p=.003, respectively), but patients who received asct did not show significant differences in survival (p=.221, p=.323, and p=.343, respectively) (fig. 2). when we stratified patients according to age, sex, and iss, low alc was associated with poor os in patients who were < 65 years - old (p=.009), high amc was associated with poor survival in the iss stage i group (p<.0001). low alc / amc ratio was associated with poor survival in patients who were < 65 years - old (p=.008), male (p=.018), or had iss stage i (p=.017) or stage iii disease (p=.010) (table 4). the aim of the present study was to investigate the role of amc as a prognostic biomarker for mm and to determine its relationship with alc, an established prognostic factor for mm. to our knowledge, this is the first study to demonstrate the prognostic value of amc and the alc / amc ratio in newly diagnosed mm. low alc (< 1,400 cells/l), high amc (490 cells/l), and low alc / amc ratio (< 2.9) were associated with poor os and poor prognostic factors such as high iss stage, plasmablastic morphology, hypoalbuminemia, and high 2 m. however, on multivariate analysis, only low alc / amc ratio was an independent prognostic factor for mm. although alc did not have statistical significance in multivariate analysis due to the relatively small sample size, our results are in agreement with previous reports showing that a high alc is associated with better prognosis in newly diagnosed mm (cut - off point, 1,400 cells/l ; the same as that used our study),3 relapsed mm after vel - dex therapy (cut - off point, 1,100 cells/l)23 and other lymphomas.24 we also found that shorter os in mm patients was associated with high amc and low alc / amc ratio, which is similar to the findings of previous studies of malignant lymphomas. amc has been shown to be a reliable prognostic marker for dlbcl (cut - off point, 630 cells/l),16 fl (cut - off point, 570 cells/l),17 and hl (cut - off point, 900 cells/l),18 and in classical hl, the alc / amc ratio at diagnosis is an independent prognostic factor for survival (cut - off point, 1.1).18 recently it has been reported that tams are a prognostic marker for the survival of patients with mm.13 tams, which constitute a significant proportion of tumor - infiltrating inflammatory cells, have been linked to the growth, angiogenesis and metastasis of a variety of cancers.7 in mm, macrophages are an abundant and important component of bm stromal cells and contribute to tumor angiogenesis.25,26 tams, which are continually being recruited and activated both in an autocrine manner and by cytokines secreted by myeloma cells, adapt functionally, phenotypically and morphologically to collaborate with endothelial cells in vessel formation.27 moreover, bm macrophages protect myeloma cells from apoptosis.28 tams therefore promote tumor growth not only by supporting angiogenesis, but also by protecting tumor cells from apoptosis, via the induction of immunosuppression.29 the important role tams play in the biology of mm, thereby affecting patient outcome, makes them a potential target for anti - vascular therapy in mm.26 as tams are derived from circulating monocytes and are recruited to the tumor site by tumor - derived chemotactic factors,14,15,30 obtaining an amc from a cbc at diagnosis is simple and could be more easily applied in clinical practice than counting tams. in this study, moreover, the alc / amc ratio, which may be a reflection of both the tumor microenvironment and host immune status, could provide prognostic information independently. we found that the patient group with high amc and low alc / amc ratio trended toward worse os whether or not they received asct, although statistical significance was not reached in the asct group because of the small sample size. a larger prospective cohort study of uniformly treated patients is required to validate these findings. in conclusion, the results of the present study suggest that amc, and, in particular, alc / amc ratio, which was found to be an independent prognostic factor for patient survival, could be used to determine the prognosis of mm. | backgroundabsolute lymphocyte count (alc) in peripheral blood has recently been reported to be an independent prognostic factor in multiple myeloma (mm). previous studies indicated that the absolute monocyte count (amc) in peripheral blood reflects the state of the tumor microenvironment in lymphomas. neither the utility of the amc nor its relationship with alc has been studied in mm.methodsthe prognostic value of alc, amc, and the alc / amc ratio at the time of diagnosis was retrospectively examined in 189 patients with mm.resultson univariate analysis, low alc (< 1,400 cells/l), high amc (490 cells/l), and low alc / amc ratio (< 2.9) were correlated with worse overall survival (os) (p=.002, p=.038, and p=.001, respectively). on multivariate analysis, the alc / amc ratio was an independent prognostic factor (p=.047), whereas alc and amc were no longer statistical significant. low alc, high amc, and low alc / amc ratio were associated with poor prognostic factors such as high international staging system stage, plasmablastic morphology, hypoalbuminemia, and high 2-microglobulin.conclusionsunivariate analysis demonstrated that changes in alc, amc, and the alc / amc ratio are associated with patient survival in mm. multivariate analysis showed that, of these factors, the alc / amc ratio was an independent prognostic factor for os. |
it is classified into type 1 (monoclonal due to paraprotein disorders), type 2 (mixed polyclonal and monoclonal secondary to hepatitis c), and type 3 (polyclonal due to lymphoproliferative disorders). the most common type described in the literature is secondary to hepatitis c, with a prevalence described as high as 50%. renal disease in mixed cryoglobulinaemia is present in approximately 20% of patients at the time of diagnosis. clinical presentation encompasses a wide variety of features from generalised bruising, hepatosplenomegaly, and acute renal failure to peripheral neuropathy and hypocomplementaemia [3, 4 ]. endocapillary proliferation, intraluminal thrombi, and thickening of the glomerular basement membrane are the most specific histological findings for type 2 cryoglobulinaemia. the current mainstay of management includes plasmapheresis with high - dose prednisolone and cyclophosphamide and treatment of the underlying cause. rituximab is a monoclonal anti - cd20 antibody used in the treatment of a variety of autoimmune diseases. we describe a case of type 2 mixed cryoglobulinaemia associated with non - hodgkin 's lymphoma successfully treated with rituximab. a 61-year - old female presented with severe community - acquired pneumonia complicated by acute renal failure. the renal failure was attributed to acute tubular necrosis secondary to sepsis and hypotension. at that time the patient was discharged home after 45 days of hospital stay with normal renal function. she represented with acute pulmonary oedema and skin rash, requiring readmission to the intensive care unit within 48 h of discharge. physical examination was consistent with pulmonary hypertension, splenomegaly, hepatomegaly, and bilateral pleural effusion. her positive investigations were : dysmorphic red cells in urine, proteinuria (24-hour urine protein 1 g), hypoalbuminaemia (serum albumin 24 g / l), low complement levels, and positive serology for mixed cryoglobulinaemia. rheumatoid factor was weakly positive and hcv rna was negative, as were all other autoimmune screens. renal biopsy revealed diffuse mesangiocapillary glomerulonephritis accompanied by hyaline capillary thrombi with focal mild endarteritis affecting the intimal zone of one small artery consistent with cryoglobulinaemic vasculitis (fig. 1, fig. the patient was treated with 5 sessions of plasma exchange and 2 sessions of haemodialysis over 1 week, followed by prednisolone and cyclophosphamide. there was a good initial response with a decrease in cryoglobulin levels within 3 weeks and symptomatic improvement. the patient was discharged on prednisolone 50 mg daily, and her renal function was normal on discharge. however, she became symptomatic again on prednisolone after 2 weeks, with reappearance of rash. the patient 's urea level was disproportionately high (with steroids and diuretics on board) compared to her creatinine level. she required 5 sessions of plasmapheresis and was recommenced on reduced cyclophosphamide with a dose of 50 mg/100 alternate days. cyclophosphamide was ceased due to recurrent thrombocytopenia, sepsis, and herpes zoster of the chest wall on the left side. she was commenced on intermittent haemodialysis (for better control of uraemia and fluid retention) and plasmapheresis on alternate days for 1 month. mg / l), but negative bence jones protein in urine, normal skeletal survey, and no light chains in the serum. her bone marrow biopsy showed a low - grade lymphoma with predominant cd20 expression on b cells. as the patient was not showing any clinical improvement and was requiring regular dialysis / ultrafiltration, second - line treatment was discussed. she was given 600 mg rituximab / week for 3 weeks in addition to prednisolone and responded within 1 week, with dramatic improvements in symptoms, absence of cryoglobulins, and normal complement levels. in view of her multiple prior infections (pneumonia, herpes zoster, and urosepsis), it was decided to administer only 3 doses of rituximab. her renal function gradually improved, and she became dialysis independent after 7 months, with complete renal recovery. cryoglobulin titres have remained negative and serum complements normal over the current follow - up of 24 months. non - hodgkin 's lymphoma - associated cryoglobulinaemia presenting with acute renal failure is uncommon. to our knowledge, this is the first case report of a patient with mixed cryoglobulinaemia associated with non - hodgkin 's lymphoma who presented with nephritis and acute renal failure [7, 8 ]. our patient was responsive to prednisolone, plasmapheresis, and cyclophosphamide but had a recurrence which was resistant to cytotoxics, steroids, and plasma exchange. rituximab has been shown to be useful in essential and secondary cryoglobulinaemia [7, 9 ]. it is a human - mouse chimeric monoclonal antibody that reacts with cd20 antigen, a transmembrane protein present in different maturation steps of b lymphocytes (from early pre - b to mature lymphocyte), thus directly and selectively targeting b cells and suppressing the production of rheumatoid factor [7, 10 ]. rituximab has proved effective and very well tolerated in b - cell non - hodgkin 's lymphomas [7, 11, 12 ], but there are conflicting case reports as well [13, 14 ]. rituximab has the advantage of allowing avoidance of serious immunosuppression and side effects [7, 11 ] and it lacks direct oncogenetic properties that could favour the progression of indolent lymphomas into an aggressive lymphoma. although a complete and long - term remission of lymphoma is obtained in a minority of cases after rituximab monotherapy, this case is unique for the fact that our patient has not shown signs of clinical or serological recurrence for 24 months after treatment with rituximab. she has not required maintenance therapy, although concerns about rituximab not being curative and need for ongoing rituximab maintenance therapy have been raised. the patient showed complete recovery with regard to renal function, although acute renal failure on presentation in mixed cryoglobulinaemia implies poor prognosis. the usual regimen consists of 375 mg / m with 1 dose per week for 4 weeks. the fourth dose was not given because of pancytopenia secondary to cytotoxics and sepsis predating the use of rituximab. our case report highlights the ongoing necessity for further exploration of new therapeutic agents in the treatment of resistant cryoglobulinaemia associated with lymphoma. no study has yet examined the efficacy of rituximab in mixed cryoglobulinaemia in a prospective controlled manner. the current literature is restricted to case reports and case series, and further prospective study is needed to fully determine the role of rituximab. resistant cryoglobulinaemia is a life - threatening condition, and the optimal management is unknown at this moment. rituximab has emerged as a well - tolerated and effective treatment with no long - term oncogenetic potential, which was confirmed by this case report. | cryoglobulinaemia is a systemic inflammatory condition characterised by immune complex - mediated small - to - medium - sized vasculitis. it has a wide variety of presentations ranging from bruising, neuropathy, and hepatosplenomegaly to acute renal failure. mixed cryoglobulinaemia is the most common type and is strongly associated with hepatitis c. management approaches include use of cyclophosphamide, prednisolone, and plasmapheresis, with differing views on alternative treatments in resistant cases. rituximab has emerged as an attractive option in resistant cases on account of its potent immunosuppressive effects on b cells. we describe a case of type 2 mixed cryoglobulinaemia in association with non - hodgkin 's lymphoma resistant to standard treatments which responded well to rituximab. this case is remarkable as mixed cryoglobulinaemia associated with non - hodgkin 's lymphoma presenting with nephritis is unusual, and, contrary to the high rate of recurrence in lymphoma - related cryoglobulinaemia, our patient has not shown any recurrence over 24 months. this highlights an alternative treatment modality which can be used in patients not responsive to existing managements for this condition with benefits of minimal side effects and no oncogenetic potential. |
folate is essential to the carbon transfer necessary for dna synthesis, cell division, and tissue growth. the mthfr gene is located at chromosome 1p36.3 and is 2.2 kb in length with a total of 11 exons. within the mthfr gene a common c to t polymorphism exists in exon 4 at position 677, it is a point mutation that converts a cytosine (c) to a thymine (t), resulting in an amino acid substitution of alanine to valine. the t variant codes for a thermolabile enzyme leading to an activity of 65% in the heterozygous state (ct) and ~30% in the homozygous state (tt), respectively. several association studies have been conducted to assess whether an association exists between the polymorphism in the mthfr gene (mim 607093). mthfr gene mutation has been related to many diseases including colon cancer, leukemia, vascular disease, depression, schizophrenia, migraine with aura, glaucoma, down syndrome, and neural tube defects. skull development is a complex process that involves on going interaction between the bones of the skull and cranial soft tissues. the cranial vault is comprised of intramembranous bones joined by the sutures of dense fibrous tissue that accommodate the growing brain. bone is added at these sutures during growth, and the skull eventually ossifies completely. well - known mutations in the fibroblast growth factor receptor-2 (fgfr2), twist1, frem1, lrit3, efna4 and runx2 have not shown constant results with different ethnic population with nonsyndromic craniosynostosis. around ~20% of craniosynostosis cases are syndromic, occurring with one or more additional major malformations caused by single - gene mutations in one of at least eight genes (fgfr1, fgfr2, fgfr3, twist1, efnb1, por, msx2 and rab23), involving primarily the coronal sutures. recently, rajagopalan., have reported that common foliate gene variant, mthfr c677 t, is associated with brain structure in two independent cohorts of people with mild cognitive impairment. they found that a very commonly carried variant in the mthfr gene, which is associated with high homocysteine levels in the blood, is significantly associated with brain structure variation, in particular with lower regional brain volumes, as per our knowledge, none of the studies had solely examined the association of mthfr gene c to t polymorphism in craniosynostosis patients in any part of the world. as a first step for a comprehensive genetic study on craniosynostosis this family - based association approach has the advantage that it avoids possible ethnic stratification that may affect the conventional case - control design. to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients. to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients. to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients. this was a cross - sectional study in which 30 patients classified as apert syndrome, pfeiffr syndrome and nonsyndromic craniosynostosis patients with their family were recruited after obtaining clearance from the institutional ethics committee. in clinically suspected cases of craniosynostosis an x - ray or a ct scan of the child 's skull was done. a complete prenatal and birth history of the child, including family history of craniosynostosis or other craniofacial abnormalities was recorded. three milliliter intravenous blood was collected from patients and from their family members (father and mother) in ethylenediaminetetraacetic acid - anticoagulated vacutainer for the purpose of the study after their written informed consent. children with primary microcephaly (secondary craniosynostosis) and postural plagiocephaly and those with any chronic diseases or associated syndromic disorders, parents and relatives not giving consent were excluded from the study. primers for mthfr gene were designed as described by reutter. and custom - synthesized primers (sigma aldrich chemicals pvt., bengaluru, india). polymerase chain reaction (pcr) for each sample was performed in 0.2 ml, thin - walled tubes using 20 g of dna, 2 - 5 pm of each primer, 200 mm dinucleotide triphosphates, 10 pcr buffer, 1.5 mm mgcl2, and 0.5 units of dynazyme ii dna polymerase (thermo scientific). the pcr reaction was carried out in a t-100 dna engine (bio - rad, hercules, ca, usa) thermal cyclers under the following conditions initial - denaturation - 94c for 8 min, denaturation - 94c for 1 min, annealing - 63c for 1 min, extension - 72c for 1 min, final extension - 72c for 7 min 4c, forever repeated for 40 cycles. the primer sequences were : modified primers (f5-tcttcatccctcgccttgaac-3 ; r5-aggacggtgcggtgagagtg-3) according to frosst. amplicons sized were verified by gel electrophoresis by running the pcr product on 2% agarose gel with the 100 bp maker [figure 1 ]. after successful amplification, a small aliquot (5 l) of the mthfr reaction mixture was treated with 1 units of hinf i restriction enzyme (neb). two percent agarose gel, polymerase chain reaction (pcr) product before hinf i restriction fragment length polymorphism ; lane 1 - 100 bp ladder ; lane 2 - blank ; lanes 3, 4, 5, 6, 7 and 8 - pcr product of 315 bp the amplified pcr products (mthfr) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide. gel photography was done with bio - red gel doc system. for mthfr 677, the pcr yielded a 315 bp product, which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition (homozygous polymorphic) and a 315,176 and 139 bp fragments for ct condition (heterozygous polymorphic). an undigested product length of 315 bp was retained by the wild types [figure 2 ]. three percent agarose gel, hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2, 3, 4, 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic the amplified pcr products (mthfr) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide. gel photography was done with bio - red gel doc system. for mthfr 677, the pcr yielded a 315 bp product, which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition (homozygous polymorphic) and a 315,176 and 139 bp fragments for ct condition (heterozygous polymorphic). an undigested product length of 315 bp was retained by the wild types [figure 2 ]. three percent agarose gel, hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2, 3, 4, 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic the amplified pcr products (mthfr) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide. gel photography was done with bio - red gel doc system. for mthfr 677, the pcr yielded a 315 bp product, which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition (homozygous polymorphic) and a 315,176 and 139 bp fragments for ct condition (heterozygous polymorphic). an undigested product length of 315 bp was retained by the wild types [figure 2 ]. three percent agarose gel, hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2, 3, 4, 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic the allele and genotype frequencies of the mthfr of offspring and parents were as tabulated in table 1. these results suggest no significant association of markers with craniosynostosis in the cases. the distribution of mthfr 677 polymorphisms among mothers and babies. it was observed that for mthfr c677 t, three out of four cases were in the group pertaining to both mother and baby being carriers of polymorphic variant. in two cases, both fathers and mother carried a polymorphic variant to the patient suggesting that maternal mthfr c677 t polymorphism may be a genetic risk factor for craniosynostosis. the folate metabolism pathway plays an important role in dna methylation, dna synthesis, cell division, and tissue growth, especially in the rapidly developing cells. thus, a defective folate metabolism could result in an impaired dna synthesis or dna methylation involved in the craniosynostosis. t + c. 677c > t in the methylene tetrahydrofolate reductase (mthfr) gene is associated with higher than normal levels of homocysteine, which may increase risk of thrombosis (mthfr thermolabile variant thrombophilia). in addition, this mutation / polymorphism is considered a mthfr thermolabile variant and is associated with hyperhomocysteinemia, increased cardiovascular risk, increased risk of neural tube defects, and increased risk of preeclampsia. furthermore, mutations in the mthfr gene may cause mthfr deficiency / homocystinuria. to the best of our knowledge, the present study is the first family - based association study between the mthfr gene and craniosynostosis. the study of single nucleotide polymorphism mthfr c677 t polymorphism in craniosynostosis has not been reported yet. our results showed that, based on single - marker frequency analysis, c677 t polymorphism was not associated with craniosynostosis. in summary, mthfr is associated with higher plasma homocysteine, a well - known mediator of neuronal damage and brain atrophy. our results do not support the hypothesis that the mthfr 677crt polymorphism plays any role in the development of craniosynostosis. however our results do suggest that most mutations were transferred through the mother to their babies. when taking the results of posterior power calculations into account, they even contradict a causative role of this polymorphism within the bounds of its estimated effect size level. in complex genetic traits, exogenous factors often do play a relevant etiologic role, and if these factors are not prevalent in the population under investigation, the role of interacting co - causative genetic factors might be missed. our findings revealed that c677 t polymorphism of the mthfr gene was not directly involved in the pathogenesis of craniosynostosis in our indian population. from the viewpoint of the findings of animal, clinical and pharmacological studies, however, the studies performed until date have produced inconsistent results. to shed light on the potential etiological role of mthf genetic variants in the pathophysiological mechanism of craniosynostosis, c667 t polymorphism of the mthfr gene is unlikely to play a role in the pathogenesis of craniosynostosis though maternal mthfr c677 t polymorphism may be a genetic risk factor. | background:677c to t allele in the 5, 10-methylenetetrahydrofolate reductase (mthfr) gene has been implicated in the etiology of various syndromes and nonsyndromic diseases but till date no direct studies have been reported with craniosynostosis.objectives:the aim was to study the family - based association of mthfr polymorphism in different categories of craniosynostosis patients.materials and methods : this was a cross - sectional study in which 30 patients classified as apert syndrome, pfeiffr syndrome and nonsyndromic craniosynostosis patients with their family were recruited. a sample of 3 ml intravenous blood was taken from patients and from their family members (father and mother) in ethylenediaminetetraacetic acid - anticoagulated vacutainer for the purpose of the study. genomic dna was extracted from peripheral blood lymphocytes by phenol chloroform extraction method. primers for mthfr gene were designed. the polymerase chain reaction was carried out. after successful amplification, a small aliquot (5 l) of the mthfr reaction mixture was treated with 1 units of hinf i restriction enzyme (neb). results were obtained and compiled.results:a total of 30 patients / participants with craniosynostosis of indian descent and their parents formed the study group. the genotyping did not confirm an association between the mthfr 677c to t polymorphism and between different categories of craniosynostosis. when comparing the offspring of mothers statistically significant differences were found.conclusion:c667t polymorphism of the mthfr gene is unlikely to play a role in the pathogenesis of craniosynostosis though maternal mthfr c677 t polymorphism may be a genetic risk factor. |
owing to the aging of the global population, the prevention and treatment of chronic diseases in the elderly have become important health issues. the increase in the number of cases of osteoporosis - induced fractures in the elderly is noteworthy.1,2 the world health organization has defined osteoporosis as a disease characterized by low bone density and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture.3 more than 200 million postmenopausal women around the world suffer from osteoporosis.4 in the united states, osteoporosis affects 2% of men and 10% of women aged 50 years and above. in addition, 49% of older women and 30% of older men have osteopenia.5 in taiwan, 1.63% of men aged 50 years and above suffer from osteoporosis, and 11.35% of women suffer from it.6 osteoporosis and falls are related to fractures, which can lead to increased morbidity and mortality, as well as decreased functional ability. the mortality of patients with hip fractures within 1 year is 20%, and only one - third of the patients have recovered their original functions.7 a large - scale, multinational study on vertebral compression fractures in asia found that the incidence of fractures in women aged 6574 years ranges from 9.2% to 18.8%, and that in women aged 75 years and above ranges from 18% to 28.7%.8 most strategies for treating bone loss have focused on dietary and pharmacologic interventions;9 however, drug treatments can have adverse effects and poor long - term adherence, despite their effectiveness.10 weight - bearing and resistance exercises can be an alternative therapy. some studies have shown that these exercises can increase bone mineral density (bmd).11,12 in comparison with pharmacological interventions, the compliance with exercise for treating osteoporosis is better.13 furthermore, fall incidence is multifactorial ; it may strengthen the case for exercise interventions, and exercise itself is effective in reducing fall incidence, whereas pharmacological and other interventions are not.14 whole body vibration (wbv) is a popular exercise where individuals stand on an oscillating plate, and the motor transmits vertical acceleration to muscle and bone.15 wolff stated the bone will increase where the load is placed, which leads to the remodeling of bone ; it was also found that the morphology (density, size, and width) of a bone will be changed by the external forces acting on it hence, he proposed the famous wolff s law.16 wbv can produce osteogenic effects by changing the flow of bone fluid through direct bone stimulation and mechanotransduction, or it can generate indirect bone stimulation through skeletal muscle activation by means of tone stretch reflex.1719 the results of animal trials had shown that vibration stimulation can increase the anabolic activity of bone tissue, as well as increase the bone volume and area.20,21 in addition, the study by wenger found that mice exhibited a shift toward higher bone density in the femur and an increase in mineralizing surface in the radius after vibration. studies and systemic reviews on postmenopausal women have found that wbv has a significant effect on femoral neck bmd ; however, it does not have a significant effect on lumbar spine bmd.3,2233 these studies have also found that the frequency and magnitude of the applied wbv used in these studies differ greatly. the aim of this study was to determine whether 6 months of high - frequency and high - magnitude wbv training at a neutral full standing position would be effective for the bmd of the lumbar spine in postmenopausal women. this study was a randomized clinical trial, in which subjects that met the inclusion criteria in community volunteer groups were recruited through advertisements from january 2010 to october 2011. as shown in figure 1, a total of 40 postmenopausal women were recruited, and 32 of them met the inclusion criteria. in the end, a total of 28 subjects completed this study. the inclusion criteria were : postmenopausal ; nonsmokers ; adequate nutritional status (body mass index [bmi ] 18.5), a lack of regular exercise at least three times per week, and the ability to follow the protocol. the exclusion criteria were : acute hernias or thrombosis ; kidney or bladder stones ; epilepsy or seizures ; pregnancy ; arrhythmia ; use of a pacemaker ; serious cardiovascular or pulmonary disease ; dizziness ; undergoing surgery or being hospitalized for treatment within the last 6 months ; and receiving any osteoporosis drugs within the last year. the subjects were fully informed of the research purpose, possible adverse events, and expected health benefits, and all subjects signed the approved informed consent form for this study after being verbally informed of the relevant information. this study was approved by institutional review board at the taichung hospital (taichung, taiwan) (irb-05 - 06). a total of 32 subjects who met the inclusion criteria were randomized into two groups using computer - generated numbers : the wbv group and the control group (con group). during the study, the two groups were asked to maintain their daily life habits and not to use any osteoporosis drugs, including calcium and vitamin d. the wbv group received vibration training three times per week at a sports center in a hospital. the subjects stood on the platform in a natural full standing posture with their bare feet. the stimulation source of the wbv device (lv-1000 ; x - trend fitness equipment, luntai enterprise co., ltd, taichung, taiwan) was a horizontal vibration with a frequency of 30 hz (1 hz = 1 oscillation / second) and a magnitude (acceleration) of 3.2 g (gravity ; 1 g = 9.81 m / second2) for 5 minutes each time. a well - trained physical therapist was responsible for executing the vibration training and for monitoring the safety of the subjects (figure 2). the subjects all underwent bmd (g / cm) tests of the lumbar spine before and after the 6-month intervention. the first to fourth lumbar spine bmd was assessed using dual - energy x - ray absorptiometry (dexa) (qdr4500 ; hologic inc, bedford, ma, usa). a physician who was certified by the international society for clinical densitometry interpreted the test data to ensure the consistency of the dexa quality. the day - to - day precision coefficient of variation percentage of this dexa machine was about 1% at the lumbar spine. this study used the statistical package for the social sciences, windows version 14 (spss inc., descriptive statistics included the mean and standard deviation and the chi - square test for the baseline characteristics. this study used a paired samples t - test to compare the change in bmd of the two groups before and after the intervention. the effects between the two groups were tested using analysis of covariance, and were adjusted by body weight, age, and baseline data to compare the change in bmd of the two groups. this study adopted p 1.0 is normal, while a t - score = 1.0 to 2.5 indicates osteopenia (low bone mass or low bone density). a t - score 2.5 is used to diagnose osteoporosis.34 the incidence of osteopenia and osteoporosis of the two groups in the trial was 100% for the wbv group and 85% for the con group. the bmd of the wbv group and the con group after 6 months was 0.8350.098 g / cm (compared to the pretest, p=0.047) and 0.8150.076 (compared to the pre - test, p=0.188), respectively. there was a significant increase in the bmd of the lumbar spine of the wbv group, while there was a decrease in that of the con group (table 2). the variables (age, bmi, and number of postmenopausal years) that might affect the bmd were further adjusted using analysis of covariance. the comparison of the change in the bmd between the two groups before and after the 6-month intervention indicated that the bmd of the lumbar spine in the wbv group increased by 2.032%3.332%, while that of the con group decreased by 0.046%1.245%. the difference between the two groups reached statistical significance (p=0.016), as shown in table 3. osteoporosis has become one of the most important health issues for postmenopausal women, and it has been found that multicomponent exercise programs based on strength, aerobic, high impact, and/or weight - bearing training are beneficial to postmenopausal women.35 however, some weight - bearing exercises are not suitable for patients with muscle weakness or joint and nerve diseases ; therefore, wbv training can be provided as an alternative exercise. wbv had negative effects if the exposure was of large intensity or long duration, which could damage the peripheral nerves and blood vessels. on the other hand, the side effects, including dizziness, headache, and fall, could be minor when exposure includes low intensity and is of short duration.3 thus, the choice of the vibration model and the duration of the intervention are important. the oscillating plate of a wbv machine can be adjusted to alter the exercise stimulus. according to the frequency / magnitude of the applied vibration, the oscillating plate can be divided into high - frequency (hz > 20) or low - frequency plates (hz 20), and they can be categorized as high magnitude (1 g) or low magnitude (< 1 g), according to the strength of the exercise.15,17 this study used high - frequency (30 hz) and high - magnitude (3.2 g) horizontal wbv to conduct a trial on postmenopausal women. after the 6-month intervention, the wbv group showed significantly improved lumbar spine bmd (p=0.016). several randomized controlled studies have compared wbv training groups with con groups and found that there is no significant effect on lumbar spine bmd or volumetric bone density in postmenopausal women.24,25,27,28 the study by rubin used a quiet standing posture to receive high - frequency and low - magnitude wbv. in the study by von stengel,30 the patients received a multifunctional training program at the high - frequency and low - magnitude wbv platform. the subjects in this study received high - frequency and high - magnitude wbv, which was different from the two studies mentioned above. al25 used the same high - frequency and high - magnitude wbv as that used in this study ; however, the subjects engaged in static and dynamic knee - extensor exercises and osteoporosis cases were excluded. in the study by gusi,27 the subjects stood on a wbv platform and maintained a 60 angle of knee flexion. the posture used on the wbv platform will affect the transmissibility of wbv. an erective posture can enhance the transmissibility of vibration through the hip and spine.36 the neutral full - standing position used in this study could enhance the effect of wbv on the bones in the lumbar spine. in recent large - scale studies, ruan enrolled 91 postmenopausal women with osteoporosis in a study and provided a 6-month intervention of high - frequency (30 hz, five times / week) wbv, and found that the wbv group s lumbar spine bmd increased by 4.3% (p=0.000) ; conversely, the con group s lumbar spine bmd decreased by 1.9% (p<0.05). in the study by beck and norling,37 following an 8-month intervention of high - frequency (30 hz) and low - magnitude (0.3 g) wbv twice per week, the con group experienced bone loss at the lumbar spine (6.6% ; p=0.02), while the wbv group did not. the results of the aforementioned studies were similar to those of this study ; however, the study by ruan required a series of five, 10-minute sessions / week, and only three, 5-minute sessions / week were performed in this study, which is more reasonable for the participants to adhere to. this would be strengthened by highlighting the dropout rate in the ruan study,28 which was 23%, as compared to the 88% retention rate in this study. in contrast, slatkovska conducted a study on 202 postmenopausal women who were taking calcium and vitamin d supplements, in which high - frequency (30 hz and 90 hz) and low - magnitude (0.3 g) wbv was used. comparisons to the con group showed that there was no significant change in the lumbar spine bmd in the wbv training group. however, the author noted some limitations, including inconsistent medical adherence (65%79%) and the fact that participants self - administered the wbv at home. in this study, the participants who received high - frequency and high - magnitude wbv did not take calcium or vitamin d supplements, and they exhibited good adherence. the entire wbv training program was performed at the sports center of a hospital and was supervised by a well - trained physical therapist. although this study observed a significant increase in the lumbar spine bmd from baseline, there were some limitations. first, the overall results may not be applicable to the general population, because the samples were low and only consisted of postmenopausal women, not from a random sampling of the general population. second, blank wbv was not provided to the con group and a double - blind design could not be implemented in this study. moreover therefore, it was impossible to identify the effect of wbv on preventing or improving osteoporosis it has been generally speculated that the increased rate of osteoporosis and osteopenia could result in a greater increase of bmd due to a low baseline bmd.39,40 however, the mechanism of wbv on lumbar spine bmd remains unclear. this study found that high - frequency (30 hz) and high - magnitude (3.2 g) wbv training could be used by postmenopausal women to improve bone loss at the lumbar spine. in order to determine guidelines for the use of wbv, including the posture used on the platform, and the oscillation type (amplitude, frequency, and duration), a large - scale study should be conducted on elderly participants, or on patients with disabilities who are unable to engage in resistant exercise or other weight - bearing exercises. this study concluded that 6 months of high - frequency, high - magnitude wbv using a neutral full standing posture is a feasible exercise for reducing bone loss at the lumbar spine for postmenopausal women. | backgroundthe issue of osteoporosis - induced fractures has attracted the world s attention. postmenopausal women are particularly at risk for this type of fracture. the nonmedicinal intervention for postmenopausal women is mainly exercise. whole body vibration (wbv) is a simple and convenient exercise. there have been some studies investigating the effect of wbv on osteoporosis ; however, the intervention models and results are different. this study mainly investigated the effect of high - frequency and high - magnitude wbv on the bone mineral density (bmd) of the lumbar spine in postmenopausal women.methodsthis study randomized 28 postmenopausal women into either the wbv group or the control group for a 6-month trial. the wbv group received an intervention of high - frequency (30 hz) and high - magnitude (3.2 g) wbv in a natural full - standing posture for 5 minutes, three times per week, at a sports center. dual - energy x - ray absorptiometry was used to measure the lumbar bmd of the two groups before and after the intervention.resultssix months later, the bmd of the wbv group had significantly increased by 2.032% (p=0.047), while that of the control group had decreased by 0.046% (p=0.188). the comparison between the two groups showed that the bmd of the wbv group had increased significantly (p=0.016).conclusionthis study found that 6 months of high - frequency and high - magnitude wbv yielded significant benefits to the bmd of the lumbar spine in postmenopausal women, and could therefore be provided as an alternative exercise. |
presently in the united states, it is estimated that one in five people will develop some form of skin cancer due to a combination of family history, environment, and genetic factors (see the american cancer society for more information, http://www.cancer.org/cancer/skincancer-melanoma/detailed guide / melanoma - skin - cancer - risk - factors). dna damage caused by ultraviolet irradiation is thought to be the main environmental culprit behind the development of this disease. likewise, genetic disruption of genes necessary for the appropriate balances between survival, proliferation, and cell death are frequently associated with the development and persistence of cells that result in skin cancer. these cancerous cells may also develop mechanisms to evade the immune system resulting in unimpeded cell proliferation, survival, and metastases. malignant melanoma is the deadliest form of skin cancer, and it arises from the uncontrollable cell division of the basal layer melanin - producing cells called melanocytes (for in - depth review, see ibrahim and haluska, 2009). melanoma commonly involves the skin, but it can occur at other sites such as the eyes, ears, and gastrointestinal tract. while melanoma only accounts for 5% of skin cancers, it is responsible for the majority of skin cancer deaths (jemal., 2009). the prognosis of melanoma is favorable when limited to the skin and wide excision is curative. however, the prognosis of metastatic melanoma is very poor with a 5-year survival rate of less than 20%. presently, the most used anti - tumor strategy for stage iv metastatic melanoma is dacarbazine (ditc), which as a single treatment is not very effective, and is associated with < 20% response rate (tsao., 2004). adjuvant and combination treatments are also used ; for example, cisplatin, vinblastine, and dtic (the regimen is referred to as cvd), in combination with interferon alpha-2 (ifn) and interleukin-2 (il-2), but none have proven to be significantly effective, yet they demonstrate substantial toxicity (legha., 1989, 1996 ; phan., 2001). in the majority of cases where a response does occur, there is often recurrence of the cancer within months. a better understanding of the pathways that are activated to promote melanoma, and targeted during treatment, will hopefully reveal novel therapeutic opportunities. toward the end of our discussion, we will propose that manipulating the bcl-2 family and the mitochondrial pathway may offer a potential therapeutic inroad to treatment success. in response to chemotherapeutics, cells attempt to eliminate the damage or recover from it by engaging stress response pathways (e.g., the p53 pathway), but often the damage is overwhelming, and the treated cells induce a form of programmed cell death known as apoptosis (green and evan, 2002). apoptotic signaling can initiate from outside the cell via plasma membrane receptors (referred to as the extrinsic pathway ; e.g., cd95/fas or tumor necrosis factor receptor, tnfr), or through stress that originates from within the cell (referred to as the intrinsic or mitochondrial pathway ; e.g., macromolecular damage ; guicciardi and gores, 2009 ; chipuk., the extrinsic apoptotic pathway is engaged when pro - apoptotic ligands such as tnf or cd95l / fasl trigger death receptor signaling which directly leads to the activation of caspases, which are the cysteine aspartic proteases responsible for inducing the apoptotic phenotype (e.g., dna laddering, loss of plasma membrane asymmetry, and cellular blebbing ; pop and salvesen, 2009). the intrinsic pathway is triggered by intracellular stress signals such as dna damage, oncogenes, hypoxia, and growth factor withdrawal (chipuk., these cellular stressors transcriptionally and post - transcriptionally regulate the b cell cll / lymphoma-2 (bcl-2) family of proteins, which are responsible for mitochondrial integrity, subsequent caspase activation, and apoptosis. while these two general pathways are distinct, there are situations of cross - talk where the extrinsic pathway promotes apoptosis through the intrinsic pathway and vice versa. in these situations, the bcl-2 family of proteins is also crucial control points of cellular fate. for the most part, cancer cells utilize and respond to chemotherapeutic agents by regulating the mitochondrial pathway of apoptosis, so our discussions will be focused on these signaling mechanisms (figure 1). the mitochondrial pathway of apoptosis. following cellular stress such as dna damage, the bcl-2 family of proteins is regulated by transcriptional (increased or decreased) and post - translational (e.g., phosphorylation, cleavage, and relocalization) mechanisms. if the cellular stress is irreparable, the culmination of pro - apoptotic signaling will lead to mitochondrial outer membrane permeabilization (momp), which allows for the release of soluble intermembrane space proteins, including cytochrome c. apaf-1, cytochrome c, and datp then coordinate to promote apaf-1 oligomerization and recruitment of pro - caspase-9 ; this complex is referred to as the apoptosome. the apoptosome allows for pro - caspase-9 dimerization and activation, and the subsequent cleavage and activation of executioner caspases (caspase-3, -6, and -7). these caspases promote the characteristic phenotypes of apoptosis, e.g., chromatin condensation, dna laddering, and plasma membrane blebbing, by cleaving numerous intracellular substrates. within the body, apoptotic cells are rapidly cleared by phagocytosis to ensure tissue homeostasis. once the apoptotic program has been initiated by compromising the outer mitochondrial membrane (omm), pro - apoptotic factors are released from the mitochondrial intermembrane space (ims), including cytochrome c (figure 1 ; liu., 1996). monomeric apoptotic protease activating factor 1 (apaf-1) cooperates with cytochrome c and datp to form the oligomeric caspase activation platform referred to as the apoptosome (li., 1997 ; the apoptosome recruits, dimerizes, and activates pro - caspase-9, which then promotes the downstream function of pro - caspases-3, -6, and -7 (figure 1). often, this step in the mitochondrial pathway is referred to as the point of no return, as mitochondrial function and atp generation gradually wane, and proteolytic cleavage of caspase substrates ensues. caspase activity is responsible for the morphological phenotypes of apoptosis, including dna laddering, loss of plasma membrane asymmetry, and phagocytosis (figure 1 ; logue and martin, 2008). regulation of the mitochondrial pathway of apoptosis plays a crucial role in the development and treatment of melanoma ; and it has been shown that defects in this pathway can result in resistance to tumor suppressor pathways and treatments (for an in - depth review, see soengas and lowe, 2003). the involvement of mitochondria in apoptosis was first suggested when caspase activity resulted from xenopus oocyte extract co - incubation with purified mitochondria (newmeyer., 1994). this activity was blocked by the addition of bcl-2, suggesting that bcl-2 could prevent mitochondrial engagement of the cytosol. since then, the bcl-2 family has grown to include almost 20 members that are divided into two functional classes of proteins : anti - apoptotic and pro - apoptotic (figure 2). most cells express a variety of anti - apoptotic and pro - apoptotic bcl-2 proteins, and through the regulation of their interactions command survival or commitment to apoptosis (for an in - depth review, see chipuk and green, 2008). the bcl-2 family of proteins is divided into anti - apoptotic and pro - apoptotic members. the anti - apoptotic members include : a1, bcl-2, bcl - w, bcl - xl, and mcl-1, all of which contain four bcl-2 homology domains (termed bh1 - 4). the pro - apoptotic proteins are subdivided into effector and bh3-only members. the effector proteins, bak and bax, also contain bh1 - 4 ; while the bh3-only proteins contain only one domain, a bh3, that is required for interactions with anti - apoptotic and effector proteins. the bh3-only proteins include : bad, bid, bik, bim, bmf, bnip3, hrk, noxa, and puma. bcl-2 core structural unit, which includes a hydrophobic groove that binds to bh3 domains. anti - apoptotic bcl-2 proteins are comprised of four bcl-2 homology domains (bh1 - 4) and are generally integrated within the omm, but may be present in other membranes like the endoplasmic reticulum (petros., 2004). bcl-2, bcl - xl, and myeloid cell leukemia 1 (mcl-1) are the major members of the anti - apoptotic bcl-2 repertoire that function to preserve omm integrity by directly binding and inhibiting the pro - apoptotic bcl-2 proteins (chen., 2005 ; willis. the pro - apoptotic bcl-2 members are divided into effectors (which also contain bh1 - 4) and the bh3-only proteins (figure 2). the effector proteins bak (bcl-2 antagonist killer 1) and bax (bcl-2 associated x protein) homo - oligomerize into proteolipid pores within the omm and are required to promote momp and cytochrome c release (lindsten., 2000 ; wei., 2001). however, these effectors require an activation step, upon which they oligomerize and gain the capacity to permeabilize membranes composed of mitochondrial lipids (kuwana., 2002). activation of bak and bax occurs through interaction with so - called direct activators (see below), or by physico - chemical effects of detergents, mild heat, or elevated ph (hsu and youle, 1997 ; khaled., 2001 ; letai., 2002 ; pagliari., the bh3-only proteins act to regulate both the anti - apoptotics and effectors (figures 2 and 3). two of these, bid (bh3 interacting domain death agonist) and bim (bcl-2 interacting mediator of cell death), are direct activators of bak and bax, acting via their bh3 domains to induce oligomerization and the permeabilization function of bak and bax (wei., 2000 ; kuwana., 2002, 2005 ; letai., the process by which bak and bax permeabilize the omm is commonly referred to as mitochondrial outer membrane permeabilization, or momp. mechanisms of action within the bcl-2 family leading to momp. the upstream involvement of the bcl-2 family in apoptosis can be divided into two parallel activities : de - repression and sensitization. the de - repression model begins at cellular status quo with anti - apoptotic bcl-2 proteins binding and sequestering bh3-only direct activators (e.g., bcl-2 binds bim). in response to stress, a de - repressor bh3-only protein is induced (e.g., bad), which can then displace the direct activator and liberate it to activate bak and bax. the sensitization model involves the preemptive binding and inactivation of anti - apoptotic proteins by sensitizer bh3-only proteins (e.g., bcl-2 binds bad). this sensitization prevents the inhibition of direct activator bh3-only proteins induced following apoptotic stimulation (e.g., bim), thus lowering the threshold level of direct activator necessary to activate the effectors bak and bax. unbound direct activator bh3-only proteins function in the direct activation of bak and bax through a transient interaction. these interactions lead to conformational changes involving n - terminal rearrangement, which exposes the bh3 domain of bak and bax. activated bak monomers pair symmetrically, and oligomerize as sets of dimers to permeabilize the outer mitochondrial membrane leading to momp. recent in vitro studies show that activated bid and bax bind to one another only in association with membranes, and this precedes bax oligomerization, which is followed by membrane permeabilization (lovell., 2008). the accepted viewpoint is that the expression, stability, and activation of the bh3-only proteins and their interactions with the anti - apoptotic and effector proteins sufficiently links pro - apoptotic signal transduction to momp(chipuk., 2010). broadly speaking, regulation of the bh3-only proteins at the transcriptional and translational levels determines the omm integrity. when an apoptotic signal is initiated, the anti - apoptotic bcl-2 proteins are inhibited by the sensitizer / de - repressor bh3-only proteins (e.g., bad, bmf, hrk, noxa, and puma), which allows for direct activator - induced bak and bax activation to promote momp, and subsequent apoptosis (figure 3). non - bcl-2 family direct activator proteins are also described ; several reports suggest a direct activator function for cytosolic p53, map-1, and asc (tan., 2001 ; furthermore, controversy exists regarding the role of puma (p53 upregulated modulator of apoptosis) in mediating bak and bax activation, as conflicting results suggest that puma is either a de - repressor / sensitizer or direct activator bh3-only protein (chipuk., 2005 ; kuwana., finally, the steps leading to bak and bax activation have been unveiled through studies that focused on structural consequences of bim and bak / bax interactions ; these structural details are discussed in figure 3 (dewson. in terms of chemotherapeutic success, pushing a cell to undergo momp is suggested to enhance clinical outcomes, and this is highlighted by the development of small molecule inhibitors to the anti - apoptotic bcl-2 proteins, which lower the cellular threshold for momp and apoptosis (letai, 2008). in response to chemotherapeutics, cells attempt to eliminate the damage or recover from it by engaging stress response pathways (e.g., the p53 pathway), but often the damage is overwhelming, and the treated cells induce a form of programmed cell death known as apoptosis (green and evan, 2002). apoptotic signaling can initiate from outside the cell via plasma membrane receptors (referred to as the extrinsic pathway ; e.g., cd95/fas or tumor necrosis factor receptor, tnfr), or through stress that originates from within the cell (referred to as the intrinsic or mitochondrial pathway ; e.g., macromolecular damage ; guicciardi and gores, 2009 ; chipuk. the extrinsic apoptotic pathway is engaged when pro - apoptotic ligands such as tnf or cd95l / fasl trigger death receptor signaling which directly leads to the activation of caspases, which are the cysteine aspartic proteases responsible for inducing the apoptotic phenotype (e.g., dna laddering, loss of plasma membrane asymmetry, and cellular blebbing ; pop and salvesen, 2009). the intrinsic pathway is triggered by intracellular stress signals such as dna damage, oncogenes, hypoxia, and growth factor withdrawal (chipuk., 2010). these cellular stressors transcriptionally and post - transcriptionally regulate the b cell cll / lymphoma-2 (bcl-2) family of proteins, which are responsible for mitochondrial integrity, subsequent caspase activation, and apoptosis. while these two general pathways are distinct, there are situations of cross - talk where the extrinsic pathway promotes apoptosis through the intrinsic pathway and vice versa. in these situations, the bcl-2 family of proteins is also crucial control points of cellular fate. for the most part, cancer cells utilize and respond to chemotherapeutic agents by regulating the mitochondrial pathway of apoptosis, so our discussions will be focused on these signaling mechanisms (figure 1). the mitochondrial pathway of apoptosis. following cellular stress such as dna damage, the bcl-2 family of proteins is regulated by transcriptional (increased or decreased) and post - translational (e.g., phosphorylation, cleavage, and relocalization) mechanisms. if the cellular stress is irreparable, the culmination of pro - apoptotic signaling will lead to mitochondrial outer membrane permeabilization (momp), which allows for the release of soluble intermembrane space proteins, including cytochrome c. apaf-1, cytochrome c, and datp then coordinate to promote apaf-1 oligomerization and recruitment of pro - caspase-9 ; this complex is referred to as the apoptosome. the apoptosome allows for pro - caspase-9 dimerization and activation, and the subsequent cleavage and activation of executioner caspases (caspase-3, -6, and -7). these caspases promote the characteristic phenotypes of apoptosis, e.g., chromatin condensation, dna laddering, and plasma membrane blebbing, by cleaving numerous intracellular substrates. within the body, once the apoptotic program has been initiated by compromising the outer mitochondrial membrane (omm), pro - apoptotic factors are released from the mitochondrial intermembrane space (ims), including cytochrome c (figure 1 ; liu., 1996). monomeric apoptotic protease activating factor 1 (apaf-1) cooperates with cytochrome c and datp to form the oligomeric caspase activation platform referred to as the apoptosome (li., 1997 ; zou., the apoptosome recruits, dimerizes, and activates pro - caspase-9, which then promotes the downstream function of pro - caspases-3, -6, and -7 (figure 1). often, this step in the mitochondrial pathway is referred to as the point of no return, as mitochondrial function and atp generation gradually wane, and proteolytic cleavage of caspase substrates ensues. caspase activity is responsible for the morphological phenotypes of apoptosis, including dna laddering, loss of plasma membrane asymmetry, and phagocytosis (figure 1 ; logue and martin, 2008). regulation of the mitochondrial pathway of apoptosis plays a crucial role in the development and treatment of melanoma ; and it has been shown that defects in this pathway can result in resistance to tumor suppressor pathways and treatments (for an in - depth review, see soengas and lowe, 2003). the involvement of mitochondria in apoptosis was first suggested when caspase activity resulted from xenopus oocyte extract co - incubation with purified mitochondria (newmeyer., 1994). this activity was blocked by the addition of bcl-2, suggesting that bcl-2 could prevent mitochondrial engagement of the cytosol. since then, the bcl-2 family has grown to include almost 20 members that are divided into two functional classes of proteins : anti - apoptotic and pro - apoptotic (figure 2). most cells express a variety of anti - apoptotic and pro - apoptotic bcl-2 proteins, and through the regulation of their interactions command survival or commitment to apoptosis (for an in - depth review, see chipuk and green, 2008). the bcl-2 family of proteins is divided into anti - apoptotic and pro - apoptotic members. the anti - apoptotic members include : a1, bcl-2, bcl - w, bcl - xl, and mcl-1, all of which contain four bcl-2 homology domains (termed bh1 - 4). the pro - apoptotic proteins are subdivided into effector and bh3-only members. the effector proteins, bak and bax, also contain bh1 - 4 ; while the bh3-only proteins contain only one domain, a bh3, that is required for interactions with anti - apoptotic and effector proteins. the bh3-only proteins include : bad, bid, bik, bim, bmf, bnip3, hrk, noxa, and puma. bcl-2 core structural unit, which includes a hydrophobic groove that binds to bh3 domains. anti - apoptotic bcl-2 proteins are comprised of four bcl-2 homology domains (bh1 - 4) and are generally integrated within the omm, but may be present in other membranes like the endoplasmic reticulum (petros., 2004). bcl-2, bcl - xl, and myeloid cell leukemia 1 (mcl-1) are the major members of the anti - apoptotic bcl-2 repertoire that function to preserve omm integrity by directly binding and inhibiting the pro - apoptotic bcl-2 proteins (chen., 2005 ; willis., 2005, 2007 ; the pro - apoptotic bcl-2 members are divided into effectors (which also contain bh1 - 4) and the bh3-only proteins (figure 2). the effector proteins bak (bcl-2 antagonist killer 1) and bax (bcl-2 associated x protein) homo - oligomerize into proteolipid pores within the omm and are required to promote momp and cytochrome c release (lindsten., 2000 ; however, these effectors require an activation step, upon which they oligomerize and gain the capacity to permeabilize membranes composed of mitochondrial lipids (kuwana., 2002). activation of bak and bax occurs through interaction with so - called direct activators (see below), or by physico - chemical effects of detergents, mild heat, or elevated ph (hsu and youle, 1997 ; khaled., 2001 ; letai., 2002 ; the bh3-only proteins act to regulate both the anti - apoptotics and effectors (figures 2 and 3). two of these, bid (bh3 interacting domain death agonist) and bim (bcl-2 interacting mediator of cell death), are direct activators of bak and bax, acting via their bh3 domains to induce oligomerization and the permeabilization function of bak and bax (wei., 2000 ; kuwana., 2002, 2005 ; letai., 2002). the process by which bak and bax permeabilize the omm is commonly referred to as mitochondrial outer membrane permeabilization, or momp. mechanisms of action within the bcl-2 family leading to momp. the upstream involvement of the bcl-2 family in apoptosis can be divided into two parallel activities : de - repression and sensitization. the de - repression model begins at cellular status quo with anti - apoptotic bcl-2 proteins binding and sequestering bh3-only direct activators (e.g., bcl-2 binds bim). in response to stress, a de - repressor bh3-only protein is induced (e.g., bad), which can then displace the direct activator and liberate it to activate bak and bax. the sensitization model involves the preemptive binding and inactivation of anti - apoptotic proteins by sensitizer bh3-only proteins (e.g., bcl-2 binds bad). this sensitization prevents the inhibition of direct activator bh3-only proteins induced following apoptotic stimulation (e.g., bim), thus lowering the threshold level of direct activator necessary to activate the effectors bak and bax. unbound direct activator bh3-only proteins function in the direct activation of bak and bax through a transient interaction. these interactions lead to conformational changes involving n - terminal rearrangement, which exposes the bh3 domain of bak and bax. activated bak monomers pair symmetrically, and oligomerize as sets of dimers to permeabilize the outer mitochondrial membrane leading to momp. recent in vitro studies show that activated bid and bax bind to one another only in association with membranes, and this precedes bax oligomerization, which is followed by membrane permeabilization (lovell. the accepted viewpoint is that the expression, stability, and activation of the bh3-only proteins and their interactions with the anti - apoptotic and effector proteins sufficiently links pro - apoptotic signal transduction to momp(chipuk., 2010). broadly speaking, regulation of the bh3-only proteins at the transcriptional and translational levels determines the omm integrity. when an apoptotic signal is initiated, the anti - apoptotic bcl-2 proteins are inhibited by the sensitizer / de - repressor bh3-only proteins (e.g., bad, bmf, hrk, noxa, and puma), which allows for direct activator - induced bak and bax activation to promote momp, and subsequent apoptosis (figure 3). non - bcl-2 family direct activator proteins are also described ; several reports suggest a direct activator function for cytosolic p53, map-1, and asc (tan. furthermore, controversy exists regarding the role of puma (p53 upregulated modulator of apoptosis) in mediating bak and bax activation, as conflicting results suggest that puma is either a de - repressor / sensitizer or direct activator bh3-only protein (chipuk., 2005 ; kuwana., 2005 ; kim., finally, the steps leading to bak and bax activation have been unveiled through studies that focused on structural consequences of bim and bak / bax interactions ; these structural details are discussed in figure 3 (dewson., 2008, 2009 ; in terms of chemotherapeutic success, pushing a cell to undergo momp is suggested to enhance clinical outcomes, and this is highlighted by the development of small molecule inhibitors to the anti - apoptotic bcl-2 proteins, which lower the cellular threshold for momp and apoptosis (letai, 2008). melanoma is notorious for chemoresistance, and there continues to be tremendous research focused on identifying the molecular mechanisms to explain this phenotype (for an in - depth review, see hocker., unlike many other cancers, melanoma rarely displays mutations within the tumor suppressor p53 pathway, which does afford the opportunity to pharmacologically regulate the p53 pathway through chemotherapy - induced dna damage and stress - responses, and we will discuss this throughout (hocker and tsao, 2007). however, mutations in n - ras [neuroblastoma ras viral (v - ras) oncogene homolog ] and b - raf (v - raf murine sarcoma viral oncogene homolog b1) are common events in melanoma, which are observed in approximately 20 and 70% of patients, respectively (shukla., 1989 ; davies., 2002 ; reifenberger., 2004 ; edlundh - rose., 2006). additionally, the loss of pten (phosphatase and tensin homolog) expression, activation of the akt / pi3k (phosphatidylinositol 3-kinases) pathway, and loss of cdkn2a (cyclin - dependent kinase inhibitor 2a) often synergize with b - raf mutations leading to the progression from benign nevus, primary tumor, to metastatic disease (haluska and hodi, 1998 ; kim, 2010 ; figure 4). melanoma originates from melanocytes in the skin that acquire a series of changes that promote the progression from a normal phenotype to a nevus, primary tumor, and eventual metastatic disease. epigenetically silenced, mutated, or deleted genes that promote disease progression are listed and discussed throughout the text. in brief, common mutations observed early in melanoma development are in the genes encoding n - ras and b - raf, and these are often complemented by mutations in the melanocyte regulators such as mitf and bcl-2. subsequent changes to pten, cdkn2a, and mh2a along with activating mutations of the akt / pi3k pathway synergize with early mutations. subsequently, alterations in pro - apoptotic sensitivity upstream (e.g., p53) and downstream (e.g., apaf-1) of the mitochondrial pathway of apoptosis are suggested. in parallel to these events, the expression and function of the bcl-2 family is altered to establish marked resistance to pro - apoptotic stimulation : there is increased expression of anti - apoptotic members, and coordinated downregulation of pro - apoptotic members. activating mutations in n - ras and b - raf lead to positive influences on the cell cycle, mainly by promoting mitosis and preventing cells from engaging their repair and stress machineries, which leads to increased stress and accumulated mutations. studies also suggest that the expression of akt / pi3k is directly correlated with melanoma progression ; and pten expression is subsequently lost by genetic and epigenetic mechanisms to further promote akt / pi3k survival pathways (steck., 1997 ; indeed, a recent mouse model of combined b - raf v600e and pten loss provides in vivo evidence that these two pathways are necessary and sufficient to promote metastatic melanoma formation (dankort., cells can engage inhibitors to the cell cycle, for example cdkn2a, which encodes for the proteins p16 (cyclin - dependent kinase inhibitor 2a) and p14 (alternative reading frame) ; yet cdkn2a is also mutated, genetically lost, or epigenetically silenced in a significant percentage of melanomas (castellano and parmiani, 1999). there are numerous other genes that are targeted during the transition between primary tumor and metastasis ; more recently, the histone variant macroh2a was shown to suppress tumor progression of malignant melanoma (kapoor., 2010). in brief, the sum of these mutations leads to an aggressive disease that is highly resistant to chemotherapeutics (figure 4). for this discussion, we are focused on understanding how melanomas with significant alterations in n - ras, b - raf, and the other pathways listed above fail to engage apoptosis despite aberrations in cellular signaling leading to unregulated proliferation (figure 4). more importantly, can we pharmacologically regulate these aberrant pathways to produce better responses in the clinic ? the answers to both of these questions point in the direction of the bcl-2 family of proteins. here, we suggest that this family of anti- and pro - apoptotic members may be what allows for melanoma development, survival, and chemoresistance, yet it offers a pharmacologically tractable achilles heel for successful combination / adjuvant therapies to treat malignant melanoma. in general, there are two approaches to block apoptosis that is normally engaged following deregulated cellular proliferation : the cell may decrease the expression of pro - apoptotic bcl-2 proteins (e.g., bax or bim) ; or the cell can over - express anti - apoptotic bcl-2 proteins (e.g., bcl-2 or mcl-1). in either case, the resulting phenotype is the failure to promote bak / bax activation, momp, and apoptosis following deregulated cellular proliferation cues or chemotherapeutic treatment. this ultimately leads to metastatic disease with poor prognosis and decreased long - term survival despite chemotherapeutic intervention. returning to the pathways that promote melanoma development and chemoresistance, it is widely regarded that b - raf v600e is the most commonly observed mutation in patients (davies., 2002 ; pollock and meltzer, 2002). this gain of function mutation is a valine to glutamic acid substitution at codon 600, which results in a hyper - activated kinase and subsequent marked increases in mapk / erk signaling (wan., 2004). increased b - raf v600e activity is demonstrated to promote bim phosphorylation, leading to its proteasome - mediated degradation in several melanoma models (cartlidge., 2008 ; sheridan., 2008 ; boisvert - adamo., 2009 ; goldstein., 2009). the b - raf v600e mutation is observed very early in melanoma development starting at the benign nevus stage. therefore, reducing bim levels and all apoptotic pathways that bim directly regulates (e.g., cytokine / growth factor withdrawal) likely leads to marked resistance to apoptosis (bouillet., 1999). likewise, since bim is a direct activator of bax and bak, chemotherapies that utilize a bim - dependent mechanism will likely not be successful (letai., 2002 ; kuwana., 2005 ; dai., 2008). multiple other pro - apoptotic bcl-2 proteins are also targeted by the b - raf v600e mutation. several studies suggest that excessive signaling through b - raf and mapk promotes bad (bcl-2 antagonist of cell death) phosphorylation and inactivation, and decreased bmf (bcl-2 modifying factor) and puma expression, and all these events lead to abrogated bak / bax activation, momp, and apoptosis (cartlidge., 2009 ; goldstein., 2009 ; keuling., 2010 ; shao and aplin, 2010). there is also a growing literature connecting ras and b - raf mutations to the induction of autophagy. autophagy is a catabolic process which promotes the degradation of cytoplasm and organelles ; and this plays a key role in tumorigenesis as it often helps cells cope with stress (for in - depth review, see kondo., 2005). activating mutations in ras and b - raf promote autophagy, which can restrict tumor cell growth (chen and karantza - wadsworth, 2009 ; maddodi., 2010 ; elgendy., 2011). in contrast, some tumor cells display an addiction to autophagy, which can promote glucose metabolism and mitochondrial function, leading to enhanced tumorigenesis (guo., 2011 ; lock., 2011). interestingly, anti - apoptotic bcl-2 proteins function at the intersection between autophagy and apoptosis by directly regulating beclin-1 activity, a key protein that is required for the initiation of the formation of the autophagy machinery. therefore, integrating the mechanistic interplay between the pathways that promote melanoma development, autophagy, and the cell death machinery is of critical importance to understanding this disease and potential therapeutic strategies (levine., 2008 ; chipuk., importantly, the regulation of anti - apoptotic bcl-2 proteins by bh3-only proteins and bh3 mimetics (discussed later) impacts not only on apoptosis, but also autophagy, and subsequent changes to stress signaling, proteostasis, and metabolism ; yet we will focus our discussion on cellular commitment to apoptosis. the role of the anti - apoptotic bcl-2 proteins in melanoma is highlighted by the phenotype of the bcl-2 knockout mouse which displays dramatic graying of the hair because of diminished follicular melanocyte survival (yamamura., 1996). in support of this phenotype, microphthalmia associated transcription factor (mitf), a key transcription factor necessary for melanocyte development and survival, directly induces bcl-2 promoter activity (mcgill., 2002). elevated levels of bcl-2 and mitf can promote chemoresistance and are associated with melanoma progression ; furthermore, antisense against bcl-2 or mitf promotes a decrease in melanoma cell survival and sensitivity to chemotherapy (jansen. perhaps this cell type does not express other anti - apoptotic bcl-2 proteins, so small increases in bh3-only protein activity may promote premature momp and apoptosis, whereas this normally tolerated level of bh3-only protein activity would be actively sequestered by bcl-2 to allow for stress resolution. such a limited repertoire of anti - apoptotic reserve would not appear to favor chemoresistance. however, the malignant transformation and chemoresistance seen in melanomas has also been attributed to increased levels in a1 (bcl-2 related gene a1), bcl - xl, and mcl-1, allowing for a rather substantial shift in the apoptotic threshold (tang., 1998 ; leiter., 2000 ; zhang and rosdahl, 2006 ; placzek., 2010 ; zhuang., 2010). the upregulation of these anti - apoptotic proteins during the transition from nevus to melanoma may indicate their active role in melanoma progression (wong. several additional transcription factors such as ets-1 (v - ets erythroblastosis virus e26 oncogene homolog 1), and the chromatin remodeling factor dek, promote mcl-1 expression, and may present novel mechanisms (and perhaps chemotherapeutic strategies) as to the mechanism of melanoma survival and chemoresistance (khodadoust., 2009 ; dong.. from both in vitro and in vivo melanoma model systems, several lines of evidence suggest that bcl-2 over - expression correlates with a malignant phenotype and a higher metastatic potential (grover and wilson, 1996 ; takaoka., 1997 ; leiter., 2000 ; utikal., 2002 ; zhang and rosdahl, 2006). this seems logical, as a cell expressing more anti - apoptotic proteins should resist apoptosis despite cues to die following events from benign status to malignancy and metastasis. however, there are conflicting results in the literature that describe decreased bcl-2 expression during melanoma progression (saenz - santamaria., 1994 ; ramsay., 1995 ; tron., 1995 ; tang., 1998 ; korabiowska., 1999 ; similarly, bcl-2 is described to be constitutively expressed in melanocytes, nevi, and melanoma, with little change in expression levels (cerroni., 1995 ; collins and white, 1995 ; morales - ducret., 1995 ; plettenberg., 1995 ; the observations in both these scenarios can be reconciled if we consider that a cell may have the opportunity to express alternative anti - apoptotic bcl-2 members, such as bcl - w, bcl - xl, mcl-1, or a1. another possibility is downregulation of the pro - apoptotic effectors bak and bax, which is also associated with tumor progression and decreased patient survival (fecker., 2006 ; tchernev and orfanos, 2007). the balance between pro - apoptotic and anti - apoptotic bcl-2 members could also be considered to control apoptosis. for example, the bax / bcl-2 ratio in chemotherapy - sensitive cells is relatively higher than in cells displaying chemoresistance (raisova., 2001). however, great caution should be employed with monitoring only a few proteins, as momp and apoptosis proceed when multiple bh3-only proteins collaborate to promote bak / bax activation. understanding the dynamic and total proteinprotein interactions within the bcl-2 family is more indicative of apoptotic sensitivity and chemotherapeutic responses. this has been recently applied to tumors of lymphoid origin, and should be developed to better delineate which patients have the best chance of chemotherapeutic responses (deng., 2007 ; ryan. returning to our original questions, it appears that melanoma has developed numerous strategies to block apoptosis despite heightened cellular proliferation cues : the loss of bh3-only protein function, along with collateral increases in anti - apoptotic bcl-2 protein expression and diversity. the marked increase in anti - apoptotic bcl-2 proteins suggests that these cells can engage a pro - apoptotic response, and that the increased bcl-2 expression favors a selection process where increased bcl-2 (or other anti - apoptotic members) confers a survival advantage. strategies that promote bh3-only protein function, while also decreasing the functional anti - apoptotic bcl-2 repertoire, will likely yield the best clinical responses ; indeed, we will discuss the results of such strategies in the following sections. standard treatments for patients diagnosed with stage iv metastatic malignant melanoma are dtic and il-2. the fda approved dtic in 1975, and it remains the most commonly used single agent chemotherapeutic for metastatic melanoma. unfortunately, the response rate is less than 20% and resistance nearly always occurs (stein and brownell, 2008). other chemotherapeutic agents are currently being studied to improve late stage melanoma treatment but few are proving to be more effective than dtic. for example, temozolomide, a imidazotetrazine - derivative of dtic, was tested in clinical trials but did not improve the overall survival of patients compared to dtic (patel., 2011). in addition to the standard combination chemotherapy regimens [e.g., cvd (cisplatin, vinblastine, dtic), dartmouth (dtic, cisplatin, carmustine, and tamoxifen), and bold (bleomycin, vincristine, lomustine, and dtic) ], a number of novel adjuvant therapies are currently being evaluated to improve the efficiency of dtic - based treatments (seigler., 1980 ; legha., 1989 ; lattanzi., 1995). in particular, ipilimumab, a human igg1 monoclonal antibody that recognizes cytotoxic t - lymphocyte - associated antigen 4 (ctla-4), enhances t - cell activation, proliferation, and attack of cancer cells (melero. fong and small, 2008 ; robert and ghiringhelli, 2009 ; hodi., 2010). the combination of dtic and ipilimumab was recently shown to be associated with a significant increase in survival among patients with untreated metastatic melanoma (robert., 2011). the mechanism of action for dtic - based therapies is to kill tumor cells by dna damage, and these treatments likely signal through the p53 pathway to induce apoptosis (igney and krammer, 2002 ; vousden and lane, 2007). fortunately, p53 is rarely targeted in melanoma, so the pro - apoptotic arm of the p53 pathway has the potential to be engaged. dtic also directly impacts on the bcl-2 family from several angles : there is downregulation of anti - apoptotic proteins, and coordinated upregulation of bh3-only proteins and effector molecules, like bim and bax, respectively (weber., 2009 ; jiang., 2010b) importantly, dtic has been studied in multiple cellular models of melanoma, and the consistent observation is that dtic promotes apoptosis through a marked regulation of the bcl-2 family leading to momp. but what accounts for the low response rate of melanoma patients to dtic ? can the threshold for bak / bax activation, momp, and apoptosis be lowered to increase dtic responses in patients ? there is minimal information regarding how combination chemotherapy regimens (e.g., cvd) impact on the bcl-2 family. it is therefore essential to better understand the mechanisms of action to design appropriate co - administered adjuvant therapies. a non - exhaustive list of several chemotherapeutic drugs (some discussed below) and their impact on the bcl-2 family in common melanoma cell lines is provided in table 1. a list of chemotherapeutics that induce changes to the bcl-2 family in various melanoma cell lines. in terms of a targeted therapeutic approach, the frequency of the b - raf v600e mutation in melanoma presents a novel and unique drug target (davies., 2002 ; sheridan., 2008). early studies with b - raf v600e mutated melanoma lines suggest that silencing b - raf v600e expression leads to greater sensitivity to apoptosis, which is likely mediated by the combined stabilization and function of bim to promote bak / bax activation in recent years, treatment of melanoma patients harboring the b - raf v600e mutation with small molecules (e.g., plx-4720, plx-4032) that specifically inhibit the b - raf v600e kinase has been successful through phase iii clinical trials, and appears to offer greater response rates and increased overall survival compared to dtic (flaherty., 2010). however, melanoma cells treated with these drugs have a means of becoming resistant to declining levels of b - raf signaling. for instance, if pten expression or function is lost, there are suggestions in the literature that melanoma cell lines can develop resistance to plx-4720 induced death due to decreased bim (paraiso., 2011). the loss of pten results in constitutive activation of the pi3k / akt pathway, which has been described in melanoma suggesting b - raf inhibitors will only work for a subset of patients carrying the b - raf v600e mutation and functional pten (vanbrocklin., 2009). dual requirements for the pi3k / akt pathway and constitutive mapk signaling are also supported by combination treatments such as sorafenib [small molecular inhibitor of several tyrosine protein kinases (vegfr and pdgfr) and b - raf ] and nanoliposomal ceramide, which have been shown to synergistically decrease pi3k / akt and mapk signaling leading to increased sensitivity to the mitochondrial pathway of apoptosis in vitro (tran., furthermore, several mek pathway inhibitors, such as uo126 and ci-1040, have also been shown to cause upregulation of pro - apoptotic bcl-2 protein function like bim, bmf, noxa, and puma, and occasionally can lead to the downregulation of anti - apoptotic proteins like bcl-2 and mcl-1 (wang., 2007 ; vanbrocklin., 2009). perhaps combinations of b - raf and mek inhibitors can promote more robust apoptosis due to dual regulations of numerous anti- and pro - apoptotic bcl-2 members (wang., 2007). from our discussion, we suggest that melanoma likely develops because of altered bcl-2 family function, which allows for survival and proliferation despite signals to die. likewise, literature supports that chemotherapies engage apoptosis by directly promoting the function of pro - apoptotic bcl-2 members. if so, are there other drugs that can be used in combination with dtic to alter the balance toward death, perhaps by decreasing anti - apoptotic bcl-2 protein expression, or by inhibiting their function by inducing bh3-only proteins ? indeed, there is already an abundant literature to show that many drugs can directly regulate the bcl-2 family to favor turning on the mitochondrial pathway of apoptosis (table 1). numerous dna damaging agents, such as cisplatin, directly impinge on the mitochondrial pathway of apoptosis by lowering anti - apoptotic bcl-2 protein expression, while also increasing effector levels, and presumably, activation and momp (shibuya. endoplasmic reticulum stress induced by tunicamycin or thapsigargin can also induce apoptosis of melanoma cells, which has been shown to directly up - regulate puma expression (jiang., 2008). proteasome inhibitors such as bortezomib have been shown to induce bid, noxa, and puma in melanoma cells (nikiforov. drugs with diverse mechanisms of action including dihydroartemisinin (promotes oxidative stress), adi - peg20 (arginine deiminase), and aminooxyacetate (transaminase inhibitor) appear to directly regulate noxa expression, which has been shown to directly impact on cellular sensitivity to both intrinsic and extrinsic forms of apoptosis (qin. the overall outcome of such studies suggests that the bcl-2 family determines the chemotherapeutic response to either engage apoptosis or continue with survival. indeed, several drugs are suggested to promote apoptosis via bim, which nicely parallels existing clinical data that shows melanoma progression and poor survival rates are associated with decreased bim expression (cartlidge. are there strategies to sensitize melanoma cells to chemotherapeutics by using this information ? in the following section, we provide evidence that depleting cellular anti - apoptotic bcl-2 function may be the optimal combination therapy to promote chemotherapeutic responses despite over - expressed anti - apoptotic bcl-2 members or decreased pro - apoptotic bcl-2 engagement. as mentioned, the bcl-2 family of proteins can influence both the progression and chemotherapeutic responses of melanoma. while conventional chemotherapeutic regimens can modulate the levels of individual anti- and/or pro - apoptotic bcl-2 proteins, changes in one or two proteins furthermore, since anti - apoptotic proteins are numerous, there are multiple members with the potential to inhibit pro - apoptotic signaling leading to chemoresistance. while there are notions to suggest that loss of bid / bim or bak / bax can lead to cancer development and/or chemoresistance, most data suggest that these proteins are not commonly targeted, which affords the over - expressed anti - apoptotic members a unique opportunity for pharmacological regulation (letai, 2008). with this understanding, directly targeting the anti - apoptotic bcl-2 proteins, and indirectly the pro - apoptotic machinery they control, is an attractive tool with potential to generate new therapies, as well as complement existing treatments. in this endeavor, the greatest successes have come through the identification of small molecules that mimic the function of bh3-only proteins to bind within the hydrophobic pocket of the anti - apoptotic bcl-2 proteins. these small molecules, referred to as bh3 mimetics, functionally neutralize several anti - apoptotic proteins, thus inhibiting their ability to sequester pro - apoptotic proteins. this strategy lowers the cellular threshold leading to bak / bax activation, momp, and subsequent apoptosis. the notion that regulating bcl-2 function in melanoma could yield enhanced responses to chemotherapeutics arose from a study that showed bcl-2 antisense improved treatment responses and tumor burden in a xenograft mouse model of human disease (jansen., however, the use of bcl-2 antisense adjuvant therapy (oblimersen sodium) with dacarbazine failed to meet expectations in several trials (conducted by genta incorporated), although there continues to be some controversy as to whether or not bcl-2 antisense is beneficial (jansen. independent of those results, pharmacological regulation of anti - apoptotic bcl-2 proteins continues to be of tremendous interest. a bh3 mimetic that has been studied with considerable success in melanoma is the abbott compound abt-737 (and the bio - available form abt-263). published in 2005, abt-737 was identified using a structural activity relationship by nuclear magnetic resonance (sar by nrm) screen for compounds that could bind within the hydrophobic groove of bcl - xl (oltersdorf., 2005). upon its discovery, abt-737 was determined to have high affinity for the anti - apoptotic bcl-2 proteins bcl-2, bcl - w, and bcl - xl, but not a1 or mcl-1. abt-737 was immediately cast as a promising new drug candidate since it could engage multiple anti - apoptotic bcl-2 family members, and it demonstrated minimal toxicity as a single agent. however, the inability of abt-737 to inhibit mcl-1 (or a1, however, a1 has restricted expression so the influence in cancer is far less than mcl-1), which is commonly over - expressed in melanomas, highlighted one weakness for using abt-737 in melanoma, and suggested a possible mechanism by which abt-737 resistance may be conferred (yecies., 2010). indeed, melanoma survival and sensitivity to chemotherapeutics appears to be mediated by a combination of anti - apoptotic bcl-2 members as preclinical studies using antisense oligonucleotides against bcl-2, bcl - xl, or mcl-1 sensitized to chemotherapy - induced apoptosis (gautschi., 2001 ; del bufalo., 2003 ; thallinger., a recent study examined the result of reduced mcl-1 expression in combination with abt-737 treatment in melanoma cells (keuling., 2009). indeed, the results showed an increased induction of apoptosis, and opened up the possibility for pro - apoptotic synergy between abt-737 and classical chemotherapeutic treatments that decrease mcl-1 levels. for example, abt-737 in combination with the standard clinically approved anti - melanoma drug, dacarbazine, promoted apoptosis through the induction of noxa, which likely synergizes with abt-737 to fully inhibit the anti - apoptotic repertoire (weber., 2009). in a similar scenario, abt-737 synergized with the proteasome inhibitor mg-132, which was also suggested to induce noxa expression and inhibition of mcl-1 function leading to increased rates of apoptosis (fernandez., 2005 ; qin., 2005 ; miller., 2009). alternatively, because of frequent mutations in the mapk pathway in melanoma, abt-737 has also been studied in combination with mek and p38 inhibitors, both of which synergize to enhance the apoptotic machinery (cragg., 2008 ; keuling. tw-37 is another bh3 mimetic, but it has a broader range of targets including bcl-2, bcl - xl, and mcl-1(verhaegen., 2006). due to the ability of tw-37 to inhibit multiple members, in particular mcl-1, it was proposed that tw-37 could synergize with chemotherapeutics to promote apoptosis independently of noxa regulation. indeed, when melanoma lines were treated with tw-37 and a mek inhibitor (u0126), significant apoptosis resulted. interestingly, the observed synergy did not occur in normal melanocytes, suggesting that the mechanism of action was specific to tumor cells. curiously, the observed apoptosis was also dependent upon the p53 pathway, which is functionally intact in the majority of melanoma cell lines and patients. obatoclax is also an inhibitor to the anti - apoptotic bcl-2 family proteins that has been studied in melanoma. first described in 2007, obatoclax is believed to inhibit all of the anti - apoptotic bcl-2 proteins, including mcl-1, and thus shows less resistance to treatment than abt-737 (nguyen. while obatoclax has been shown to induce apoptosis as a single agent treatment in other types of cancer, such efficacy has not been observed in melanoma, leading to the study of combination treatments (trudel., 2007). when combined with the endoplasmic reticulum stressors tunicamycin or thapsigargin, increased apoptosis was observed (jiang., 2009). this synergy is likely due to obatoclax functioning like a sensitizer bh3-only protein to inhibit the anti - apoptotic bcl-2 proteins, lowering the cellular threshold for bak / bax activation, and allowing the mitochondrial apoptotic machinery to be engaged when signaled. however, it should be noted that despite its confirmed inhibiting interaction with the anti - apoptotic bcl-2 proteins, albeit at a higher concentrations than abt-737, the mechanism by which obatoclax elicits a response is not definitive, as studies have suggested alternative targets than the bcl-2 family (konopleva., 2008). while the above drugs target the anti - apoptotic bcl-2 proteins, none of them have proven to be as effective at binding the anti - apoptotics proteins as the bh3-only proteins themselves. given the importance of the bh3 domain anti - apoptotic interaction, which lowers the cellular threshold of apoptosis, short chemically synthesized peptides mimicking the different bh3-only proteins have been generated and tested for function (letai., 2002). indeed, bh3 domain peptides from different bh3-only proteins revealed the distinction between direct activator and sensitizer / de - repressor bh3-only proteins (figure 3). the bid and bim bh3 domain peptides promote bak / bax - dependent momp and cytochrome c release, whereas the bad bh3 promotes momp by inhibiting bcl-2, bcl - xl, and bcl - w (and revealing bid / bim activity, see figure 3), and not by direct bak / bax interactions (chen. one issue that arose with using synthetic peptides was the loss of -helicity that is required for protein - protein interactions between the bh3-only proteins and the anti - apoptotics. to address this issue, non - natural amino acids containing olefin - bearing tethers were introduced into the bh3 domain sequence, effectively stapling the peptide into an -helical form (walensky., 2004, 2006). these stapled bh3 domains are referred to as stabilized -helix of bcl-2 domains (sahbs), and these may serve as a new tool for designing cancer therapeutics to target the bcl-2 family and enhance endogenous bh3-only activity. for example, the bid bh3 sahb was shown to induce apoptosis in leukemia cells as well as inhibit growth of human leukemia xenografts in vivo (walensky., 2004). recently, a mcl-1 bh3 sahb was developed which inhibits mcl-1 with very high affinity, and indeed has also been shown to sensitize cancer cells to apoptosis (n.b., the mcl-1 sahb uniquely binds to the mcl-1 hydrophobic groove ; stewart., the identification of an exclusive and potent mcl-1 inhibitor is important, given the lack of potent inhibitors that target mcl-1 and the emergence of mcl-1 as a chemoresistance factor in a broad range of human cancers. these developments will hopefully serve as an additional platform to discover small molecule regulators of the bcl-2 family. presently, bh3 domain peptides have not been examined in the context of melanoma treatment ; efforts to elucidate their potential impact would be useful for understanding both the biology of melanoma, and hopefully its treatment. of note, bh3 mimetics also promote autophagy, alone or in combination with b - raf inhibition ; and this may be a contributing mechanism with therapeutic potential in metastatic melanoma (armstrong., 2011 ; malik. throughout our discussions, we focused on applying the mechanisms of action for the bcl-2 family to the regulation of melanoma tumorigenesis, apoptosis, and treatment. as the bcl-2 family defines the balance between life and death in normal and cancer cells, it is reasonable to seek a full understanding of the cellular mechanisms that control bcl-2 family function to establish how stressed cells undergo apoptosis, and how cancer cells resist pro - apoptotic signals to ensure survival. here we suggest that multiple anti - apoptotic bcl-2 proteins ensure survival, and that melanocytes undergoing stress during tumorigenesis and metastasis increase the expression and/or function of these pro - survival members. likewise, chemotherapeutics (single agents and combinations) must promote the inhibition of these pro - survival members by a combination of bh3-only protein functions to engage bak / bax activation and achieve desired clinical outcomes. while we have focused on the direct targeting of the bcl-2 family to promote momp, it is important to mention that there are also pharmacological opportunities to promote apoptosis both upstream and downstream of mitochondria. in particular, the development of xiap (x - linked inhibitor of apoptosis protein) inhibitors is critical as the increased expression of xiap is positively correlated with melanoma thickness, tumor progression, and chemoresistance (chawla - sarkar., 2004 ; kluger. xiap is a potent inhibitor to the apoptotic caspases, so relieving this apoptotic brake is predicted to lower the threshold leading to the cell death phenotype (eckelman., 2006). indeed, the development of smac (second mitochondria - derived activator of caspases, the cellular xiap inhibitor) mimetics to target xiap is shown to induce apoptosis in numerous melanoma cell lines (vucic., 2002 there are also numerous additional pathways that offer opportunities to enhance tumor cell killing though momp and apoptosis. for example, epigenetic mechanisms are implicated in melanoma survival as mir-149 and mir-193b enhance mcl-1 expression in nevi, patient tumor samples, and melanoma cell lines (chen., 2011 ; jin., 2011). histone deacetylase (hdac) inhibitors sensitize melanoma cells to chemotherapy through a number of mechanisms, including suppression of the ras / mapk signaling pathway and enhanced dna damage signaling leading to apoptosis (peltonen., 2005 ; kobayashi., 2006 ; munshi., furthermore, the role of chromatin modifications may offer collateral means of pharmacological intervention as the requirement for swi / snf complexes in mitf regulated melanocyte - specific gene expression pathways directly impact on bcl-2 expression, and likely sensitivity to chemotherapeutics (de la serna. 2010). determining the appropriate combination treatments for each patient based on his / her individual tumor status will likely generate the most effective treatments fortunately, the cellular signaling pathways that intersect between the survival and apoptosis cascades are numerous (e.g., b - raf mutation and bcl-2 over - expression ; proteasome function and mcl-1 over - expression) and this affords the pharmacological opportunity to explore multiple combination strategies to cure melanoma. in the future, enhanced animal models of human disease will likely shed light upon the mechanisms that drive melanocyte tumorigenesis and metastasis. the combined b - raf v600e / pten mouse model mentioned earlier is perhaps the ideal model system to genetically evaluate the role of the bcl-2 family in melanoma progression (dankort., 2009). as further models are developed, they should be evaluated along side the standard models of bcl-2 family function (e.g., bak bax, bidbim, puma, melanocyte - specific bcl-2 and mcl-1 transgenics, etc) to fully appreciate the influence of the bcl-2 family in mediating melanoma development and treatment. likewise, a better understanding of the pathways (e.g., mitochondrial dynamics, structural studies, lipid environment etc) that regulate the bcl-2 family itself will certainly yield novel target strategies to obtain better clinical outcomes. the future shows significant promise to develop more specific and bio - available small molecule inhibitors to the anti - apoptotic bcl-2 proteins, and perhaps drugs that directly promote bak / bax activation ; this will be another significant milestone in adjuvant - based chemotherapeutic strategies targeting the mitochondrial pathway of apoptosis. the hope is that as we further dissect the mechanisms of the bcl-2 family and apoptotic pathways, novel therapeutic targets and combination strategies will emerge and provide significant benefits to those afflicted with melanoma. 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. | the global incidence of melanoma has dramatically increased during the recent decades, yet the advancement of primary and adjuvant therapies has not kept a similar pace. the development of melanoma is often centered on cellular signaling that hyper - activates survival pathways, while inducing a concomitant blockade to cell death. aberrations in cell death signaling not only promote tumor survival and enhanced metastatic potential, but also create resistance to anti - tumor strategies. chemotherapeutic agents target melanoma tumor cells by inducing a form of cell death called apoptosis, which is governed by the bcl-2 family of proteins. the bcl-2 family is comprised of anti - apoptotic proteins (e.g., bcl-2, bcl - xl, and mcl-1) and pro - apoptotic proteins (e.g., bak, bax, and bim), and their coordinated regulation and function are essential for optimal responses to chemotherapeutics. here we will discuss what is currently known about the mechanisms of bcl-2 family function with a focus on the signaling pathways that maintain melanoma tumor cell survival. importantly, we will critically evaluate the literature regarding how chemotherapeutic strategies directly impact on bcl-2 family function and offer several suggestions for future regimens to target melanoma and enhance patient survival. |
he3286 (17-ethynylandrost-5-ene-3,7,17-triol) is a chemical derivative of the natural mammalian sterol androst-5-ene-3,7,17-triol (aet). aet exhibits anti - inflammatory activity in rodent models, is elevated in plasma of obese subjects with normal glucose disposal, and may play a compensatory role in preventing development of metabolic syndrome (reviewed in). aet is pharmaceutically unsuitable, due to poor oral bioavailability and its propensity for oxidative inactivation by 17-hydroxysteroid dehydrogenase. he3286 is stabilized against oxidation at position 17 and consequently orally bioavailable, does not bind to any known nuclear steroid hormone receptors, and is pharmacologically unrelated to androgens, estrogens, corticosteroids, or peroxisome proliferators. he3286 has shown broad anti - inflammatory activity in animal models of rheumatoid arthritis, ulcerative colitis, multiple sclerosis, lung inflammation, autoimmune type 1 diabetes, and neuroinflammation (reviewed in). in these models, nuclear factor kappa b (nfb) activation and proinflammatory cytokine production furthermore, he3286 was not markedly immunosuppressive in rodent models of ovalbumin immunization, klebsiella pneumoniae or pseudomonas aeruginosa infection, coxsackievirus b3 myocarditis, delayed - type hypersensitivity, and mitogen - induced proliferation, or in the human mixed lymphocyte reaction assay (reviewed in). obesity induces an insulin - resistant state in adipose tissue, liver, and muscle and is a strong risk factor for the development of type 2 diabetes mellitus. in adipose tissue, mcp-1 and tumor necrosis factor alpha (tnf) play dominant proinflammatory roles. adiposity - induced inflammation - stimulated kinases phosphorylate insulin receptor substrate-1 on serine residues and inhibit insulin signaling. two recent publications report the activity of he3286 against in vitro inflammatory responses and in vivo rodent models of obesity - induced inflammation and insulin resistance [5, 6 ]. he3286 suppressed endotoxin - induced nfb activation, reporter gene expression, nuclear localization, and p65 phosphorylation in mouse macrophages and decreased phosphorylation of the proinflammatory extracellular signal - regulated (erk1/2), ikappab (ikk), jun n - terminal (jnk), and p38 mitogen - activated protein (p38 mapk) kinases. he3286 also attenuated tnf-stimulated inflammation and tnf-induced adipocyte - stimulated macrophage chemotaxis [5, 6 ]. he3286 treatment of diabetic db / db mice, insulin - resistant diet - induced obese mice, and genetically obese ob / ob mice suppressed progression to hyperglycemia and markedly improved glucose clearance. this effect appeared to be consequent to reduced insulin resistance, since he3286 lowered blood insulin levels in both db / db and ob / ob mice. in these studies he3286 suppressed levels of the chemokine monocyte chemoattractant protein-1 (mcp-1), along with its cognate receptor, c - c motif chemokine receptor-2, in white adipose tissue. in zucker diabetic fatty rats, he3286 downregulated inflammatory cytokine and chemokine expression in both liver and adipose tissues and suppressed macrophage migration into adipose tissue. normalized fasting and fed glucose levels, improved glucose tolerance, and enhanced skeletal muscle and liver insulin sensitivity, as assessed by hyperinsulinemic, euglycemic clamp studies. in addition, he3286 reduced liver cholesterol and triglyceride content, leading to a feedback elevation of low - density lipoprotein (ldl) receptor and decreased total serum cholesterol. recently, we have reported that he3286 binds to erk1/2, lrp1, and sirt2 and proposed that the he3286-mediated decrease in hyperactivation of erk1/2 may be causal for its metabolic and anti - inflammatory activities. in a clinical study in obese, impaired glucose tolerance (igt) subjects, he3286 significantly increased the frequency of insulin - resistant subjects with improved day 29 insulin - stimulated glucose disposal, increased hdl cholesterol, and decreased day 28 crp compared to placebo - treated subjects. based on baseline glucose clamp studies, insulin - resistant subjects had elevated inflammatory biomarkers, with lower adiponectin and higher cytokine secretion in lps - stimulated pbmc. after 28 days of he3286 treatment, adiponectin levels increased significantly in insulin - resistant subjects, compared to placebo. these results support our hypothesis that obesity - induced inflammation is a significant contributor to metabolic dysregulation and that the anti - inflammatory activity of he3286 can preferentially benefit the insulin - resistant inflamed subpopulation of obese igt subjects. based on preclinical studies and these foregoing results in igt subjects, it was conjected that he3286 might benefit obese inflamed insulin - resistant individuals with type 2 diabetes mellitus (t2 dm). a widely accepted clinical endpoint for t2 dm is the change in hba1c, a surrogate marker for the extent of hyperglycemia an individual experiences over time. traditionally, erythroid hematology values are considered stable in healthy individuals, and hemoglobin and hba1c turnover is reported to reflect the normal red cell half - life of 3860 days. in t2 dm, the life span of red cells can be altered significantly by inflammation, particularly tnf-induced oxidative stress, obese low - grade systemic inflammatory response syndrome, the presence of elevated levels of advanced glycation endproducts on the surface of red cells [13, 14 ], hypoxia, and excessive erythrocytosis. there are reports of large fluctuations in hba1c in type 1 diabetes, especially in subjects with poor glycemic control [18, 19 ]. this information prompted us to also assess the association of obesity - related chronic low - grade inflammation with hemoglobin concentration and hba1c variability in uncontrolled t2 dm. we retrospectively analyzed the hematologic and metabolic clinical laboratory data for placebo groups from 10 clinical studies that were conducted between 2001 and 2010. these studies included both healthy subjects and individuals in progressive stages of metabolic disease that presented with increased chronic low - grade inflammation coincident with elevated bmi that included dyslipidemic, igt, and t2 dm participants with uncontrolled hba1c. with an understanding of the variability associated with progressive adiposity, inflammation, and metabolic disease, we assessed the activity of he3286 to decrease obesity - induced inflammation and insulin resistance in t2 dm. high metabolic and hematologic laboratory value variances were observed in these patients. for comparison, similar parameters were retrospectively analyzed from placebo subjects enrolled in 10 clinical studies conducted by harbor therapeutics, inc. all studies excluded patients with known liver disease and alcoholism. the protocols and all amendments were reviewed and approved by the relevant institutional review boards, and all studies were conducted in accordance with the declaration of helsinki and the international conference on harmonization / who good clinical practice standards. details of studies 2100 - 200, -201, -202, and -203 have been published. these four double - blind, randomized, placebo - controlled, healthy human safety studies were conducted in the netherlands (kendle international, utrecht) and the united states (parexel international, baltimore, md). two single - dose, dose - escalation studies assured safety and evaluated the pharmacokinetics of androst-5-ene-3,17-diol (he2100) (studies 200 and 202). a multidose, dose escalation study was performed to assess safety and pharmacokinetics and potential early activity of he2100 (study 201). early activity, defined by effects on peripheral blood elements, was confirmed by a follow - up study that included elderly subjects and an initial study of bone marrow hematology (study 203). details of studies 2200 - 100, -101, -120, and -130 have also been published. healthy adult and elderly subjects were randomized to receive three consecutive daily subcutaneous injections of placebo, 50, or 100 mg androst-5-ene-3,7,17-triol (he2200), followed by 2 months of periodic observation (trial 2200 - 100), or to receive placebo, 25, or 100 mg he2200 transmucosally (buccal administration) once daily for five days followed by 2 months of periodic observation (trial 2200 - 101). study 2200 - 120 was a phase ii study in healthy hepatitis b - nave, and elderly (6585 years old) volunteers, who received hepatitis b vaccine, were randomized to concomitantly receive either 100 mg of he2200 or placebo equivalent. subjects received three subcutaneous injections of study drug or placebo prior to the first and second doses of hepatitis b vaccine given 28 days apart. the third dose of vaccine was given at 6 months without he2200 or placebo treatment, and the study terminated 28 days later. study 2200 - 130 was a phase ii study in dyslipidemic subjects, ages 1870 years, with plasma triglyceride concentrations 1.72 mmol / l, total cholesterol levels of 5.78.3 mmol / l, and hdl levels of 1.2 mmol / l for males and 1.4 mmol / l for females. after informed consent was obtained, subjects initiated a step ii aha diet and discontinued all lipid lowering agents for a six - week run - in period. each subject 's lipid profile at week four of the diet was used to determine eligibility for the study. at six weeks, qualified subjects were randomized to receive 25 or 100 mg of he2200 or placebo equivalent by buccal administration for 28 days. he3286 - 0102 was a multicenter, double - blind, dose - ranging phase i study designed with 5 cohorts of obese, impaired glucose tolerance (igt), but otherwise healthy participants. subjects were screened for fasting blood glucose level of 40 pg / ml in metformin - treated subjects, and of bmi > 31 in treatment - nave subjects. heteroscedasticity (differences in variances between subgroups) was tested for changes in insulin, c - peptide, fasting glucose, homa2 % b and homa2 insulin resistance (homa2 ir), leptin, hba1c, insulin, mcp-1, and triglycerides. subgroup distributions were tested for normality (shapiro - wilks w test) for he3286 and placebo treatment. differences in dispersions between he3286 and placebo treatment were analyzed using the 2-sided f test. he3286, 17-ethynylandrost-5-ene-3,7,17-triol active pharmaceutical ingredient was manufactured by norac, azuza, ca, and formulated and filled in gelatin capsules by eminent services corporation, frederick, md. all manufacturing procedures were performed according to current good manufacturing practices. the phase ii trial design was a double - blind, randomized, placebo - controlled parallel group study of the safety, tolerance, and activity of he3286 when administered orally for 12 weeks to adult t2 dm patients (figure 1(a)). this was an adaptive design to investigate the characteristics of t2 dm subjects that respond to he3286. in cohort 1 of the study, 95 eligible patients, who consented to participate, were randomized 1 : 1 to receive study treatment (he3286 10 mg / day or placebo) in addition to a stable dose of metformin. inclusion criteria for cohort 1 included hba1c 7.5% and fasting glucose 12.5 mmol / l. in cohort 2, 69 subjects who consented to participate and who met a revised eligibility criteria as determined by cohort 1 were randomized 1 : 1 to receive study treatment (he3286 10 mg / day or placebo) as monotherapy. after the analysis of data from the first stage of the study, the population for cohort 2 was phenotypically enriched by screening for the following : hba1c 7.010.5%, fasting glucose 12.5 mmol / l, bmi 28 kg / m, insulin 27.8 pmol / l, c - peptide 0.67 nmol / l, and serum mcp-1 36 the sponsor selected sites after a site visit to determine site qualifications and the investigator 's ability to conduct clinical investigations according to the protocol and current good clinical practice regulations : clinical trial registration : he3286 - 0401 nct00694057 http://www.clinicaltrials.gov. first, the variances for selected hematologic and metabolic laboratory values, such as the mean coefficient of variation (cv) and the cv range for each individual subject, were determined and compared with those of healthy subjects. second, the intravisit changes in hba1c were compared for individual subjects for each condition with those of healthy subjects. third, intravisit and day 84 changes in hba1c and other hematology and laboratory parameters were tested for random effects. random effects were tested using residual maximum likelihood (reml) using statxact, and outliers were examined using mahalanobis distance (cytel software corporation, cambridge, ma) in conjunction with sas software (sas institute, cary, nc). correlations were tested using spearman or pearson correlations, and the hypothesis that placebo participants with clinical conditions have higher frequencies of abnormal hematology and laboratory values than healthy subjects was tested using one - tailed fisher 's exact test. heteroscedasticity (tests of different variabilities between subpopulations) was tested for normal distributions (shapiro - wilks w test), and dispersion was tested using the 2-sided f test (prism graph pad, san diego, ca). if there were significant differences in variances between groups, they were further examined using a t - test assuming unequal variances, nonparametric mann - whitney test, or fisher 's exact test. due to the exploratory nature of this hypothesis - testing study through the course of this analysis it was discovered that the inflammatory status of the selected patient population created large and rapid changes in the patient 's red cell mass that affected the whole body hemoglobin mass and consequently the fidelity of the hba1c metric. in order to investigate he3286 treatment effects on hba1c in t2 dm patients, hba1c changes were normalized to the day 84 average hb for each subject, by averaging hb values acquired at each clinic visit. this is statistically justified based on the fact that random effects have a mean of zero but are characterized with high variances. normalized hba1c (nhba1c) was applied to correct for the inflammation - induced variances found in this t2 dm study population with uncontrolled inflammation. hb is the concentration of hemoglobin (reported in units of g / dl). blood volume (male) = 0.6041 + 0.3669 (height in meters) + 0.03219 (weight in kg). blood volume (female) = 0.1833 + 0.3561 (height in meters) + 0.03308 (weight in kg). total hba1c = hba1ctotal hb mass (total hba1c units = g). 84 day average total hb mass = mean of baseline to day 84 total hb mass measurements. normalized hba1c (nhba1c) = 100 (total hba1c/84 day average total hb mass)(nhba1c units = % hb). the hypothesis that chronic low - grade inflammation leads to increased variance in laboratory values was explored by a retrospective review of hematology and metabolic clinical parameters from placebo subjects enrolled in 10 clinical studies conducted by harbor therapeutics, inc., since 2001. only placebo subject data from these studies were used for intercomparisons to exclude study drug effects. changes in variance (cv means and ranges) for hematologic and metabolic parameters sorted by medical condition are displayed in figure 2. dyslipidemic patients showed increased variances in hematocrit, hba1c, and fasting glucose compared to healthy subjects. although their lipid parameters were abnormal, their lipid variances were not significantly higher than those of healthy subjects. igt subjects had significantly higher variances for rbc, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin, and hba1c. although they had higher postprandial glucose, their fasting glucose variances were not significantly greater than those of healthy subjects. metformin - treated t2 dm patients had significantly higher variances for rbc, hematocrit, hemoglobin, hba1c, platelets, fasting glucose, cholesterol, and ldl. treatment - nave t2 dm patients had significantly elevated variances for rbc, hematocrit, mean cell volume, mean corpuscular hemoglobin, hemoglobin, hba1c, platelets, fasting glucose, cholesterol, hdl and ldl. healthy subjects hba1c values were only collected in study 2100 - 202. over 28 days, the 8 subjects showed an intravisit median change of 0 and a range from 0.2 to 0.2% hb, consistent with literature reports. hba1c was measured in dyslipidemic patients on days 1 and 28, yielding a single intravisit value for 21 patients with a median of 0 and an increased range of 0.5 to 0.5% hb. subjects with dysregulated glucose showed a median change of 0 with increased ranges : igt over 56 days (0.3 to 0.4% hb), metformin t2 dm over 112 days (2.2 to 2.0% hb), and treatment - nave t2 dm over 112 days (3.4 to 2.8% hb). figure 3(f) shows the intravisit changes in hba1c for each medical condition on the same scale. these results indicate that the intravisit variances for individual t2 dm patients are increased and distinct from the normal variances in healthy subjects. treatment - nave t2 dm patients had the greatest variance in hba1c. in this group, pearson correlations were used to investigate the hba1c and insulin variance relationships with other clinical parameter variances (table 1). individual patient hba1c cv, were correlated with cvs for hemoglobin, mcp-1, glucose, crp, hdl, ldl, triglycerides, lymphocytes, monocytes, platelets, rbc, hematocrit, and mcv, and insulin cvs were similarly correlated with cvs for glucose, wbc, and neutrophils, indicating dysregulation of multiple hematopoietic and metabolic functions within the same individual. furthermore, reml analyses demonstrated significant random changes in day 84 glucose (p 40 table 4 displays the he3286 day 84 treatment effects on clinical parameters in this subgroup. significant decreases were observed for homa2 ir (p = 0.02), c - peptide (p = 0.04), hb (p = 0.02), hct (p = 0.02), and rbc (p = 0.02) changes in the he3286 treatment group when compared to the placebo (metformin alone) group. therefore, the day 84 treatment effect on nhba1c was investigated in the more inflamed mcp-1 subgroup (table 4). the median magnitude of the nhba1c was 0.44% hb (he3286, 0.34 ; placebo, + 0.1). the he3286 data was normally distributed, but placebo was significantly abnormal (p = 0.0006, w test, data not shown). the he3286 treatment effect was found to significantly decrease nhba1c from zero (p = 0.03). the frequency of he3286 patients with decreased nhba1c was significantly greater than placebo (17/22 versus 9/25, p = 0.0008). there were no significant differences between he3286 and placebo groups at follow - up day 112. > 40 pmol / l are shown for nhba1c in figure 4(a) and for homa2 ir in figure 4(b). the majority of he3286 patients showed decreased nhba1c and homa2 ir, whereas the majority of placebos showed increases. these results are consistent with inhibition of nfb hyperactivation and consequent restoration of normal insulin signaling, consistent with the preclinical he3286 observations. the correlation between baseline bmi and change in hba1c in the he3286 group was explored by stratifying participants on the median bmi (31 kg / m). the nhba1c in the he3286, but not placebo participants (with bmi > 31 kg / m), correlated significantly with their baseline mcp-1 (p = 0.03) (table 3). this strengthens the hypothesis that he3286 benefited the obese inflamed subset of t2 dm patients. the nhba1c also correlated significantly with mcp-1 (p = 0.002) in he3286 (table 3), but not in placebo participants. thus the decrease in inflammation (mcp-1) was associated with the decrease in hba1c with he3286 treatment. the obese patients with a bmi (> 31 kg / m), demonstrated a significant treatment effect (t - test) to decrease nhba1c by 0.6% hb compared to placebo, but only after exclusion of 2 outliers (mahalanobis distance). the day 84 distribution of the nhba1c for bmi > 31 is shown in figure 4(c) (outliers circled). the variances were much higher in the treatment - nave patients ' parameters compared to uncontrolled metformin - treated patients (figure 3(f)). we speculated that these two outliers were still subject to inflammation - induced random effects, after only 84 days of treatment and that additional treatment may be necessary to observe effects in these individuals. because of the lag in hba1c following glucose excursions, we tested the treatment effects on follow - up day 112. a significant day 112 treatment effect (with no outliers) was found in the high bmi stratum, both by nonparametric and parametric tests (table 4). he3286 participants had a significant mean change from baseline (1.0% hb, p = 0.0007), whereas placebo did not. the mean change compared to placebo was also significant (0.7% hb, p = 0.03). the he3286 participants also had a significant median change from baseline (1.2% hb, p = 0.002), whereas placebo did not. the magnitude of the response in the he3286 treatment groups was significant (1.0% hb, p = 0.02) compared to placebo, as was the frequency of subjects with a 0.5% hba1c decrease (9/12 versus 4/13, p 31 kg / m are shown in figure 4(d). a significant treatment effect that lowered fasting glucose consequently, the possibility that he3286 decreased hba1c through action on postprandial glucose was investigated. serum 1,5-anhydroglucitol (1,5-ah) is a dietary human metabolite that is reabsorbed by a kidney glucose transporter. the 1,5-ah level declines when blood glucose levels are elevated above 10 mmol / l and likewise increases when the blood glucose level declines. 1,5-ah was measured in a subset of 42 participants (19 from stages 1 and 23 from stage 2) that had available day 84 retention samples. analysis of 18 patients treated with he3286 demonstrated that their 1,5-ah concentration increased significantly (+ 10.4 mol / l, p = 0.02) ; 24 treated with placebo demonstrated no significant concentration increase (+ 0.6 mol / l, p > 0.1). the majority of he3286 patients significantly increased 1,5-ah, compared to placebos (15/18 versus 11/24, p = 0.02, fisher 's exact test). this outcome indicates that he3286 had a treatment effect to decrease postprandial glucose excursions compared to placebo, which further supports that it 's pharmacologic property is to decrease insulin resistance (see and figure 4(b)) and lower hba1c. heteroscedasticity (differences in variances between groups) was investigated by analyzing data distributions for normality (shapiro - wilks w test) and analyzing dispersion (2-sided f test). in cohort 1 placebo, but not he3286, day 84 distributions (w test) were significantly abnormal for changes in insulin, c - peptide, fasting glucose, homa2 % b, homa2 ir, and leptin in all subjects, and for changes in hba1c, fasting glucose, and homa2 % b for mcp-1 > 40 pmol / l participants. variances for cohort 1 placebo subjects (f test) were also significantly higher than those of he3286 subjects for insulin, c - peptide, and homa2 ir for all subjects. cohort 2 placebo, but not he3286 distributions were abnormal (w test) for the group as a whole for changes in all the following parameters : day 84 nhba1c, fasting glucose, mcp-1, and triglycerides and day 112 nhba1c, fructosamine, and homa2 % b. in the bmi > 31 subgroup, abnormal distributions were found for changes in all the following parameters : day 84 homa2 % b and day 112 insulin, c - peptide, homa2 % b, and homa2 ir. variances for cohort 2 placebo subjects as a whole were significantly higher (f test) for changes in all of the following parameters : day 84 insulin, homa2 % b, and triglycerides, and day 112 insulin and homa2 % b. variances in placebo were also higher for the bmi > 31 kg / m subgroup for changes in day 84 mcp-1 and triglycerides and day 112 insulin (table 5). these differences in distribution and dispersion between groups were not readily evident until day 84 of treatment (data not shown). together, these findings further support an he3286 treatment effect that decreases random metabolic effects and restores homeostasis to uncontrolled t2 dm patients. this initial clinical trial of he3286 in diabetes was designed to take all eligible patients with uncomplicated t2 dm even though he3286 was only qualified in animal models of obese diabetes and subsequently only demonstrated activity in obese individuals that present with inflammation - induced insulin resistance. the strategic intent of the study was to identify the responding t2 dm population by surveying a broad swath of the constellation of syndromes that are defined by the t2 dm condition. based on findings in cohort 1, which indicated low bmi individuals were he3286 nonresponders, and the inclusion criteria in the second cohort of the trial were modified, concentrating the population to elevated weight (bmi) and inflammatory status (mcp-1). this eliminated the patient population that had progressed to lose significant -cell function and who were no longer able to produce insulin, a population clearly not indicated for treatment with an insulin sensitizer. notably, these criteria were also imposed on clinical trials with the thiazolidinediones (j. olefsky, personal communication). in addition treatment - nave patients were recruited in cohort 2 to remove the potential for metformin to blunt the he3286 treatment effect and consequently amplify the single agent treatment outcome. we designed this study to test the hypothesis, based on preclinical data and on molecular studies of he3286 binding partners, that he3286 would decrease the hyperactivation of nfkb with consequent restoration of insulin signaling [5, 6 ], dependent on its interaction with extracellular signal regulated kinase (erk) 1 and 2 in addition to other binding partners. erk1 is an important mediator of inflammation - induced insulin resistance [2325 ], insulin receptor substrate (irs)-1 serine (inhibitory) phosphorylation, and the inhibitory effect of tnf on insulin signaling. he3286 does not inhibit insulin - mediated erk activation, but inhibits lps- and tnf-stimulated erk hyper - activation, and irs-1 serine phosphorylation mediated by ikk and jnk [5, 6 ]. coincident he3286-mediated changes in erk, ikk, jnk, and p38 mapk signal transduction may explain the preferential responses observed in high adiposity inflamed t2 dm patients. signal transduction pathways in omental fat are altered in obese, compared to lean individuals. in humans, activation of jnk and p38 mapk was increased in omental fat (compared to paired subcutaneous fat) from obese, but not lean individuals, and this hyperphosphorylation correlated with clinical parameters of hyperglycemia and insulin resistance. it will be important to further clarify the role of erk in the activity of he3286. data analysis presented here demonstrated that the cohort 1 day 84 changes in the primary end point hba1c had a significant relationship with expected changes in beta - cell function, fasting glucose, and weight, and also with baseline inflammation status (mcp-1). surprisingly a relationship with hemoglobin was also detected, a biomarker that is presumed stable over several weeks. of these covariates associated with hba1c change, only fasting glucose was significant in placebo patients. in the enriched cohort 2 population, the hba1c he3286 treatment response was no longer dependent on mcp-1 but rather bmi with a statistically significant negative correlation ; the higher the bmi the larger the effect on hba1c decline. the cohort 2 outcome remained correlated with expected changes in -cell function and with fasting glucose. thus the general population enrolled in cohort 1 was a very different ensemble of participants than those enrolled in cohort 2. while the relationships of change in hba1c with changes in -cell function and fasting glucose remained, the relationship to weight loss was not seen in the cohort 2 participants selected with higher bmi inclusion criteria. cohort 1 placebo group hba1c change was dependent only on baseline inflammation status (tnf and day 84 tnf change). in cohort 2 importantly, fasting glucose change was not correlated with hba1c change in this group, indicating that glucose levels were uncoupled from the hba1c surrogate marker. rather, placebo hba1c variance (cv) was correlated strictly with inflammation in both cohorts. this was evidenced by correlation to baseline tnf in cohort 1 and dependent on both changes in mcp-1 and surprisingly hemoglobin cv in cohort 2. hb cv was in turn dependent on baseline mcp-1 (tnf was not measured). in cohort 1 this later dependency on hb cv was not detected perhaps due to the heterogeneity of the more general patient population (including nonobese and noninflamed diabetics). in cohort 2, placebo hba1c cv was negatively correlated with weight change, indicating that higher weight led to increased variance. cohort 2 placebo weight loss was unexpectedly unrelated to hba1c and glucose control. since placebo hba1c cv was correlated with tnf change in cohort 1, the weight loss associated with higher hba1c cv in the cohort 2 placebo group is presumed to be related to inflammation effects on satiety or metabolism leading to changes in caloric intake and energy balance. the significant correlation of changes in hba1c and hemoglobin was an unexpected observation as hemoglobin is considered an invariant biomass from which hba1c is formed as a reflection of total hyperglycemia and therefore its status as an fda approved biomarker. inspection of individual patient hba1c changes revealed a high degree of intravisit variance, contrary to its presumed highly controlled and stable total body mass. further exploration of the hematopoietic elements gathered with the safety data demonstrated these variant effects were not only on the hemoglobin mass but also on other components such as rbc, hematocrit, mean red cell volume, mean corpuscular hemoglobin and platelets, as well as a variety of metabolic parameters including glucose and cholesterol. unexpected variance in metabolic and hematologic parameters related to the effects of chronic low - grade inflammation in uncontrolled obese diabetes produced a significant barrier to these analyses and data interpretation. the variances that caused differing distributions and dispersions between treatment and placebo groups ' coupled with the he3286 treatment effect presented significant statistical challenges. statistically random effects in the treatment - nave placebo group were demonstrated for day 84 changes in glucose and in the key surrogate parameters hemoglobin, hba1c and homa2 % b. the increasing variances in individual hba1c change with metabolic disease progression demonstrated median changes of zero for dyslipidemic, igt, and t2 dm patients. statistically, random effects are assumed to be the realization of a normal distribution with a mean of zero and a variance that can be estimated. in order to investigate he3286 treatment effects, we were prompted to remove this random component by normalizing hba1c to the day 84 average total hemoglobin mass (mean 84-day change of zero) for each patient. in the broadly defined population of metformin - treated t2 dm patients (cohort 1), the he3286 responsive patient population was found in the upper two tertiles of the inflammation marker mcp-1 (40 pmol / l). in the inflamed treatment - nave patients ' population studied in cohort 2, the responding population was found above the median bmi (obese subjects, > 31 kg / m). the magnitude of the treatment response was indeed greater in the treatment - nave (cohort 2) than metformin - treated patients. in both cohort 1 and 2, he3286 treatment was associated with a total hb mass normalization evidenced by day 84 data distributions and decreased variances in numerous metabolic and erythroid values. for several of these dysregulated parameters, we interpret these results to indicate that he3286, via its anti - inflammatory activity, decreased inflammation - driven metabolic dysregulation. he3286 showed a significant effect to improve insulin resistance in igt subjects and to decrease homa2 ir in cohort 1 t2 dm patients, but not in cohort 2. it is possible that since cohort 2 was nave, previously untreated t2 dm patients and showed higher variances, additional improvements would be observed with longer treatment time frames or drug combination therapy. the relationship between inflammation and increased variances in erythroid and metabolic laboratory parameters was investigated in clinical settings of increasing chronic low - grade inflammation, adiposity, and metabolic dysregulation. compared to a healthy group, significantly increased variances were observed for hematocrit, and hba1c for dyslipidemic, igt, and t2 dm patients. rbc and hemoglobin values were also significantly variable, and the fasting glucose was variable in both dyslipidemic and t2 dm patients. in treatment - naive t2 dm, high variances and random effects were observed in a large number of metabolic and hematologic parameters that the medical community relies on for medical diagnoses. this data supports our hypothesis that, in obese subjects, adipose tissue inflammation contributes to both metabolic and hematologic dysregulation within the same individuals. this is the first clinical report of extreme fluctuations in the marker hba1c in patients with uncontrolled type 2 diabetes mellitus, but there are published data for type 1 diabetes mellitus (t1 dm). fluctuations in % hba1c of more than 1% occurred in 50% of the patients year to year, and over 9 years the minimum - maximum range was > 3% and > 5% hba1c in 55% and 11% of patients, respectively,. in t1 dm subjects followed for 4 years, there was high cv for intraindividual hba1c measurements (15.5 8.1%), which was lower for patients with good glycemic control. intrasubject variations of fasting glucose and hba1c (hba1c 68%, with 7 be treated with additional agents to return them to a glucose - controlled state. in this specific patient population of type 2 diabetes with hba1c that is uncontrolled according to the ada recommendations, the authors have found that the basic hypothesis of stable hemoglobin and red cell lifespan allowing extrapolation from hba1c change and glucose control is flawed, that intravisit fluctuations can be large, and that a change in hba1c values between two visits is unlikely to reflect a meaningful therapeutic effect on glucose control in this uncontrolled population. thus, in clinical efficacy studies in patients with poorly controlled hba1c, the authors recommend that additional tests of glucose control be used for determination of efficacy of new antidiabetic therapies. numerous publications argue for the improved management by using continuous glucose monitoring, and for the time - averaged effects of using 1,5-anhydroglucitol to better understand variation in glucose control. the hypotheses tested in this study appear to be borne out in the high adiposity t2 dm patient. since inflammatory changes were driving hba1c changes in the placebo group for both cohorts 1 and 2, the changes observed with he3286 treatment appear to be due to its anti - inflammatory activity (i.e., to break the cycle of inflammatory kinase - mediated inhibition of insulin receptor signaling). furthermore, t2 dm subjects that lack chronic, low - grade inflammation lack the specific lesion in the insulin receptor signaling pathway that he3286 was developed to interdict. their glucose intolerance arises for other reasons, and therefore they are unaffected by he3286. obese type 2 diabetic incidence is increasing at an alarming rate. regaining glucose control and metabolic regulation and preventing or delaying macrovascular and microvascular complications could help to contain rising health care costs for end - stage diabetes complications. understanding which patients are to benefit from a new therapy is now a regulatory consideration. the fda has published the critical path initiative, with personalized medicine, or the patient - specific information to individualize therapy and disease management as a major theme, and published on the importance of clinical validation of personalized medicine selection criteria in diabetes. based on estimates of obese, inflamed diabetics in the future at approximately 50% (j. olefsky, personal communication) he3286 is active at low (hormonal level) doses and is an anti - inflammatory insulin sensitizer with a toxicology profile conducive for chronic daily use. in the responsive subpopulations he3286 significantly decreased hba1c compared to placebo, by day 84 in metformin - treated subjects with high mcp-1, and by day 112 in treatment - nave subjects with high bmi. the data presented here in uncontrolled t2 dm patients make a compelling argument for further testing of he3286 in the high adiposity, inflamed t2 dm patient subset, using oral glucose tolerance testing, 1,5-ah, or continuous glucose monitoring to assess treatment effects. the correlation or lack thereof with the surrogate marker hba1c should be confirmed in these uncontrolled patients. | obesity - related inflammation - induced insulin resistance and metabolic dysregulation were investigated in retrospective analysis of placebo hematologic and metabolic laboratory data from trials associated with increasing chronic low - grade inflammation and body mass index. studies included healthy subjects and those with progressive stages of metabolic dysregulation, including type 2 diabetes mellitus with uncontrolled hemoglobin a1c. intrasubject variances in erythroid and metabolic values increased with metabolic dysregulation. random effects were demonstrated in treatment - nave diabetes for erythroid, glucose, and hba1c fluctuations. the anti - inflammatory insulin sensitizer, he3286, was tested for its ability to decrease obesity - related inflammation - induced insulin resistance and metabolic dysregulation in diabetes. he3286 significantly decreased erythroid and metabolic variances and improved 1,5-anhydroglucitol (a surrogate of postprandial glucose) compared to the placebo group. he3286 hba1c decrease correlated with weight loss and inversely with baseline monocyte chemoattractant protein-1 (mcp-1) in metformin - treated diabetics. normalization of hba1c to the 84-day average hemoglobin revealed that he3286 hba1c decrease correlated with high baseline mcp-1 and mcp-1 decrease in treatment - nave diabetics. he3286 decreased insulin resistance, increased the frequency of decreased day 84 hba1c in metformin - treated subjects, and decreased day 112 hba1c in treatment - nave diabetics. he3286 may be useful to restore metabolic homeostasis in type 2 diabetes. |
glycogen synthase kinase-3 (gsk-3) is a cmgc serine / threonine protein kinase initially described as one of the kinases that phosphorylates and inhibits glycogen synthase. it is now widely accepted though that gsk-3 plays an important role in various essential physiological processes, such as development, cell cycle, or apoptosis. apart from glycogen synthase, a plethora of different substrates has been identified in all cellular compartments, that is, metabolic proteins, cytoskeletal proteins, and transduction and transcription factors (see table 1). in neuronal development, gsk-3 has been reported to control morphogenesis and axonal polarity, synaptogenesis, and survival [9, 10 ]. in addition, gsk-3 dysfunction has been associated with brain pathological conditions, such as alzheimer 's disease (ad) [11, 12 ] or prion neurotoxicity. thus, the deep knowledge of the role of both gsk-3 isoforms in brain metabolism will allow us to understand their contribution to neurodegenerative processes. gsk-3 unique position in modulating the function of a diverse series of proteins in combination with its association with a wide variety of human disorders has attracted significant attention to the protein both as a therapeutic target and as a means to understand the molecular bases of these disorders. furthermore, gsk-3 appears to be a cellular nexus, integrating several signalling systems, including numerous second messengers and a wide selection of cellular stimulants. gsk-3 has been highly conserved during evolution, and homolog genes have been identified in virtually every eukaryotic genome investigated, including species, such as dictyostelium discoideum, xenopus laevis, or drosophila melanogaster [1416 ]. in mammals, gsk-3 is encoded by two genes known as gsk-3 and gsk-3 [17, 18 ] encoding gsk-3 (483 aa in humans) and gsk-3 (433 aa) proteins with apparent molecular masses of 51 and 47 kda, respectively. both isoforms are almost identical (98%) within their atp binding pocket but differ at their n- and c - terminal domains. a neuron - specific splicing isoform (2) having an insertion of 13 aa within the substrate - binding domain has also been described. mammalian gsk-3 and are each widely expressed although some tissues show preferential levels of some of the two proteins. crystallographic studies have revealed the three - dimensional structure of gsk-3 [21, 22 ]. its overall shape is shared by all kinases, with a small n - terminal lobe mostly consisting of -sheets and a large c - terminal lobe essentially formed of -helices. the atp binding pocket is located between the two lobes and although, is well conserved among kinases, it is possible to obtain selective inhibitors by taking advantage of the small differences that exist between the different kinases. current availability of crystal structures of complexes of gsk-3 with a variety of ligands, together with molecular modelling approaches, provides the necessary clues for enhancing selectivity towards gsk-3. some gsk-3 substrates do not require a very specific sequence, but rather a previous (primed) phosphorylation by a priming kinase on a ser or thr residue located four aminoacids, c - terminal to the ser or thr residue to be modified by gsk-3 (see below for regulation through primed phosphorylation). the crystal structure of human gsk-3 has provided a model for the binding of prephosphorylated substrates to the kinase. according to it, primed ser / thr is recognized by a positively charged binding pocket formed by residues arg96, arg180, and lys205 that facilitates the binding of the phosphate group of primed substrates. gsk3 uses the phosphorylated serine or threonine at position + 4 of the substrate to align of the two domains for optimal catalytic activity [21, 22 ]. furthermore, crystal structures of gsk-3 complexes with interacting proteins frat / gbp and axin have allowed defining the molecular basis for those interactions, which play critical role in some signalling pathways (see below for regulation through protein complex formation). these studies confirm the partial overlap of the binding sites of axin and frat1/gbp predicted from genetic and biochemical studies [2, 25 ] but reveal significant differences in the detailed interactions and identify key residues mediating the differential interaction with both proteins. this ability of gsk-3 to bind two different proteins with high specificity via the same binding site is mediated by the conformational plasticity of the 285299 loop, while some residues in this versatile binding site are involved in interactions with both axin and frat ; others are involved uniquely with one or the other. as already mentioned above, one of the main characteristics of gsk-3 is that its activity is high in resting, unstimulated cells while regulated by extracellular signals that typically induce a rapid and reversible decrease in enzymatic activity. glycogen synthase kinase-3 is a dual specificity kinase differentially regulated by tyrosine and serine / threonine phosphorylation. and for many years, it was believed to be a constitutively active kinase ; however, it has become apparent that the activity of gsk-3 may be regulated by a variety of means. in fact, control of gsk-3 activity occurs by complex mechanisms that are each dependent upon specific signalling pathways. the first regulatory mechanism described of gsk-3 activity involved the phosphorylation of specific residues of gsk-3 by other kinases, and more recently through autophosphorylation [17, 28 ]. the first one corresponds with a serine residue at positions 21 in gsk-3 and 9 in gsk-3. it has been clearly established that phosphorylation of serine 21 or 9 correlates with the inhibition of its kinase activity [2931 ]. many protein kinases are capable of phosphorylating gsk-3 at this residue, such as akt, ilk, pka, and p90rsk [32, 33 ], and many physiological situations of inhibition of gsk-3 correlate with serine phosphorylation, such as insulin / igf1, ngf, or estradiol treatments, not only in neurons. one is the threonine 43, present only in the isoform gsk-3, which may be phosphorylated by erk. second, serine 389 and threonine 390 present in gsk-3 have been shown to be phosphorylated by p38 mapk. in both cases, the data suggested that this phosphorylation may increase the capacity of ser-9 to be phosphorylated rather than promote a direct inhibition (see figure 1). in contrast, tyrosine phosphorylation present in positions 279 in gsk-3 or 216 in gsk-3, appears to correlate with an increase of its kinase activity. different candidates such as pyk-2 and fyn kinases have been reported to be able to phosphorylate gsk-3 in vitro on tyrosine. in addition, mek1/2 has been showed to have this capacity only in fibroblasts [38, 39 ]. this data contrast with those reported in dictyostelium discoideum where there is compelling evidence indicating that zak 1 is responsible for generating tyrosine phosphorylation in gsk-3 [14, 40 ]. more recently, an alternative hypothesis has been proposed for the regulation of gsk-3 tyrosine phosphorylation. this hypothesis suggests that in mammalian systems phosphotyrosine in gsk-3 corresponds to an intramolecular autophosphorylation event and may be regulated by hsp90. molecular dynamics and crystallographic studies clearly suggest that tyr216 renders the kinase active through interactions with arg220 and arg223, stabilizing the activation loop and allowing full substrate accessibility [42, 43 ]. however, this hypothesis still lacks a cellular demonstration. however, our data indicated that not all pharmacological inhibitors of gsk-3 decrease the level of phosphotyrosine. therefore, lithium chloride inhibits gsk-3 activity, but this inhibition does not alter its ptyr content. moreover, in neuronal cells, tyrosine phosphorylation of residue 216 or 279 increased following exposure to lpa and even upon exposure of neurons to -amyloid or prp [13, 45, 46 ] in a clear correlation with an increase in gsk-3 activity. in addition, in many neuronal cells, the pharmacological inhibition of tyrosine phosphatases with ortho - vanadate increases the basal level of gsk-3-ptyr. thus, considering all these data, in addition to this tantalizing autoregulatory system proposed, we hypothesized that some as - yet - unidentified tyrosine kinases and phosphatases may also regulate this kinase (see figure 2). one regulatory mechanism that is still not fully understood involves the interaction of the gsk-3 with structural proteins (scaffold proteins). it is well known that gsk-3 contributes to a multiprotein complex formed by axin and adenomatous polyposis coli (apc), among others (for review see, i.e.,). indeed, in the absence of ligand, gsk-3 is able to phosphorylate -catenin for targeting it for proteasome degradation. more recently, some data suggests that this complex may be specific for gsk-32 isoform, which opens the possibility of a deeper analysis of specific functions of gsk-3 isoforms. another system of protein - kinase interaction was denoted as gsk-3-binding protein (gbp or frat) [25, 50 ]. three different frats have been cloned and characterized ; however, their mechanism of action is not well understood. frat1 appears to act as an inhibitory system, whereas frat2 appears to preferentially increase gsk-3-mediated phosphorylation in some residues. surprisingly, recent data demonstrated that frat is dispensable because the triple frat - knockout mouse lacks any major defect in brain development. all these data indicated that the precise role of frat in gsk-3 regulation is still to be defined. using the binding site on gsk-3 for frat / gbp, gskip can block phosphorylation of different substrates and functions as a negative regulator of gsk-3 beta. as previously mentioned, the specificity of many kinases is governed by a consensus sequence of aminoacids sequence. however, as almost general rule, gsk-3 substrates do not require a very specific sequence, but a previous (primed) phosphorylation residue modified by a priming kinase located four aminoacids, c - terminal, to the ser or thr residue to be modified by gsk-3. the crystal structure of human gsk-3 provides a model for the binding of prephosphorylated substrates to the kinase (pdb i d are 1i09 and 1h8f). according to it, primed ser / thr is recognized by a positively charged binding pocket formed by residues arg96, arg180, and lys205 that facilitates the binding of the phosphate group of primed substrates. some priming kinases have been identified, such as cdk-5 [5557 ], par-1, casein kinase i, pk - c, or pk - a. however, it is not clear so far whether a second set of nonprimed substrates may define a different group of functions. in addition, different glycogen synthase kinase-3 isoforms appear to exhibit distinct substrate preference in the brain. in developmental brain, the presence of gsk-3 was high at e18 and peaked on p8, decreasing after that period. in addition, this report showed that the developmental profile of gsk-3 and gsk-3 is different, having downregulated after birth which suggested a differential role in neuronal development. however, the putative differential role of each isoform has been explored in few reports, that is,. it is important to indicate that a portion of gsk-3, mostly, has been reported to be associated in the growth cone. this gsk-3 pool appears to respond rapidly, being modified by phosphorylation and/or relocated in the growth cone by external signals such as semaphorins or ngf. gsk-3 activity is also dependent on its subcellular localization ; some data illustrated the presence of gsk-3 and in many neuronal compartments and in primary neurons, either in axon, dendrite, or in nucleus [66, 67 ]. in addition, gsk-3 has been found in the cytoplasm, nucleus and the mitochondria. considering the list of gsk-3 substrates reported, it is evident that most of its activity should occur in the cytoplasm and in the nucleus, while we have less information about gsk-3 potential targets in the mitochondria. recent data suggested that proteins such as mcl-1 and hexokinase may be regulated by gsk-3 activity. it has been suggested that nuclear gsk-3 may be involved in phosphorylation of many transcription factors such as cyclin d1, -catenin, hsf-1, nfat, and camp - response element - binding protein, among others (table 1), for review see [28, 70, 71 ]. also, it has been proposed that gsk-3 in the nucleus may have a role in alternative splicing. in addition, proapoptotic stimuli induce nuclear accumulation of gsk-3 ; however, this hypothesis has been not established in other neuronal death paradigms (d. simon, unpublished observations). further insight into gsk-3 regulation has been gained very recently by revealing an essential role of multivesicular endosomes in the wnt signalling pathway. a combination of protease protection assays, detergent permeabilization, and cryoimmunoelectron microscopy demonstrated that wnt activation of the frizzled and lrp6 receptors triggers sequestration of gsk-3 into these membrane - bounded organelles, leading to decreased gsk-3 levels in the cytosol. this process seems to require -catenin, forming a feed - forward loop by facilitating gsk-3 sequestration. this regulation involves the removal by calpain of a fragment from the n - terminal region of gsk-3, including the regulatory serines 9/21. the same study showed that both isoforms and are cleaved by calpain, although with different susceptibility. it is noteworthy to consider that a similar mechanism has been described for -catenin in hippocampal neurons, where after nmda - receptor - dependent activation, calpain induced the cleavage of -catenin at the n terminus, generating stable and truncated forms which maintain its transcriptional capacity. likewise, gsk-3 truncation is mediated by extracellular calcium and can be inhibited by memantine, an nmda antagonist used for the treatment of alzheimer 's disease. interestingly, gsk-3 has also been recently shown to be cleaved at the n - terminus (and subsequently activated) by matrix metalloproteinase-2 (mmp-2) in cardiomyoblasts. the regulation of gsk-3, as previously mentioned, is an essential regulatory key controlling many physiological processes in neurons. many external signals may trigger pathways that finally may activate or inhibit gsk-3 activity, either transiently or in more sustained way. these physiological pathways could be subdivided in two major clusters, those that essentially have to inhibit gsk-3 activity, and second, those that may, at least transiently, trigger gsk-3 activity. among these pathways, the signalling triggered by insulin or igf-1 [19, 79, 80 ] and ngf / bdnf / nt3 [81, 82 ] has similar features. these tyrosine kinase receptors initiated cytoplasm signals in which the inhibition of gsk-3 activity is a common feature. it is generally accepted that the kinase implicated in this inhibition is pkb / akt [2931 ], even though kinases such as pka or ilk have also been implicated [32, 33 ]. in all cases, phosphorylation on serine 21 and 9 (and, resp.) the second well - documented pathway is wnt / wingless signalling [83, 84 ]. this signalling has been widely studied, and it has been shown to be essential in early embryonic patterning, cell fate, cellular polarity, and cell movement in both vertebrates and invertebrates [47, 85 ]. in many if not all cell systems, the canonical wnt pathway is formed by a set of phylogenetically conserved proteins including the wnt receptor frizzled (fz), and a coreceptor lrp5/6 ; dishevelled (dsh), and a scaffolding protein that activates a complex formed by axin / apc / gsk3-/-catenin [47, 8588 ]. in this pathway, in the absence of ligand, gsk-3 phosphorylates -catenin, among other proteins, this phosphorylation constituting part of a degradation signal for -catenin. however, in the presence of wnt, the receptor complex triggers a signal in which dsh inhibits the activity of gsk-3 by a mechanism not completely understood, so far. this system appears to be specific for gsk-3 as no counterpart has been described for gsk-3 to date ; however, some gsk-3 activity appears to be necessary for wnt signalling [89, 90 ]. more recently, a bioinformatics - based screen for proteins whose stability may be controlled by gsk-3 has led to the identification of a number of multiple wnt signalling target proteins, suggesting that this pathway controls a broad range of cellular activities apart form -catenin - mediated transcriptional activation. furthermore, gsk-3-mediated wnt signalling seems to regulate the turnover of many cellular proteins [74, 91 ], indicating that gsk-3 phosphorylation - dependent protein degradation may be a widespread cellular mechanism to regulate a variety of cellular processes in response to extracellular signals. functional segregation of the insulin / growth factor and wnt roles requires either that there be no exchange between the subsets of the cellular gsk-3 pool committed to each role, or that the recruitment of gsk-3 to the axin - apc complex can reverse or override inhibitory ser9 phosphorylation present in a recruited gsk-3 molecule. phosphatases capable of removing extant ser9 phosphorylation are certainly known to be associated with the axin - apc complex [92, 93 ]. alternatively, the very substantial enhancement in activity towards -catenin afforded by the axin scaffolding may simply allow a primed -catenin substrate to outcompete a pser9-gsk-3 n - terminal peptide for access to the substrate - binding site. estrogens regulate many physiological processes and fulfil a wide range of functions during development and differentiation in mammals of both sexes. the actions of estrogens are mediated by estrogen receptors and have been classified as either genomic actions or nongenomic, rapid actions. the genomic actions are based on the capacity of the estrogen receptors (ers) to modulate transcriptional activity either directly or through coactivators or corepressors, that is, [94, 95 ]. more recently, it has been shown that in addition to its direct transcriptional activity, estrogen receptors activate a set of cytoplasm signals in a similar manner to some growth factors. hence, it has been reported that estradiol acts synergistically with igf-1 in the brain or in neurons, activating the pi3k / akt pathway [34, 96, 97 ]. this inhibitory phosphorylation is time- and concentration dependent, and an antagonist of estradiol prevents this event. the kinase responsible is sensitive to the inhibition of the pi3k pathway, and for this reason, it seems that the best candidate would be akt [98, 99 ]. a more detailed analysis of these new signals will give us clear evidence whether this pathway is completely convergent with those using pi3k / akt / gsk3, as previously mentioned. in neurons, lpa has been shown to induce neurite retraction and the rounding up of neuroblastoma cell lines. in some primary neurons, it also promotes growth cone collapse and neurite retraction [37, 101 ]. this bioactive lipid acts as a growth factor through specific seven transmembrane domain receptors, denoted as lpa 14 [102, 103 ]. we described that gsk3 activity was increased after lpa treatment in diverse neuronal cells of different species in correlation with the neurite retraction process [104, 105 ]. this activation correlated with an increase in gsk3-ptyr and may be downstream g12 or g13 [101, 105, 106 ]. the previous inhibition of gsk3 activity prevents, at least in part, the growth cone collapse response. similarly, it has been reported that three different gsk-3 antagonists (licl, sb-216763, and sb-415286) can inhibit the growth cone collapse response induced by sema 3a. however, the exact mechanism of how this activation of gsk3 occurs is not known, so far. many reports indicate that in dictyostelium discoideum gsk-3 activity may increase in response to camp binding to a heptahelical g - protein - coupled receptor. in this system, a tyrosine kinase and a tyrosine phosphatase have been described as regulators of gsk-3 activity [14, 40 ], but similar kinase and phosphatase have not been found in mammals. furthermore, it has been reported that reelin and netrin increased gsk-3 activity, similar to what lpa did. this netrin or reelin - dependent gsk-3 activation seems not to be a particular characteristic of the cell line or neuron used but rather a more general physiological process [107109 ]. indeed, even in situations where the final balance is an inhibition of gsk-3 kinase activity, such as following the addition of igf1/insulin or after estradiol addition, a transient activation of gsk-3 could be observed [34, 38 ]. all these data suggest that the upregulation and downregulation of this kinase is more complex than might initially have been considered. deregulation of gsk-3 has been linked to a wide range of human pathological conditions including type ii diabetes, muscle wasting, cancer and neurological disorders such as bipolar disorder, schizophrenia, depression, stroke, sleep disorders, and alzheimer 's disease (ad), among others, for a review see. lithium and valproic acid are mood stabilizers widely used in the chronic treatment of bipolar disorders. lithium ions directly inhibit gsk-3, most likely by competing with magnesium, while valproic acid is able to inhibit gsk-3 activity in relevant therapeutic concentrations in human neuroblastoma cells although in vivo direct inhibition of gsk-3 by valproic acid remains a matter of debate. the precise mechanism of action by which lithium exerts its therapeutic effects is not known, but it is conceivable that the acute effects on gsk-3 result in changes in gene regulation and cellular changes which could affect the neuronal plasticity over time. actually, lithium is also an inhibitor of several phosphomonoesterases and phosphoglucomutases, but the fact that gsk-3 has been shown to be significantly inhibited at therapeutic lithium concentrations [118120 ] suggests that at least a significant proportion of lithium 's therapeutic actions in bipolar disorder results from the inhibition of gsk-3, underlying its importance as a therapeutic target for this disorder [121, 122 ]. lymphocytes of patients with schizophrenia show impaired gsk-3 protein levels and activity, whereas gsk-3 has been reported to be reduced in the frontal cortex of postmortem schizophrenic brains. since the wnt family of genes plays a central role in normal brain development, it is possible that gsk-3 impairment may lead to abnormal neuronal development. more recently, a direct association has been shown between gsk-3 and the n - terminal region of disrupted - in - schizophrenia-1 (disc1), a strong genetic risk factor associated with schizophrenia. moreover, mounting evidence suggests that gsk-3 is a crucial node that mediates various cellular processes that are controlled by multiple signalling molecules such as disc-1, par3, par6, and wnt proteins that regulate neurodevelopment. interestingly, increased levels of gsk-3 have also been reported in postmortem analysis of brains from ad patients compared to age - matched control samples, whereas a spatial and temporal pattern of increased active gsk-3 expression correlates with the progression of nft and neurodegeneration. apart from being the major kinase to phosphorylate tau both in vitro and in vivo, gsk-3 has been recently proposed as the link between the two major histopathological hallmarks of ad, the extracellular amyloid plaques and the intracellular nft [129, 130 ]. exposure of primary neuronal cultures to a induces activation of gsk-3, tau phosphorylation, and cell death [133, 134 ], whereas blockade of gsk-3 expression by antisense oligonucleotides or its activity by lithium inhibits a-induced toxicity [135, 136 ]. gsk-3 inhibition per se decreases a production in cells and in an animal model of amyloidosis [61, 120 ], most likely through a mechanism involving inhibition of -secretase. furthermore, amyloid precursor protein (app) itself is a substrate for gsk-3 in vitro and in vivo. finally, modulation of the gsk-3 signalling pathway by chronic lithium treatment of transgenic animals might also have neuroprotective effects by regulating app maturation and processing. in tauopathies such as frontotemporal dementia with parkinsonism (ftdp) linked to chromosome 17, the presence of some mutations in tau protein correlates with the onset of the disease [139, 140 ]. treatment of transgenic mice overexpressing mutant human tau (p301l, 4ron), with the gsk-3 inhibitor lithium, has been shown to significantly decrease the levels of tau phosphorylation and significantly reduce the levels of aggregated, insoluble tau. administration in this model of a second gsk-3 inhibitor, ar - a014418, also correlated with reduced insoluble tau levels, supporting the notion that lithium exerts its effect through gsk-3 inhibition. more recently, chronic lithium administration has also been shown to reduce tau phosphorylation in the 3xtg - ad mice, but did not significantly alter the amyloid load. an increase in gsk-3 activity has also been shown to coincide with cell death following middle cerebral artery occlusion in mice which results in cortical infarcts, and a reduction in infarct volume with the gsk-3 inhibitor lithium was demonstrated, indicating that gsk-3 inhibition may be beneficial in stroke. in fact, pharmacological inhibition of gsk-3 reduced infarct volume and improved behaviour in a focal cerebral ischemia model. besides deregulation of its activity in neurodegenerative processes, mounting evidence further suggests a potential role for gsk-3 as a therapeutic target in a range or other pathologies, including pancreatic cancer, parenchymal renal diseases, and hiv-1-associated dementia, among others. the recent discovery that glycogen synthase kinase-3 (gsk-3) promotes inflammation through nuclear factor kappa b (nfb) has revealed new functions on regulating inflammatory processes. furthermore, gsk-3 inhibition provides protection from inflammatory conditions in different animal model, suggesting that gsk-3 inhibitors may have multiple effects influencing these conditions. finally, recent developments suggest an active role of gsk-3 in various human cancers, although its role in tumourigenesis and cancer progression remains controversial. it may function as a tumour suppressor for certain types of tumours, whereas it seems to promote growth and development for some others. deregulation of gsk-3 has been shown to promote gastrointestinal, pancreatic, and liver cancers and glioblastomas. furthermore, gsk-3 inhibition attenuates cell survival and proliferation, induces cell senescence and apoptosis, and sensitizes tumour cells to chemotherapeutic agents and ionizing radiation. nevertheless, an attractive target for a variety of human diseases, its therapeutic potential on tumourigenesis, and cancer chemotherapy still needs to be carefully evaluated. the close involvement of gsk-3 activity in different human pathologies has sparked intense efforts in developing inhibitors as therapeutic agents. thus, the discovery of small molecule gsk-3 inhibitors in the last few years has not only attracted significant attention to the protein as a therapeutic target but also has provided a means to further understand the physiological functions of gsk-3 and to gain further insight into the molecular basis of those disorders. in fact, at least one small molecule gsk-3 inhibitor program has made it to the clinic. tideglusib (np-12) is a synthetic small molecule form the tdzd chemical class which is currently in phase ii development for two cns indications : alzheimer 's disease and progressive supranuclear palsy (psp), a tauopathy. three decades after its discovery as a protein kinase involved in glycogen metabolism, gsk-3 was revealed as a key enzyme in regulating many critical cellular processes, providing a link between many different substrates and various signalling pathways as well as gene expression. modulation of its activity has also turned out to be much more complex than originally thought, as evident from what has been reviewed here. furthermore, its role in a variety of highly relevant human pathological conditions has drawn significant attention to this enzyme as a potential therapeutic target, and the recent development of specific inhibitors has granted us new tools to dissect out its molecular and physiological functions while providing novel therapeutic agents. taken all together, the next few years will certainly bring us further insights into the cellular functions of this fascinating enzyme. | glycogen synthase kinase-3 (gsk-3) unique position in modulating the function of a diverse series of proteins in combination with its association with a wide variety of human disorders has attracted significant attention to the protein both as a therapeutic target and as a means to understand the molecular basis of these disorders. gsk-3 is ubiquitously expressed and, unusually, constitutively active in resting, unstimulated cells. in mammals, gsk-3 and are each expressed widely at both the rna and protein levels although some tissues show preferential levels of some of the two proteins. neither gene appears to be acutely regulated at the transcriptional level, whereas the proteins are controlled posttranslationally, largely through protein - protein interactions or by posttranslational regulation. control of gsk-3 activity thus occurs by complex mechanisms that are each dependent upon specific signalling pathways. furthermore, gsk-3 appears to be a cellular nexus, integrating several signalling systems, including several second messengers and a wide selection of cellular stimulants. this paper will focus on the different ways to control gsk-3 activity (phosphorylation, protein complex formation, truncation, subcellular localization, etc.), the main signalling pathways involved in its control, and its pathological deregulation. |
a major goal is to determine what are the biochemical / physiological factors in the wound that can reconstruct the damaged parts more effectively. wound healing is a dynamic interactive process involving many precisely interrelated phases that overlap in time and lead to the restitution of tissue integrity. the healing process reflects the complex and coordinated body response to tissue injury resulting from the interactions of different cell types and extracellular matrix components. failure of coordinated regulation can result in tissue fibrosis with excessive collagen production and, if highly progressive, the fibrotic process may eventually lead to organ malfunction and death. most chronic wounds are associated with fibrosis of various organs, ischemia, or diabetes mellitus and affect from 3 to 6 million people in the usa, with older persons (> 65) accounting for 85% of these events. nonhealing wounds result in enormous health care expenditures, with the total cost estimated to be more than $ 3 billion per year. the importance of the ecm in the complex processes of wound healing is that it provides architectural support for the tissues and a platform for cells and molecules that regulate inter- and intracellular signaling. ecms are secreted molecules that constitute the cell microenvironment and are composed of a dynamic and complex array of glycoproteins, collagens, glycosaminoglycans (gags), and proteoglycans (pgs). among these, the gag hyaluronan (ha) and the pgs such as versican and aggrecan are all partners in the control of the wound healing process. it is now well accepted that the ecm not only provides architectural support for resting tissues, but also undergoes important alterations after injury that are essential for directing cell behavior during the wound healing process. the function of the ecm facilitates repair of the wound either directly by modulating important aspects of cell behavior such as adhesion, migration, proliferation, metabolism, differentiation, and survival, or indirectly by modulating extracellular protease secretion / activation, or by modulating growth factor activity or bioavailability. cells have specific transmembrane receptors that recognize ecm components and interact with the intracellular cytoskeleton and signaling pathways. classic examples of ecm interactions with cells that fulfill the criteria of anchoring and adhesion to receptors that modulate intracellular signaling pathways involve cell surface receptors such as integrins and the ha receptor cd44 [35 ]. receptors on ecm are involved in many pathological processes, including inflammation, fibrosis diseases, and cancer [68 ]. although it is clear that a cascade of ecm molecules, including gags, pgs, connective tissue glycoproteins, and cell surface adhesion receptors, are involved in wound healing, we will primarily address the problem of wound healing with abnormal fibrosis by focusing on the role of the cell - adhesion molecule cd44 and its principal ligand ha in wound healing and tissue fibrosis. wounds are injuries to a living tissue. the cellular, molecular, biochemical, and physiological events associated with this process consists of a highly orchestrated sequence of events that require the collaborative efforts of many different cell types, including blood cells, epithelial and connective tissue cells, inflammatory cells, and many soluble factors, such as coagulation factors, growth factors, and cytokines. the behaviour of each of the participating cell types during the phases of proliferation, migration, matrix synthesis, and contraction, as well as the soluble factor and matrix signals present at a wound site, is crucial for repairing the tissue injury. it is a dynamic and strongly regulated process that starts immediately after the initial lesion, and it will last until complete closure of the wound and regeneration of the tissue as functional as possible occurs. fibroblasts are the principal biosynthetic cells producing interstitial collagens, fibronectins, and other matrix components. they also differentiate into myofibroblasts, a specialized contractile cell type responsible for closure of the wound. in the setting of repetitive trauma or certain pathological states, increased ecm deposition of abnormal matrix (scarring ; fibrosis) occurs in a variety of fibrotic diseases in tissues, including liver, kidney, lung, and heart [12, 13 ], and in scleroderma [14, 15 ]. collagen deposition in the matrix is a requisite and, typically, reversible part of wound healing. however, in fibrosis, normal tissue repair can evolve into a progressively irreversible fibrotic response with fibroblast differentiation to excessive numbers of myofibroblasts and increased collagen deposition. wound healing involves integrated and overlapping phases : (a) haemostasis, (b) inflammation, (c) proliferation, and (d) remodelling (figure 1). immediately after the injury, vascular constriction and platelet aggregation at the injury site form a fibrin clot, which reduces leakage of blood from damaged blood vessels in the wound. the fibrin clot is a temporary shield containing many important molecules : fibronectin (fn), sparc (secreted protein, acidic and rich in cysteine), thrombospondin, vitronectin, and growth factors such as transforming growth factor- (tgf-), platelet - derived growth factor (pdgf), fibroblast growth factor (fgf), epidermal growth factor (egf), and insulin - like growth factor-1 (igf-1) released by platelets and monocytes. components of the fibrin clot also bind to cells and to other ecm proteins simultaneously. the clot then provides a provisional matrix for migration of the cells to pass over and through during the wound repair process [16, 18 ]. once the bleeding is controlled, sequential infiltration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes into the wound (chemotaxis) promote the inflammatory phase [1921 ]. a critical function of neutrophils is the clearance of invading microbes and cellular debris in the wound area, although these cells also produce substances such as proteases and reactive oxygen species (ros), which can cause additional damage. unless a wound is grossly infected, the neutrophil infiltration terminates within a few days, and expended neutrophils will be phagocytosed by tissue macrophages, which then degrade nonviable tissue and dead bacteria. however, inflammation can lead to the damage of tissue if it lasts too long. thus, the reduction of inflammation is frequently a goal in therapeutic settings. by clearing the apoptotic cells, macrophages help the resolution of inflammation, and they undergo a phenotypic transition to a reparative state that stimulates keratinocytes, fibroblasts, and angiogenesis to promote tissue regeneration [22, 23 ]. t - lymphocytes migrate into wounds following the inflammatory cells and macrophages, and they peak during the late - proliferative / early - remodelling phase. although the role of t - lymphocytes is not completely understood, studies have reported that cd4 + cells (t - helper cells) have a stimulatory role while cd8 + cells (t - suppressor - cytotoxic cells) have an inhibitory role in wound healing [24, 25 ]. blood factors are released into the wound that cause the migration and division of cells, which prepares them for the proliferative phase. in this way the reparative phase and remodeling is characterized by the formation of the granulation tissue that fills the wound before reepithelialization where epithelial cells migrate across the new tissue to form a barrier between the wound and the environment. granulation tissue contains fibroblasts and endothelial cells in an ecm that contains gags and pgs, which supports capillary growth, fibronectin, and collagen formation at the site of injury so that vascular density of the wound can return to normal. thus, following robust proliferation and ecm synthesis, wound healing enters the final remodelling phase, where the wound also undergoes physical contraction mediated by contractile fibroblasts (myofibroblasts) that appear in the wound [20, 21 ] (figure 1). the time - dependent sequence of events in wound healing includes regulation of cell - ecm interactions that are controlled by soluble mediators that act synergistically to direct wound remodelling by regulating ecm synthesis and degradation. subsequently, the myofibroblast population is also expanded as a result of epithelial cells undergoing epithelial - to - mesenchymal transition (emt) and of the activation of resident fibroblasts that leads to ecm deposition and tissue remodeling. the types of soluble mediators released during tissue injury are described below. following tissue injury, platelets aggregate and release platelet - derived growth factor - ab (pdgf - ab) from the granules. pdgfs are potent mitogens and chemoattractants for many cells, including fibroblasts, smooth muscle cells, mesenchymal cells, neutrophils, and monocytes, and they upregulate fibronectin, procollagen, and collagen activities. pdgfs have crucial roles in fibrotic disorders such as kidney, lung, and skin fibrosis [10, 2729 ]. healing of the wounds involves increased infiltration of inflammatory cells and fibroblasts followed by a marked increase in collagen deposition at the wound site. tgf-1 influences collagen degradation by stimulating tissue inhibitor of metalloproteinase (timp), which inhibits protease activity and decreases degradation of newly synthesized collagen [3033 ]. we and others showed that blocking tgf-1 decreases ecm deposition, scar formation, and fibrosis [14, 34 ]. like pdgf, the fibrogenic potential of tgf-1 makes it a prime candidate for drug therapy in settings of tissue fibrosis. fgfs are strongly mitogenic for endothelial cells and are involved in angiogenesis, directing endothelial cell migration, proliferation, and plasminogen activator synthesis. igfs are produced by several cell types including macrophages and fibroblasts [37, 38 ], and they have the potential to activate fibroblasts by either stimulating replication or increasing the production of connective tissue components such as collagen, elastin, and pgs, including versican [39, 40 ]. egf acts as a mitogenic factor for cells including fibroblasts, keratinocytes, smooth muscle cells, and epithelial cells [4144 ] and increases skin wounds. however, exaggeration of this process of repair and the subsequent increased reorganization of the tissue matrix can lead to the development of fibrotic scar tissue that is characterized by excessive accumulation of ecm components, including fibronectin, pgs, ha, and interstitial collagens. pgs have core proteins or glycoproteins with large gag side - chains (figure 2), and they participate in cell - cell and cell - matrix interactions, cell proliferation, and migration, and in cytokine and growth factor signaling associated with wound healing. small leucine - rich pgs (slrps) and the chondroitin sulfate pg versican are found in the dermis of wounds, the pg perlecan in the basement membrane, and the heparan sulfate pgs, syndecans, and glypicans on the cell surfaces. the versican - v3 isoform promotes transition of normal dermal fibroblasts to myofibroblasts [46, 47 ]. expression of syndecans-1 and syndecans-4 in wounds stimulates keratinocyte and endothelial cell migration and angiogenesis in mice. decorin, a member of the slrp family, negatively regulates tgf-1 and demonstrates effects of antifibrosis in various tissues, including kidney, muscle, and lung. pgs can maintain the ecm in a hydrated condition, exclude other macromolecules, and allow permeability of low molecular weight solutes. thus, by interacting with other ecm components, pgs are critical to organize the matrix [56, 57 ]. of the various ecm macromolecules, gag chains (figure 3) exhibit considerable structural diversity resulting from a complex biosynthesis that is tightly regulated in biological systems, enabling the modified gags to selectively interact with a variety of ligands in a spatially and temporally controlled manner [56, 57 ]. during the proliferation phase of wound healing, fibroblasts and other mesenchymal cells enter the inflammatory site of the wound in response to growth factors that are necessary for stimulation of cell proliferation. the fibroblasts synthesize collagen and pgs, which continues for several weeks with proportional increases of collagen. during this time, endothelial cells form capillaries, and the gags (ha, chondroitin sulfate (cs), and dermatan sulfate (ds)) also change in their levels. initially, ha is synthesized in large amounts by the fibroblasts for 2 weeks, followed by increased levels of ds and cs pgs. gradually, when the proliferation of cells reaches a plateau, heparan sulfate (hs) pgs are elevated in the wound. sulfated pgs with cs and ds assist in collagen polymerization, and hs pgs on cells can create anchors to surrounding matrix. pg degradation by proteases in the wounds can release gag - peptide fragments, which may modulate the wound healing process. for instance, cs and ds can regulate growth factor activity and may stimulate nitric oxide production, which, in turn, can modulate angiogenesis, whereas hs can stimulate the release of il-1, il-6, pge2, and tgf- and contribute to the modulation of its proangiogenic effects in the tissues [63, 64 ]. studies have demonstrated colocalization of the large cs pg versican with ha in cables in smooth muscle cells and in an epithelial cell system. of the gags, ha has a key role in each phase of wound healing as well as in regulating ecm organization and metabolism. ha is omnipresent in the human body and in all vertebrates, occurring in almost all biological fluids and tissues, with the highest amounts in the ecm of soft connective tissues. ha is a linear, naturally occurring, nonsulfated gag of the ecm (figure 3). ha has a repeat of disaccharides consisting of d - glucuronic acid and n - acetylglucosamine [6870 ]. native ha has a very high molar mass, usually in the order of millions of daltons, (10 to 10 da) before being progressively degraded into smaller fragments in the ecm [14, 67, 70, 71 ]. it possesses interesting viscoelastic properties based on its polymeric and polyelectrolyte characteristics. despite its relatively simple structure, ha is an extraordinarily versatile gag and is involved in several key processes, including early emt in development and morphogenesis, cell signaling, wound repair and regeneration, matrix organization and pathobiology. a pattern has emerged ; following tissue injury, inflammatory cells, keratinocytes, fibroblasts, endothelial cells, and pluripotent stem cells undergo interactions with ecm macromolecules or their fragments to heal the wound. during the inflammatory phase of wound healing, ha accumulates in the wound bed and acts as a regulator of early inflammation. the major functions of ha in this phase are to modulate inflammatory cell and fibroblast cell migration, proinflammatory cytokine synthesis, and the phagocytosis of invading microbes. in this inflammatory phase, ha degradation products (low - mw ha presumably ~2.5 10 da) can promote early inflammation. at sites of inflammation and tissue injury, these low - mw ha fragments that accumulate from degradation of high molecular weight ha can initiate toll - receptor-2 and toll - receptor-4 (tl - r2 and tl - r4) induction of proinflammatory cytokines il-6, tnf-, and il-1. these cytokines, in turn, induce ha production in vitro by various cell types, including endothelial cells, dendritic cells, and fibroblasts. the proliferative phase overlaps with the remodeling phase where keratinocytes differentiate to fibroblasts. during these events, the growth factors and cytokines released by the inflammatory cells induce fibroblast and keratinocyte migration and proliferation. furthermore, the levels of ha synthesized by both fibroblasts and keratinocytes are elevated during reepithelialization where epithelial cells migrate across the new tissue to form a barrier between the wound and the environment (figure 1). the levels of ha and its degradation products are abundant in patients with scleroderma fibrosis and in the animal models of bleomycin - induced lung injury [76, 77 ]. the excessive production of ha is one of the major events in scleroderma fibrosis [78, 79 ]. furthermore, increased ha levels are observed in bronchoalveolar lavage (bal) fluid and/or plasma from patients with pulmonary fibrosis, interstitial lung disease, and idiopathic pulmonary injury. however, failure to remove ha fragments from the site of tissue injury contributes to the unremitting inflammation and destruction observed in tissue fibrosis. clearance of ha fragments depends both on its receptor cd44 and on recognition by the host via tl - r2 and tl - r4 (figure 1). most cells synthesize ha at some point during their life cycles implicating its function for fundamental biological processes. unlike all of the sulfated gags, biosynthesis of ha does not require a core protein and is not done in the cell 's golgi networks. ha is naturally synthesized by a class of integral membrane proteins called ha synthases, of which vertebrates have three types : has1, has2, and has3 [8688 ]. the expression of various has isozymes is likely to be a fine control system critical for the effective mediation of different cell behaviors. while has1 and has2 are able to produce large - sized ha (up to 2000 kda), ha produced by has3 for example, a number of studies have defined the details of transcriptional regulation of the has2 gene promoter in response to a variety of cytokines and growth factors that are released as a result of wounding [92, 93 ]. some of the most dramatic effects of cytokines on ha regulation occur in epidermal keratinocytes of the skin, in which ha production is boosted many - fold by exposure to a variety of growth factors including egfr [94, 95 ]. interestingly, wounding of keratinocytes releases hb - egf, which itself has been shown to upregulate ha synthesis in neighboring cells [96, 97 ], an example of the paracrine effects (cell - cell cross - talk) that now appear to have a central role in mechanisms of fibrosis (discussed more below). has2 activity can also be governed by posttranslational pathways, such as regulation of o - glcnacylation. once in circulation most cells do not have this option but do have a metabolically active pericellular matrix (glycocalyx). (figure 4) for example, keratinocytes catabolize hyaluronan by a mechanism that involves the cd44 ha receptor [86, 99 ] and a hyaluronidase, most likely gpi - anchored hyaluronidase 2. the presence of a protease, such as adamts5 (aggrecanase) is likely also involved in order to remove associated proteoglycans (aggrecan and versican). cd44 rapidly transports (t1/2 of ~15 min) the fragmented ha (2030 kda) with any remaining bound proteins into an endosomal compartment distinct from coated pits and pinocytotic uptake pathways. the fragments are then transported to lysosomes for complete degradation (t1/2 of ~3 h) (figure 4) [86, 99 ]. therefore, distinct sites for biosynthesis and catabolism of ha on the surface of cells could effectively cooperate in controlling its dynamic metabolism. the stability of cytosolic has2 is significantly increased when serine 221 on has2 is o - glcnacylated [86, 101 ]. recent studies from our laboratory indicate that the matricellular protein periostin regulates has2 activation at a serine residue in embryonic heart valve remodelling. it is possible that o - glcnacylation of this serine is a key for regulating whether or not has2 remains inactivated in response to periostin during development of the heart valve, which would allow the enzyme to migrate to the cell surface after its synthesis in the er. there is increasing evidence that phosphorylation of serine and threonine residues in has2 to control hyaluronan synthesis whether or not it is activated [86, 103 ]. the phosphoserine increases when ha synthesis increases and phosphothreonine increases when ha synthesis decreases, as it is expected from the data discussed by hascall 's group. ha, either alone or more often through its interaction with its binding partner cd44 on the cell membrane, is crucial for the tissue morphogenesis. for example, while the has1 and has3 null mice are developmentally normal, has2 deletion results in lethal defects in cardiac development and vascular abnormalities. tgf-2-induced has2 expression and subsequent ha - cd44 signaling are required for endocardial cushion formation in has2-null mice [104107 ]. recent studies demonstrate that the balance of ha produced by distinct has enzymes is important for regulating inflammatory responses and wound contraction in the skin after injury. at physiological ph, ha is a highly polyanionic molecule associated with counter ions, such as na+, k+, ca2 +, and mg2 +. ha is characterized by its ability to occupy large hydrophilic solvent domains due to its very large size, which helps maintain the extracellular space and facilitates the transport of small molecular weight solutes through its domain. solutions of high molecular mass ha exhibit time - dependent viscoelasticity because of polymer chain entanglement. during rapid growth and tissue remodelling, the hydrated domain and the viscoelastic properties of ha are relevant for the application of ha in tissue repair as has been known for decades. in addition to the physiochemical effects of ha, ha also mediates the migration of fibroblasts to the wound site [110, 111 ]. in vitro studies have demonstrated that, in the presence of specific growth factors, the higher the levels of ha, the greater the cell migration in cell cultures [14, 102, 112116 ]. most of the effects of ha upon cell behavior are mediated via interactions between ha and the ha receptors, cd44 [7, 14, 111, 113, 117120 ] and rhamm [121124 ], through which intracellular signalling pathways are activated. in skin wound healing, the differentiation of fibroblasts to myofibroblasts is very important for the closure of wounds and for the formation of the collagen - rich scar. in this regard, various studies have pointed to an important role of ha and has enzymes in regulating fibroblast - to - myofibroblast conversion. work by the group of steadman and phillips has shown that the pericellular ha coat that surrounds human dermal fibroblasts appears to regulate profibrotic behavior of these fibroblasts, such that inhibition of ha synthesis significantly reduces tgf-1-driven fibroblast proliferation and transformation to myofibroblasts. furthermore, the mechanism by which ha regulates tgf- signaling effects in the fibroblasts appears to involve changes in colocalization of the ha receptor (cd44) and the epidermal growth factor receptor (egfr), both of which interact in the plasma membrane within lipid rafts [127129 ]. strong evidence for an important link between ha, cd44, and fibrotic processes is also found in the lung (as discussed later in section 4.2.1). at another level, ha in the skin appears to regulate cytokine production and secretion in healing wounds by regulating the influx of leukocytes into the wound area. for example, selective loss of has1 and has3 (in has1/has3 double knockout mice) leads to a proinflammatory milieu that favors recruitment of neutrophils and macrophages in the connective tissue (dermis). in the has1/has3 double knockout mice, the rate of wound closure is accelerated (rather than inhibited), despite loss of ha - synthetic capacity in the skin epithelium and a reduction in overall ha levels in the dermis. one possible explanation for this rapid wound closure is the observation that neutrophils and macrophages are recruited in greater numbers from small cutaneous vessels at the wound sites. the abundant leukocytes secrete higher amounts of cytokines (e.g., tgf-1), which probably activate local fibroblasts, making them more contractile and promoting their transformation into myofibroblasts, which thereby contracts the wounded. the mechanism for robust neutrophil / macrophage recruitment in the has1/has3 mice is currently unknown. in a third example of how ha is important in fibrosis, overactive fibroblast behavior contributes to the pathogenesis of progressive fibrotic disorders such as scleroderma [71, 130132 ]. recent studies have shown that a critical element in the etiology of scleroderma is the presence of abnormal paracrine signaling involving signal - amplification loops between skin fibroblasts and the overlying keratinocytes. when keratinocytes from scleroderma patient skin are cocultured with fibroblasts, the fibroblasts were stimulated to produce more ecm due to dysregulated paracrine signaling involving il-1 and tgf-. given the importance of ha in regulating fibroblast responses to tgf- and other cytokines, the potential for involvement of ha and cd44 in fibrotic processes of the skin appears to be ripe for future investigation. in the lung, has - mediated ha synthesis also has a vital role in repair after tissue injury. in the human disease idiopathic pulmonary arterial hypertension, increased has1 and decreased has2 levels are observed in pulmonary artery smooth muscle cells isolated from the patients, in whom total lung ha concentrations are also increased. in a mouse model of asthma, expression of has1 and has2 is increased in lung tissue. conditional deletion of has2 in mesenchymal cells in -smooth muscle actin (-sma)-has2 transgenic mice abrogated the invasive fibroblast phenotype, impeded myofibroblast accumulation, and inhibited the development of lung fibrosis. high molecular weight ha has many crucial structural and physiological functions in wound repair following injury on the basis of its molecular weight and accessibility to various ha - binding proteins (habps). ha is removed from the ecm as a consequence of local catabolism. in mammals, the enzymatic degradation of ha results from the action of 5 functional hyaluronidases (hyals), hyal1 and hyal2 are considered to be the main active haases in tissues in almost all somatic tissues. no haase activity for human hyal3 has been shown, and, in mice, hyal3 does not seem to have a major role in constitutive ha degradation. recently, a novel haase (kiaa1199) has been described, which is also detectable in human skin. the larger isoform of hyal1 is often secreted by the cell, while the smaller isoform is retained in acidic intracellular vesicles and lysosomes. hyal2 is often found as a glycosylphosphatidylinositol- (gpi-) anchored form, tethered to the extracellular side of the plasma membrane [138, 139 ]. by catalyzing the hydrolysis of ha, a major constituent of the interstitial barrier, hyals lower the viscosity of ha, thereby increasing tissue permeability. hyal1 has the maximal ha - degrading activity at ph 3.53.8 and cleaves ha to small oligosaccharides, which is consistent with its role of activity within lysosomes. hyal2 shows optimal activity at ph 6.0 - 7.0 and cleaves high molecular weight ha into intermediate size fragments of ~20 kda. ha degradation products stimulate endothelial cell proliferation, migration, and tube formation following activation of specific ha receptors, in particular cd44 and rhamm. ha fragments are implicated in the progression of lung diseases, and hyals are elevated in scleroderma, a fibrotic lung disease [138, 143 ]. ha catabolism by hyals and ros creates products that have biological activities distinct from native high molecular weight ha. low and intermediate molecular weight ha (2 104.5 10 da) can stimulate gene expression in macrophages, endothelial cells, eosinophils, and certain epithelial cells [146149 ]. ha at ~200 kda represents an interesting therapeutic strategy as it promotes reconstruction of a functional epithelium monolayer in vitro. on the other hand, excessive ha degradation products also promote fibrotic scar tissue formation [151, 152 ]. skin wounds on early mammalian embryos heal perfectly with no signs of scarring and with complete restitution of the normal skin architecture, and the wound fluid ha is of high molecular weight. during tissue injury and inflammation, ha that is normally present as high molecular weight (> 1000 kda) is modified into monocyte - adhesive matrices that stimulate immune cells at the injury site to express inflammatory genes through interactions with cell surface receptors. this leads to the release of enzymes and free radicals, which break the long chain molecules to lower molecular weight forms that have extraordinarily wide - ranging and often opposing biological functions, owing to the activation of different signal transduction pathways. studies have shown that ha fragments of lower molecular weight (~50200 kda) are proinflammatory, immunostimulatory, and proangiogenic, and they competitively bind to ha receptors on cell surfaces (figure 4). while ha fragments may be important in initiating the inflammatory response, removal of these fragments is also critical for the resolution of the repair process. however, studies of cd44-null macrophages indicate that there are other signaling pathways, notably through toll - like receptors, tl - r2 and tl - r4. biological functions of ha and ha fragments are manifested through its interactions with a large number of ha - binding proteins (habps or hyaladherins) that exhibit significant differences in their tissue expression, specificity, affinity, and regulation [4, 7, 84, 118, 158163 ]. a number of habp bind ha through binding motifs, known as the link module, which consists of a span of ~100 amino acids that binds ha when oriented in the correct tertiary structure. habps are constituents of the ecm, stabilize its integrity, and are involved in cellular signal transduction dependent on the molecular weight of ha and the cell phenotype. therefore, a single chain of high molecular weight ha can theoretically accommodate on the order of 1000 habps. the habp link module family includes the link proteins, the pgs aggrecan, versican, brevican and neurocan, cd44 standard and variants, tumor necrosis factor- stimulated gene 6 (tsg-6), and lymphatic vessel endothelial receptor 1 (lyve-1) [167169 ]. studies have shown that, in response to ha of 40400 kda, the nf-b - mediated gene expression is activated by ha binding with ha receptor for endocytosis (hare). the rhamm receptor is an unrelated ha - binding protein with a ha - binding site peptide motif (b(x7) b) of minimal size of interaction with ha. cd44 and rhamm are well - studied receptors associated with tissue injury, repair, cancer cell growth, and metastasis [4, 14, 159, 163, 171173 ]. in addition, the binding of ha to intracellular adhesion molecule (icam-1) may affect its binding to other receptors at early stages of inflammatory activation. the constitutive expression of cd44 and ha by a wide variety of cells implies that the interaction between these molecules is regulated. cd44 is the best characterized transmembrane ha receptor and because of its wide distribution it is considered to be the major ha receptor on most cell types. cd44 is a structurally variable and multifunctional cell surface glycoprotein encoded by a single gene (figure 5). the genomic structure of cd44 consists of 21 exons and the cd44 gene expression varies in size due to insertion of alternatively spliced variable exons derived from exon6exon14 to form cd44v1cd44v10 that are located in the membrane - proximal extracellular cd44 domains, approximately where n - terminal sequence homology between cd44 molecules from different species ends. the standard cd44 (cd44s) has a molecular weight ~90 kda and exhibits extensive n - linked and o - linked glycosylation of the extracellular region, emphasizing the glycoprotein nature of cd44. cd44 can be induced to bind ha in cells activated with inflammatory stimuli, including cytokines, such as tnf-, il-, il-1, il-3, granulocyte - macrophage colony stimulating factor (gm - csh), and interferon- (ifn) [84, 177, 178 ]. the molecular mechanisms underlying the induction of cd44-mediated ha binding include increased expression, variable glycosylation, receptor clustering, gag attachment, phosphorylation, and inclusion of variant exons in the receptor [6, 7, 177, 179184 ]. we and others have shown that malignant cells produce ha in order to activate their tumorigenic functions [7, 113, 117, 119, 120, 182, 185190 ], while smaller oligosaccharides (~2 - 3 kda) can ameliorate these effects in vitro [191, 192 ]. the variant 6 isoform, cd44v6, is of particular interest because it is overexpressed in many cancers, and ha - cd44v6 promotes growth [6, 7, 118, 193198 ], which has a significant role in disease onset and progression. an increase in serum soluble cd44v6 due to mmp cleavage, along with serum hgf and ha levels, may serve as companion biomarkers for the presence of tumors and their responsiveness to cd44v6 [199205 ]. we have shown that ha - cd44v6 signaling promotes tumor cell survival and tumor growth. in addition, ha binding to cd44v6 is more avid than to cd44s and results in altered signaling [206208 ]. in addition, cd44v6 mediates cross - talk between cd44v6 and receptor tyrosine kinases (rtks), including c - met [14, 209 ]. we also demonstrated that periostin, a fibrogenic matricellular protein, also activates has2 thus releasing free ha, which interacts with cd44 to regulate phenotypic transitions of lung fibroblasts to an invasive myofibroblastic phenotype, characterized by the overexpression of cd44, collagen 1, and -sma (figure 1). overexpression of has2 by -sma positive myofibroblasts produced fatal lung fibrosis, whereas conditional knockout of has2 in myofibroblasts reduced the development of lung fibrosis. moreover, cd44 contributed to the progressive fibrotic phenotype because lung fibrosis was reduced by either crossing the -sma - has2 transgenic mouse with the cd44 deficient mouse or by treatment with a blocking antibody to cd44. all the functional effects of ha in inflammation and fibrosis may not be mediated by cd44. the role of cd44 in ha binding and signaling has recently been investigated in hematopoietic cells from cd44-null mice. cd44-null mice develop normally and exhibit minor abnormalities in hematopoiesis and lymphocyte recirculation, indicating that the lack of cd44 can be compensated for in cd44-null mice. the induction of inflammatory gene expression in response to ha was observed in the cd44-null bone marrow cultures and in dendritic cells. it has been shown in wound healing or tissue injury that there is a potent mechanism for clearing ha following the injury. however, cd44-null mice challenged with bleomycin in an experimental model of lung injury accumulate extensive ha matrix that is not removed by the recruited macrophages with the resulting compromise of oxygen exchange, which results in death. studies with the cd44-null mice and tissues have discovered the differential effects of cd44 in the predominant cell types that mediate host injury, suggesting potential roles for cd44 in mediating pathogenesis of host injury [172, 211 ]. for example, administration of il-2 to wild - type mice triggered a significant vascular leak syndrome (vls) in the lungs and liver. in contrast, in cd44-null mice, vls induced by il-2 was markedly reduced in the lungs and liver. future studies in cd44-null mice will elucidate the importance of ha homeostasis and provide new insights into the role of cd44 in vivo and in the tissue / cell models required to study the mechanisms of action of cd44 at the cellular and molecular levels of tissue injury and repair. the viscoelastic and hydrated domain properties of ha are relevant for exogenous application of ha in tissue repair and regeneration processes. exogenous application of ha accelerates skin wound healing in various animal models, including rats and hamsters [213215 ]. laurent. showed that exogenous ha can promote scarless healing in tympanic membranes, and balasz and denlinger hypothesized that ha rich matrices can inhibit fibrous scars. later, it was shown that in utero scarless fetal tissue repair is associated with high overall levels of ha for longer periods, indicating that high levels of ha may in part reduce collagen matrix deposition and contribute to scarless tissue repair. in the older (late gestation) fetus and in adults, a reduction in ha levels is associated with fibrotic scarring. demonstrated scarless healing in adults in an animal model, hoxb13 knockout mice, in which ha levels remain elevated in adult skin. conversely, when hoxb13 is overexpressed in the epidermis, ha levels are suppressed in vitro and the skin behaves as a profibrotic wound - healing environment in vivo. although older findings in the literature regarding ha levels and wound healing were rather difficult to interpret in the past, newer ideas about the role of ha in regulating cytokine receptor signaling at the individual target cell level may help to reconcile the role of ha in fibrosis and healing in the future, as discussed further below. the effects of different ha preparations in the following studies are attributed to differences in growth factor and cytokine presentation to ha, and to ha receptor mediated molecular organization. the identification of the biological activities of various growth factors and cytokines in wound healing suggest that cells in injury models can respond to peptide factors for the long - term repair processes. for example, fibroblasts derived tgf-1, b - fgf, pdgf, and egf stimulate ha synthesis synergistically, and their effects on cell proliferation are through ha - initiated pathways, indicating the benefits of exogenous application of ha on ecm remodelling. the former produce more ecm protein when ha is added to the culture, show greater migration to ha in vitro, and are insensitive to the applied pdgf, b - fgf, and egf. pdgf induces expression of tgf-1 in adult wounds, which suggests that some of the longer term effects of pdgf are achieved indirectly by activation of tgf-1 by fibroblasts within granulation tissue. clinical studies have also demonstrated that exogenous application of pdgf - ab together with other growth factors to chronic wounds can accelerate their closure [227, 228 ]. application of egf to organotypic cultures of epidermal cells leads to increased has with increased proliferation and migration, and tgf-1 inhibits this response, a finding that shows why scarring wounds heal slowly. fetal and adult wound healing also differ with respect to the participation of various cytokines, particularly members of the tgf- family. increased canonical wnt signaling occurs during postnatal wound repair but not during fetal cutaneous wound repair. in this regard, tgf-1 and tgf-2 have been detected in adult wounds, while tgf-3 is the principal isoform found in fetal wounds in response to rwnt3a protein. moreover, increased levels of macromolecular ha lead to decreased scarring in fetal life, whereas adult fibroblasts increased scarring due to increased ha breakdown products. in addition, rapid wound closure is reported in has1/has3 double knockout mice, which have decreased amounts of ha in the skin, and wounding is accompanied by increased efflux of neutrophils into the tissue and by an earlier onset of myofibroblast differentiation. in this case, increased inflammation might compensate for the decreased ha. thus, in clinical settings, ha - protein (growth factor / cytokine) complexes may ameliorate the scarring. it is likely that addition or removal of combinations of growth factors, or other agents such as protease inhibitors, will be more beneficial in some clinical circumstances. we have shown that manipulating ha concentrations and ha - cd44 interactions can alter signaling pathways with many regulatory and adaptor molecules, including src kinases, rho - gtpases, pi3kinase, ankyrin, and ezrin [7, 118 ]. the engagement of cd44 with ha can modify cell survival and proliferation by changing intracellular engagement of erm proteins [192, 232 ]. additionally, ha may activate several receptor tyrosine kinases and ha - cd44 may promote clustering and cooperate with other growth factor receptors [188, 190 ]. additionally, we have shown that silencing variant 6 (cd44v6) inhibits tumor growth in vitro and in vivo [117, 182 ]. moreover, blocking cd44v6 inhibits fibrogenesis of fibroblasts in scleroderma lung fibrosis, and blocking ha - cd44v6 downregulates fibroblast contractility. therefore, ha - cd44 variant interactions may modify several signaling pathways not directly related to cd44, but to other receptors that may interact with cd44. thus, in clinical settings, increased ha in response to tnf-, il-, il-1, il-3, gm - csh, and ifn [84, 177, 178 ] may promote ha - cd44 clustering and synergize with the cytokine receptor signaling pathways to induce fibrotic responses. this could explain how small ha fragments not capable of bridging receptors can alter these responses. based on the studies above, there is now increasing evidence that ha can be used in biomedical applications for beneficial effects in wound healing. coculture of apligraft with neonatal foreskin fibroblasts and keratinocytes significantly alters the composition of the matrices produced. identification of ha for its ability to augment keratinocyte proliferation, fibroblast migration, and endothelial cell angiogenic responses in the wound bed makes it a useful biopolymer for wound healing, and pretreatment of ha matrices with fibroblasts has been applied to human wounds. in contrast to collagen, ha is identical between species and has been used to make biomaterials by stable chemical modifications that have been used for wound healing. furthermore, the degradation of ha matrix can have many effects on the regenerating wound, including water homeostasis, enhancement of angiogenesis, and collagen deposition and organization, which can benefit epithelial regeneration. for example, benzyl esters of ha (hyaff p80 and hyaff p100), with differing degradation profiles, were used with a laserskin method to treat both chronic and acute wounds, which showed excellent results in promoting angiogenesis in the wound bed and epithelial engraftment after 14 days and wound healing without contraction. ha scaffolding material, including thiol - functionalized derivative ha - dtph, has already been shown to be completely biocompatible in tissue engineering and implantation to provide three - dimensional templates that can improve cell growth and growth factor presentation [240243 ]. application of a cross - linked ha derivative (polyethylene glycol diacrylate- (pegda-) cross - linked ha - dtph (ha - dtph - pegda)) strongly inhibited contraction of a collagen matrix, whereas high molecular weight ha (hmw ha) facilitated collagen gel contraction. this suggests that manipulating the interaction of ha with other matrix molecules can alter ecm remodeling in wound healing. ha is known to have a very short half - life of several hours in the body, which should be overcome for tissue augmentation applications. the residence time of ha can be prolonged by cross - linking ha in cosmetic fillers by the chemical modification of carboxyl groups of ha [245, 246 ] because they are in recognition sites of hyaluronidase (hyal2) and ha receptors [248, 249 ]. fibrosis is the accumulation of ecm components in organs or tissues and is a fundamental feature of systemic sclerosis (ssc) [250, 251 ]. we are studying wound healing in ssc, which affects the skin and many internal organs, including the lungs, the gastrointestinal tract, and the heart. we will discuss a few therapeutic strategies and possible agents designed to inhibit pathologic mesenchymal phenotypes in ssc fibrosis, including treatment approaches that modulate inflammatory pathways, inhibit profibrotic growth factors, modulate epigenetic codes, and interfere with mesenchymal phenotype. as discussed in previous sections, many cytokines are involved in tissue repair, pdgf, egf, fgf, and igf1, and they can have many different roles in the healing process, ranging from regulation of cell proliferation, differentiation, and chemotaxis to directing wound remodeling by regulating ecm synthesis and degradation. these proteins may be locally synthesized and released as polypeptide growth factors and cytokines, which then have key roles in regulating cell and tissue functions. it increases profibrotic signals that promote biosynthesis of important components of the ecm, including collagens, ctgf, collagen receptor integrins, decorin, and timps. tgf-1 is secreted at sites of injury by platelets and monocytes as well as by other cells, which promotes autocrine and paracrine interactions. we showed that tgf-1 autocrine signaling in ssc fibroblasts induces a sustained expression of cd44v6, which interacts with ha and activates cell cycle progression and -sma production via erk activation that increases collagen matrix synthesis. inhibition of tgf-1, or blocking cd44v6 by cd44v6sirna, reduces these functions of ssc fibroblasts significantly. we postulated that when tgf-1 stimulation of fibroblasts is inappropriate, that is, too much tgf-1 or heightened sensitivity to tgf-1 due to autocrine signaling, pathologic fibrosis ensues with sustained ha - cd44v6 that will eventually overwhelm the system in favor of profibrotic effects. in addition, we postulated that the increase in antifibrotic hepatocyte growth factor (hgf) expression at the onset of chronic injury may initially compensate and support a regenerative process [14, 253 ], whereas repetitive lung injury results in overexpression of tgf-1 that leads to the profibrogenic effects. therefore, the balance between tgf-1 and hgf appears to have a critical role in determining whether the injured tissues undergo recovery or fibrogenesis. fresolimumab is a human monoclonal antibody that inactivates all forms of tgf-. in phase i trials, fresolimumab was safe and well tolerated in patients with primary focal segmental glomerulosclerosis, ipf, and renal cancer. similarly, phase ii trials of a human monoclonal antibody for ctgf (fg 3019) for patients with liver fibrosis (due to chronic hepatitis b infection) and pulmonary fibrosis are promising. imatinib mesylate, used for the treatment of chronic myelogenous leukemia (cml), blocks both profibrotic tgf-1 signaling and suppresses activity of the pdgf receptor [254, 255 ]. in ssc, however, the results are still inconclusive. as many profibrotic pathways are linked to tgf-1 signaling, novel antifibrotic therapies that target other pathways may indirectly act via suppression of tgf-1. for example, peroxisome proliferator - activated receptor- (ppar-) can suppress tgf-1-dependent cell activation and collagen production in fibroblasts and inhibit the development of fibrosis in murine models. recent studies also suggest that nadph oxidase 4 (nox4) is essential for tgf--induced differentiation of fibroblasts to myofibroblasts in vitro and for bleomycin - induced pulmonary fibrosis in vivo. the development of small molecule inhibitors and/or other strategies targeting nox4 or the use of ppar- agonists may abrogate fibrosis through antifibrotic mechanisms. studies suggest that the rhoa / rock pathway is a critical regulator of contractility of mesenchymal cells, including lung fibroblasts from ssc patients [258260 ]. fasudil, a small molecule inhibitor of rock, has recently been studied in us populations for other disease indications (https://www.clinicaltrials.gov/). it also reduces myofibroblast activation in lung fibrosis in an animal model, suggesting a potential use of this compound for treatment of fibrotic diseases. finally, the profibrotic pathway linked to tgf-1 signaling may directly act through a profibrotic mechanism through the augmentation of a ha - cd44 pathway. for example, ha can promote a profibrogenic activity in fibroblasts cells, as shown by changes in cellular behavior due to ha - cd44 interaction that induces biological processes. when hyaluronan synthase 2 (has2) was transgenically overexpressed by myofibroblasts in vivo, a severe fibrotic phenotype followed bleomycin - induced lung injury, presumably due to ha - cd44 function. mesenchymal fibroblasts that are derived from has2-deficient mice, or are treated with a cd44 blocking antibody, fail to show the same degree of fibrogenic function as do wild - type mice. our recent study showed that sustained cd44v6-induced signals regulate myofibroblast proliferation, activation, and matrix deposition in ssc fibroblasts in response to autocrine tgf-1 stimulation. this indicates that tissue specific blocking of ha - cd44 signaling by silencing cd44 using specific sirna can be a viable approach to attenuate profibrogenic functions. together these studies address components of wound healing processes and describe a number of different mechanisms that have been implicated in the pathogenesis of defective wound healing that leads to progressive fibrosis disorders. concepts relating wound healing to fibrogenic mechanisms have converged on a model of inflammation that coordinates with ecm components, soluble mediators that induce wound healing, and failure of tissue regeneration leading to fibrosis. ha - based novel therapeutic mechanisms that can use ha - biomaterials and antagonists to ha - cd44 signaling pathways are beginning to produce promising results in in vitro and in vivo models of both wound healing and fibrosis. considering that promising studies sometimes do not translate into patient benefit under different biological conditions and disease states, care must be taken to ensure the long - term safety of using advanced engineering strategies and well - conducted and controlled clinical trials need to be evaluated before the therapeutic agents, or ha - based biomaterials can be recommended for defective wound healing. our future studies will focus on determining the mechanisms by which ha - cd44 regulates impaired wound healing, with particular emphasis on micrornas that regulate ha synthesis and cd44 biology in normal and pathological wound healing. | a wound is a type of injury that damages living tissues. in this review, we will be referring mainly to healing responses in the organs including skin and the lungs. fibrosis is a process of dysregulated extracellular matrix (ecm) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). in tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ecm molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine - mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. this review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. first, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross - talk between cell types. second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed. |
an 81-year - old woman recognized pruritic scaly erythemas on the scalp 2 months before the visit to our hospital. her medical history included nephrosis syndrome that had been treated with oral cyclosporine 100 mg / day for 8 years. the results of routine blood work before the onset of skin eruptions had been normal. although the head erythemas appeared to be seborrheic dermatitis (sd), the lesions did not respond to topical steroidal lotion. when she visited our hospital, the eruption had disseminated to the trunk and extremities, resulting in generalized erythroderma (fig. laboratory tests showed white blood cells 13,900/l (normal 3,5009,800), hemoglobin 11.9 mg / dl (normal 11.315.5), platelets 271,000/l (normal 155,000365,000), eosinophils 19.0%, atypical lymphocytes 2.0%, lactate dehydrogenase 517 iu / l (normal 176353) and soluble interleukin-2 receptor 1,510 u / ml (normal < 550) ; human t - lymphotropic virus type i antibody was negative. a skin biopsy of the erythema of the trunk showed that lymphocytes and eosinophils had infiltrated dominantly to the superficial perivascular space. 2). polymerase chain reaction and flow cytometry of the skin specimens failed to detect malignant cells. however, atypical lymphocytes (basket cells) were detected in the peripheral blood and bone marrow (fig. 3). flow cytometry of the blood and bone marrow specimens showed that malignant cells were positive for cd2, cd3, cd4, cd5, cd7, cd25 and cd45ra. positron emission tomography - computed tomography revealed abnormal enhancement of axillary and inguinal lymph nodes and bone marrow. we consulted a hematologist and peripheral t cell lymphoma not otherwise specified (ptcl - nos), stage iv according to ann arbor was diagnosed. ptcl can cause sd - like dermatitis that gradually develops into auto - sensitization dermatitis. as chemotherapy was dismissed considering her age, we administered fexofenadine 120 mg / day and oral prednisolone 20 mg / day so as to alleviate erythroderma and pruritus. we added pregabalin 50 mg / day according to the recommendation by yosipovitch, which reduced pruritus dramatically. the amount of daily oral steroid was tapered to 5 mg over 4 months, maintaining remission of cutaneous symptoms. few publications have described sd as a paraneoplastic syndrome in cases with lung cancer and lymphoma [4, 5, 6 ]. the diagnosis of paraneoplastic syndrome usually requires concurrent onset and parallel course with malignancy at least. although we could not confirm whether the severity of the sd would parallel that of ptcl, sd seems to be a paraneoplasia since the onset of the sd coincided with that of ptcl and the sd was resistant to ordinary topical therapy. although the mechanisms of pruritus as well as the antipruritic effect of anticonvulsive agents are not clear, they probably inhibit central itch pathways. in this case, simple sd and subsequent auto - sensitization dermatitis were suspected at first. however, the sd was too resistant to ordinal therapy and developed to erythroderma. complete blood count revealed the presence of lymphoma that became an important clue to diagnosis. | seborrheic dermatitis is an inflammatory eruption that tends to distribute on the sebaceous areas of the body and is rarely described as a paraneoplasia. here we report a case with a responsive seborrheic dermatitis - like eruption of the head which resulted in generalized erythroderma. intensive examinations detected concurrent malignant lymphoma. |
n - methyl-2-pyrrolidone (nmp) (fluka, new jersey, usa) was used as received. sabouraud dextrose agar (sda), sabouraud dextrose broth (sdb), tryptic soy agar (tsa) and tryptic soy broth (tsb) (difco, usa) were used as media for antimicrobial test. the 20% w / w lf127 solutions containing 10, 20, 30, 40, 50, 60 and 80% w / w nmp were prepared using a cold method. briefly, lf127 was dissolved in distilled water at 4 until a homogeneous solution was obtained and nmp was subsequently added to with the polymer solution. the ph values of the systems were measured using a ph meter (professional meter pp-15 sartorius, goettingen, germany). the test tube inverting method as described previously was employed to roughly determine the phase boundary. gelation and gel melting temperatures were assessed using a modification of the previously reported technique. five millilitre aliquot of gel was transferred to a test tube (internal diameter of 1.8 cm) and sealed before immersing in a thermostat water bath at 4. the temperature was controlled at the heating rate of 0.2/min, and the transition temperature was monitored at an accuracy of 1. the samples were then examined for gelation, which was said to have occurred when the meniscus would no longer move upon tilting through 90 (n=3). the gel melting temperature, the temperature at which a gel started flowing upon tilting through 90, was recorded (n=3). the rheological behavior of prepared systems was investigated by recording their shear stress (f) as a function of shear rate (g) with a brookfield dv - iii ultra programmable rheometer (brookfield engineering laboratories. the measurements were conducted at three different temperatures (4, 28 and 35) (n=3). these three conditions were selected to represent the temperature in refrigerator, room temperature and temperature of human body, respectively. the continuous check for viscosity change with temperature was rather difficult to perform since there was notably different apparent viscosity of system at different temperature to measure with the same cone of the rheometer. rheological behavior of the gels is expressed as n value (flowing parameter) of the martin 's equation given by, log g = nlog f log '... (1), where ' is apparent viscosity. for n value of 1 or close to 1, a rheological behavior is newtonian and for n value not equal to 1, a rheological behavior is non - newtonian but are dilatant (if n>1) and pseudoplastics (if n1) and pseudoplastics (if n 50% nmp exhibited as cloudy gel. on the other hand, at 27 and 37, the prepared systems containing 50% w / w nmp. the sol - gel transition temperature of gel comprising 40% w / w nmp was not found because this system formed gel even at low temperature at 0. these results suggested nmp decreased the gelation temperature of the system. the prior studies reported that the gelation of thermosensitive gel was affected by various factors, such as temperature and concentrations of polymer, active ingredients / excipients and electrolytes. the gel formation related to micellar packing and volume fraction. the type of structures obtained in the presence of selective solvent appeared to be a function of the volume fraction of the polar / nonpolar components. the effects could be viewed in terms of a reduction of water activity leading to an increase in the active concentration of polymer in systems. the decreased gelation temperature was owing to the interaction between the hydrophobic portions of the polymer molecules, which could disrupt the micellar structure and increase the entanglement of micelles. most of flavors increased the viscosity of the poloxamer aqueous solution and decreased the gelling point of poloxamer 407 aqueous solution in proportion to amount added. the flavors might bind to the hydrophilic end chains of poloxamer, promoting dehydration resulting in a decrease in the gelation temperature of polymer solution. this is in agreement with previous findings on the effects of hydrophobic molecules on the gel formation of peo - ppo - peo block copolymer systems. for example, t - butylbenzene could reduce the gelation temperature of lf127 while benzoic acid and p - hydroxybenzoate esters can cause a decrease in the gelation temperature of the block copolymer. thus, the more hydrophobic the solute the greater the decrease in gelation temperature. there was a decreased gelation point of lf127 system with increasing amount of lidocaine and prilocaine. this effect was owing to the presence of hydrophobic component inducing micellization resulting in the micellar growth. the ambiguous sol - gel transition temperature of gel comprising 40% w / w nmp might be due to the equilibrium of suitable amount nmp for dehydration of the lf127 molecule, therefore the gel was formed at low temperature. flow curves of the systems comprising 20% w / w lf127 and different concentrations of nmp at different temperatures, 4, 27 and 37, are shown in figs. 2, 3 and 4, respectively. the incorporated nmp caused an apparent change in the flow curve of the lf127 system. the flow curve shifted from low to high shear stress along the temperature increased when the concentration of nmp was less than 20%. the unique flow curve with initially increased and subsequently rapidly decreased shear stresses was evident for the systems containing 50 and 60% nmp at 4 because their initial gel strength was destroyed after shear rate was enhanced. as the concentration of nmp was higher than 50%, the flow curve shifted from high to low shear stress when the temperature increased. the change of temperature slightly affected the flow of systems containing 30 - 40% w / w nmp. the flow curve of system was the non - newtonian behavior that the up curve did not coincide with the down curve indicating the presence of thixotropy, with a hysteresis loop. but the area of the hysteresis loop of all prepared systems did not increase as the nmp concentration increased. however, the flow curve moved to a higher shear stress value, indicating the higher gel compactness. the flow parameters of lf127 systems containing different concentrations of nmp at 4, 27 and 37 are shown in table 1. the n values of the l systems containing 0 - 20% nmp increased with increasing temperature. the n values of 0 - 20% nmp systems at 4 were close to 1 indicating a newtonian flow characteristic, while these values at higher temperature were>1 indicating a non - newtonian flow characteristic. the n values of the prepared gels containing 30 - 40% nmp were>1, which were not affected by the temperature. the increased nmp concentration from 50 to the n values of the prepared gels containing 50 - 80% nmp at 4 were more than one, indicating the non - newtonian flow. moreover, the n values of 50 - 80% nmp gels at 37 close to 1 indicating the newtonian flow characteristic. the viscosity coefficients () of systems containing 0 - 20% nmp increased whereas that of 30 - 40% nmp slightly increased with temperature. on the other hand, the viscosity coefficient of systems containing 50 - 80% w / w nmp decreased when the temperature increased corresponding with above obtained flow curves. flow parameters of the aqueous l systems containing different concentrations of nmp at different temperatures (n=3) antimicrobial activity of gels containing different concentrations of nmp against s. aureus, e. coli and c. albicans (fig. antibacterial activity against s. aureus and e. coli was increased as the amount of nmp was increased from 20 - 40% w / w while antifungal activity against c. albicans was enhanced by increasing the nmp amount. the inhibition zones of pure nmp against s. aureus, e. coli and c. albicans were 1.90.1, 2.00.2 and 3.50.1 cm, respectively (fig. therefore, nmp could solubilize the lipid in cell membrane and promote the leakage of microbial cell membranes. inhibition zones of nmp against three microbes (s. aureus, e. coli and c. albicans) using agar diffusion method (n= 3). lower nmp amount (30% w / w) could shift the sol - gel transition to a lower temperature but the gel - sol transition was shifted to a higher temperature. higher nmp amount (40% w / w) could shift both sol - gel and gel - sol transitions of that system to a lower temperature. the amount of nmp > 60% could reverse the phase of the lf127 system to non - newtonian flow at 4 and the newtonian flow at high temperature. aqueous lf127 system containing nmp exhibited antimicrobial activities against s. aureus, e. coli and c. albicans in a dose - dependent manner. | the purpose of this study is to investigate the effects of n - methyl-2-pyrrolidone on the thermosensitive properties of aqueous ethylene oxide - propylene oxide block copolymer (lutrol f127) system. due to the aqueous solubility enhancement and biocompatibility, n - methyl-2-pyrrolidone is an interesting solubilizer for the poorly water soluble drugs to be incorporated in the lutrol f127 system. effect of n - methyl-2-pyrrolidone on physicochemical properties of lutrol f127 system was investigated using appearance, ph, gelation, gel melting temperature and rheology. the antimicrobial activity of the thermosensitive n - methyl-2-pyrrolidone gel was also tested. lower n - methyl-2-pyrrolidone amount (30%w / w) could shift the sol - gel transition to a lower temperature but the gel - sol transition was shifted to a higher temperature. higher n - methyl-2-pyrrolidone (40%w / w) could shift both sol - gel and gel - sol transitions of the system to a lower temperature. the amount of n - methyl-2-pyrrolidone > 60% w / w could reverse the phase of the lutrol f127 system to non - newtonian flow at 4 and newtonian flow at high temperature. aqueous lutrol f127 system containing n - methyl-2-pyrrolidone exhibited antimicrobial activities against staphylococcus aureus, escherichia coli and candida albicans with the n - methyl-2-pyrrolidone in a dose - dependent manner. |
paracoccidioidomycosis (pcm) appears to be caused by a complex group of fungi within the paracoccidioides genus comprising four distinct phylogenetic lineages known as ps2, ps3, s1, and pb01-like (carvalho. based on clinical and genetic studies, the pb01 isolate differs from the other strains and has been included in a new species known as paracoccidioides lutzii (teixeira., 2009). most patients are rural workers but cases in urban centers located on the route of migration movements are also found (restrepo, 1985 ; mcewen., 1995). the infection starts by inhalation of conidia that subsequently transform into infective yeast forms in the lung. although acquisition of the fungus typically results in asymptomatic infection, it can progress in susceptible individuals and give rise to acute, subacute, and chronic clinical forms of the disease (franco., 1993). a mortality evaluation of p.brasiliensis showed that it is the 10th most common cause of death owing to chronic / recurrent infections and parasitic diseases in brazil. when analyzed as the underlying cause, 51.2% of deaths were due to pcm, which is then one of the most lethal among systemic mycoses. in the 19962006 decade, the most severe cases of pcm occurred in the 3059 years - of - age range, predominantly (87%) in men (prado., 2009). besides the mortality data, it is important to consider the morbidity associated to the disease, which invariably leads to withdrawal of the patients from labor activities or school. in the severe cases, hospitalization of patients is necessary for long periods of time with high costs. antifungal chemotherapy is required for pcm treatment, although there is no assurance, even after treatment, of complete destruction of the fungus. initial treatment depends on the severity of the disease and may last from 2 to 6 months ; it includes sulfonamides, amphotericin b, or azoles. in severe cases endovenous amphotericin b or sulfonamides are required and when there is clinical improvement, it can be switched to oral sulfonamides or azoles. extended periods of treatment are often necessary, up to 2 or more years, with a significant frequency of relapsing disease. according to brazilian guidelines, oral itraconazole is the drug of choice (shikanai - yasuda, 2005 ; shikanai - yasuda., 2006 ; travassos and taborda, 2011). although chemotherapy stands as the basic treatment of pcm, therapeutic vaccination with fungal antigens or passive transfer of specific monoclonal antibodies may boost the cell immune response and add to the protective effect of chemotherapy, eventually counteracting a relapsing disease and reducing fibrotic sequels. both the innate immune response and the adaptive immunity are important for the antifungal protective effect. the immune system recognizes fungal antigens with subsequent eliciting of antibodies and t cell protective responses. antigens of p. brasiliensis, gp43 or p10, depend on ifn- for their protective activity (reviewed in travassos., 2008). the aim of this review is to update the new concepts and methodologies used in the attempt to develop a therapeutic vaccine against pcm. the use of low - virulence yeast cells as the immunization tool has been investigated. subcutaneous infection with p. brasiliensis pb265 induced cellular immunity with high t cell reactivity in susceptible mice which resulted in immunoprotection or disease exacerbation depending on the route of a secondary infection (arruda., 2007a). immunoprotection with aseptical cure was shown in the pre - immunization procedure and required a combination of cd4 t cells and cd8 t cells and the production of endogenous ifn- and il-12 as well as increased levels of anti - p. brasiliensis - specific igg1 and igg2a antibodies (arruda., 2007b). the irradiated cells were examined by scanning, and also transmission electron microscopy. when examined 2 h after irradiation the cells showed deep folds or collapsed. the plasma membrane and cell wall were intact, but an extensive dna fragmentation was found (demicheli., 2007). the use of attenuated yeast cells by gamma - irradiation induced a long lasting protection in balb / c mice (demicheli., 2006 ; for this purpose, balb / c mice were immunized twice in the ocular plexus with 10 viable cells. animals were then challenged, by the same route, with yeast cells after 30, 45, and 60 days after the last immunization. thirty days after the challenge, a significant reduction in the fungal burden was observed in the lung, spleen, and liver. a 99.5% decrease in cfu was obtained 90 days post challenge. the animals showed high levels of ifn- and igg2a with very low production of il-10 and il-5, suggestive of a th1 immune cell response (do nascimento martins., 2007). the number of immunizations with radio - attenuated yeast cells also interferes with the immune response. authors found that mice immunized once developed a mixed th1/th2 response, which was less efficient in the control of the infection, whereas a th1 pattern was obtained with two immunizations resulting in the elimination of p. brasiliensis yeast cells (do nascimento martins., 2009). soluble antigens of p. brasiliensis and fractions obtained by ion exchange chromatography of p. brasiliensis extract (f0, fii, and fiii), showed variations in the induction of a protective immune response. mice immunized with f0 and fii developed benign chronic pcm restricted to the lung, associated with low mortality rates and the presence of compact granulomas with few fungal cells. significant enhancement of ifn- and high levels of igg2a and igg3 were found in animals immunized with f0. immunizations with fii induced significant production of ifn- and il-10 associated with high levels of igg1 and igg2a. in contrast, mice immunized with fiii developed a progressive disease with dissemination to spleen and liver. these animals did not control the spread of the fungus showing granulomas with a high number of viable fungal cells (diniz., 2004). a fraction of approximately 380 kda designated high - molecular - mass (hmm) fraction was able to induce lymphocyte proliferation and production of ifn- but not il-4 when incubated with spleen cells from balb / c mice infected intravenous with p. brasiliensis (isolate pb18). animals previously immunized s.c. with hmm and infected with virulent yeast cells showed a significant reduction of the fungal burden in the lungs and spleen (pavanelli., 2007). the major diagnostic antigen gp43 was isolated from p. brasiliensis culture supernatant fluids in 1986 (puccia., 1986). it reacted with antibodies from virtually 100% of patients with pmc, except some patients exposed to p. lutzii, who showed irregular reactivity to gp43 (batista., 2010). epitopes in gp43 that elicit a strong antibody response are peptidic in nature (puccia and travassos, 1991) and different isoforms of gp43 vary in their reactivity with patients sera. the gp43 gene was cloned and sequenced (cisalpino., 1996) ; gene expression and polymorphism have been shown (travassos. the first evidence that gp43 carried an immunodominant epitope able to elicit dth reactions was shown in guinea pigs (rodrigues and travassos, 1994) and later in patients (saraiva., the t cell epitope responsible for dth reactions, and cd4 t cell proliferation, was mapped to a peptide called p10 with the sequence qtliaihtlairyan (taborda., 1998). the hexapeptide htlair has been shown to be essential for priming the cellular immune response. in p.lutzii, there is, however, an important mutation in this hexapeptide (teixeira., 2009). peptide p10 showed to be promiscuous in its presentation by mhc class ii molecules from three different mouse haplotypes (taborda., this was extended to most caucasian hla - dr molecules (iwai., 2003). by using the tepitope algorithm neighboring peptides to p10 were also recognized with high affinity hla - dr binding (iwai., 2007). the protective effect of p10 using complete freund s adjuvant (cfa) against intratracheally infected mice is shown in figure 1. representative histopathology of lung lesions caused by p. brasiliensis pb18 strain in mice immunized with p10 in presence of freund s complete adjuvant (cfa). (a) lung section from infected mouse, with a granuloma containing multiple viable fungal cells ; (b) lung section from mouse treated with cfa, showing the extensive granulomatous lesion with intense cellular infiltration and large number of multiplying fungal cells ; (c) lung section from mouse immunized with p10 admixed with cfa showing preserved alveolar structure, absence of granulomatous lesions as well as of fungal cells. mice infected with yeast cells of the highly virulent p. brasiliensis (pb18) underwent p10 and/or drug treatment starting after 48 h or 30 days of infection. the treatment continued for 30 days, during which groups of mice received intraperitoneal doses of itraconazole, fluconazole, ketoconazole, sulfamethoxazole, or trimethoprim / sulfamethoxazole every 24 h. amphotericin b was administered every 48 h. immunization with p10 was carried out weekly for 4 weeks, once in cfa and three times in incomplete freund s adjuvant (marques., 2006). animals were sacrificed at different times of infection and significant reduction in the fungal load was observed in both groups, with an additive protective effect obtained with the combination of p10 and antifungal drugs (marques., 2006). unexpectedly, animals treated with sulfamethoxazole, showed early protection followed by relapse, but the association of sulfamethoxazole and p10 vaccination successfully controlled the infection. the detection of cytokines in lung homogenates from mice vaccinated with p10, showed a typical th1 response, rich in ifn- and il-12 but without suppression of th2 cytokines (marques. in an attempt to reproduce a general anergic state, balb / c mice were treated with dexamethasone-21 phosphate added to the drinking water. mice were then infected with virulent p. brasiliensis (pb18) and after 15 days were subjected to chemotherapy and/or p10 immunization. the association of drugs and p10 immunization conferred additive protection. a significant increase in il-12 and ifn- and decrease of il-4 and il-10 were observed in mice immunized with p10 alone or in association with antifungal drugs, indicating that also in this case of immune suppression, p10 immunization can be helpful (marques., different ways of peptide delivery using formulations that did not include cfa have also been investigated. a tetramer of truncated p10 was designated m10 and had four equal liaihtlairyan (n - terminal qt - less p10) chains synthesized on a branched lysine core containing glycine at the c - terminal position. mice immunized with a single dose of m10 without adjuvant and challenged intratracheally with p. brasiliensis showed significantly fewer lung, spleen, and liver cfus and few or no yeasts in lung sections histopathology (taborda., 2004). the therapeutic or prophylactic protective effect of p10 was also tested with the peptide admixed with different adjuvants, bacterial flagellin, aluminum hydroxide, cationic lipid in comparison with cfa. a vaccine formulation based on intranasal administrations of gp43 or p10 with animals were immunized with recombinant purified flagellins genetically fused with p10, either or not flanked by two lysine residues, or with the synthetic p10 admixed with purified flic. balb / c mice immunized with the chimeric flagellins and particularly those immunized with p10 admixed with flic had reduced fungal burden in the lungs and elicited a predominantly th1-type immune response (braga., 2009). reduction of the pulmonary fungal burden was obtained with aluminum hydroxide, cfa, flagellin, and cationic lipid in intratracheal infected balb / c mice. the cationic lipid proved to be very efficient in the clearance of fungal load and reduction of fibrotic areas in the lung (mayorga., 2012). glycolic acid) nanoparticles (plga) was tested in the experimental therapeutic protocol of pcm. the incorporation of p10 into plga reduced the amount of this peptide necessary to decrease the fungal load in the infected animals and avoid disease relapse when compared with p10 emulsified in freund s adjuvant (amaral., 2010). the potential use of the recombinant protein of 27 kda (rpb27), present in the soluble fraction f0 (reis., 2008), has been investigated. immunization of rpb27 in the presence of propionibacterium acnes and aluminum hydroxide prior to intravenous infection by the orbital plexus with virulent p. brasiliensis (pb18), was able to efficiently protect balb / c mice. recently, another recombinant protein, rpb40, was used associated with fluconazole and shown to reduce the fungal burden in the lungs of balb / c mice (fernandes., 2011, 2012) immunization of balb / c mice with a mammalian expression vector (vr - gp43) carrying the full gene of gp43 with cmv promoter induced b and t cell - mediated immune responses protective against the intratracheal challenge by virulent p. brasiliensis yeast forms. the cellular immune response in mice immunized with vr - gp43 induced ifn- and the response was maintained for at least 6 months although reduced to half of the stimulation index obtained 15 days after immunization (pinto., 2000). in order to develop a more specific dna vaccine based predominantly on a t cell - mediated immune response, a plasmid encoding the p10 minigene in pcdna3 expression vector was tested in intratracheally infected balb / c and b10.a mice. the vaccination with plasmid encoding p10 induced a significant reduction in the fungal burden in the lung. co - vaccination with a plasmid encoding mouse il-12 proved to be even more effective in the elimination of the fungus with virtual sterilization in the long - term infection and treatment assay, using the more susceptible b10.a mice. the immunization elicited significant production of il-12 and ifn- (rittner., 2012). such immunization with plasmid encoding p10 induced memory cells as well as t regulatory cells (amorim, 2010), that might help reduce the tissue cell damage of the protective immune response. the use of heterologous dna plasmid encoding hsp65 from mycobacterium leprae was used in a prophylactic protocol. intramuscular immunization with dnahsp65 induced an increase of th1 cytokine levels and reduction of the fungal burden with marked reduction of collagen and lung remodeling (ribeiro., 2009). similar results were obtained with a therapeutic model (ribeiro., 2010). a competent cd4 t cell response producing ifn- is usually the chief protective mechanism in fungal infections, particularly in pcm, and dendritic cells are able to initiate the response in nave t cells. the use of transfected dcs with a plasmid (pmac / ps - scfv) encoding a single chain variable fragment (scfv) of an anti - id antibody that is capable of mimicking gp43 from p. brasiliensis was used to subcutaneously immunize balb / c mice. after 7 days, the scfv peptide was presented to the regional lymph node cells and was capable to activate proliferation resulting in a decrease of fungal burden (ferreira., 2011). using an experimental model in balb / c mice, p10-primed dcs were administrated prior to (subcutaneous vaccination) or weeks after (subcutaneous or intravenous injection) p. brasiliensis infection, and showed to significantly reduce the fungal burden. the protective response mediated by the injection of primed - dcs was mainly characterized by increased production of ifn- and il-12 and reduction in il-10 and il-4 compared to infected mice that received saline or unprimed - dcs (magalhes., 2012). the exacerbated humoral immune response in pcm has been associated to a poor prognosis since patients with acute and subacute forms of the disease show high antibody titers. it has been reported, however, that monoclonal antibodies against gp70 were protective against experimental pcm (mattos grosso., 2003). the same group of researchers also showed that a monoclonal antibody against surface 75 kda protein was able to inhibit fungal growth (xander., 2007). infection by p. brasiliensis was examined in balb / c mice (buissa - filho., 2008). using a panel of monoclonal antibodies, protective and non - protective monoclonal antibodies with similar reactivity with gp43 on elisa, were found. the reactivity of mab 3e, the most efficient mab in the reduction of fungal burden, that was able to enhance phagocytosis, was mapped to the sequence nhvripigywav (buissa - filho., 2008). this peptide could thus represent, together with p10, another candidate for a peptide vaccine against pcm. in a p10-pre - immunization protocol, mabs were tested as protective agents. challenge with virulent p. brasiliensis and p. lutzii yeasts, resulted in additive protection using short - term protocols in comparison with a non - protective mab (data not published). while chemotherapy has the chief role of reducing the fungal burden in mycotic infections, the long - term control and eventual sterilization involve an effective immune response. the combination of chemotherapy and an effective vaccine against pcm should ideally treat the more serious cases of the systemic mycosis aiming at a shorter period of treatment, prevention of relapses and of fibrotic sequels. a highly immunogenic antigen of p. brasiliensis and the peptide containing a t cell epitope have repeatedly proved to be protective in prophylactic and therapeutic models with massively infected mice. a new peptide also from the gp43 contains a b cell epitope that reacts with a protective monoclonal antibody. recently, a dna vaccine expressing the p10 peptide showed a remarkable protective effect in a long - term infection protocol using mice highly susceptible to pcm. immunized mice had memory t cells as well as t regulatory cells that prevented tissue cell damage due to the initial pro - inflammatory protective response involving t effector cells. these encouraging results, along with other protective immunizations using new adjuvants, delivery systems, and dendritic cells, point to a next stage of experimentation aiming at the clinical use of the peptide vaccines. 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. | paracoccidioidomycosis (pcm) is an endemic latin american mycosis caused by paracoccidioides brasiliensis and also by the recently described p. lutzii. the systemic mycosis is the 10th leading cause of death due to infectious diseases in brazil. as published, 1,853 patients died of pcm in the 19962006 decade in this country. the main diagnostic antigen of p.brasiliensis is the 43 kda glycoprotein gp43, and its 15-mer peptide qtliaihtlairyan, known as p10, contains the t - cd4 + epitope that elicits an ifn--mediated th1 immune response, which effectively treats mice intratracheally infected with pcm. the association of peptide p10 with antifungal drugs rendered an additive protective effect, even in immunosuppressed animals, being the basis of a recommended treatment protocol. other immunotherapeutic tools include a peptide carrying a b cell epitope as well as protective anti - gp43 monoclonal antibodies. new delivery systems and gene therapy have been studied in prophylactic and therapeutic protocols to improve the efficacy of the recognized antigens aiming at a future vaccine as co - adjuvant therapy in patients with pcm. |
bacterial infection can evoke shock, acute respiratory failure, multiple organ failure, and disseminated intravascular coagulation (dic), resulting in a high mortality rate. lipopolysaccharide (lps), a major component of the outer membrane of gram - negative bacteria, initiates the cascade of pathophysiological reactions called endotoxin shock. a number of mediators including lipid mediators, cytokines, free radicals, complement fragments, coagulatory factors, and proteases contribute to the pathogenesis of endotoxin shock [26 ]. among them, the products generated from neutrophils are recognized to play important roles. activated neutrophils release various types of mediators including proteases and oxygen radicals. protease - antiprotease imbalance is involved in a variety of inflammatory diseases [8, 9 ]. because neutrophil elastase exerts the most injurious effects on many substrates (e.g. elastin, type i iv collagen, fibronectin, laminin, and proteoglycans), it can be a key mediator of tissue injury. indeed, neutrophil elastase and cathepsin g - deficient mice have shown resistance to lethal effects of lps. further, deficiency of secretory leukoprotease inhibitor also reportedly caused higher mortality from endotoxin shock with higher production of il-6 and high mobility group-1. in addition, fitzal and co - workers have shown that blockade of pancreatic proteases in the intestinal lumen ameliorates systemic inflammation induced by intravenous administration of lps. uti is a multivalent kunitz - type serine protease inhibitor that is found in human urine and blood. uti reportedly inhibits neutrophil elastase activity in vitro [15, 16 ] and trypsin activity in patients with pancreatitis. although the therapeutic effects of uti on circulatory shock have been recognized, especially in japan, the current understanding of the target mechanisms / pathways remains unsatisfactory. tani and colleagues reported that uti protects against septic shock induced by gram - negative bacteria in vivo, but only estimated clinical signs such as cardiac index, blood pressure, lactic acid, blood glucose, and blood base values. however, these studies have a critical limit in that the animals were treated with human - derived uti as a foreign protein ; the direct effect of uti on inflammatory diseases including systemic inflammatory response (sirs) syndrome has never been examined in knock - out mice. at first, the role of uti in systemic inflammation using mice deficient in uti gene was investigated. both uti (/) and wild - type (c57/bl6 : wt) mice were injected intraperitoneally (i.p.) with vehicle or lps at a dose of 1 mg / kg body weight. evaluation of coagulatory and fibrinolytic parameters and white blood cell (wbc) counts at 72 h after i. p. challenge showed that fibrinogen levels were significantly greater in lps - challenged wt mice (p<0.05) and lps - challenged uti (/) mice (p<0.01) than in vehicle - challenged mice with the same genotypes. in the presence of lps, they were also significantly higher in uti (/) mice than in wt mice (p<0.05). wbc counts significantly decreased after lps challenge in uti (/) mice (p<0.01 versus other groups). lps appeared to shorten pt when compared to vehicle treatment in uti (/) mice, although this difference did not reach significance. in the presence of lps, prothrombin time was significantly shorter in uti (/) mice than in wt mice (p<0.05). histopathological changes in the lung, kidney, and liver of both genotypes at 72 h after lps challenge revealed severe neutrophilic inflammation in uti (/) lungs challenged with lps. in contrast lps challenge induced neutrophilic infiltration around glomeruli and in the interstinum of the kidneys of both genotypes. however, the severity was more prominent in uti (/) mice than in wt mice in the presence of lps. lps caused wide spread centrilobular vacuolation of hepatocytes and neutrophilic infiltration in livers of both genotypes. in the presence of lps the protein expression of interleukin (il)-1, macrophage inflammatory protein (mip)-1, mip-2, macrophage chemoattractant protein (mcp)-1, and keratinocyte chemoattractant in the lung, kidney, and liver 72 h after lps administration was evaluated. in the lung, lps challenge caused significant elevation of il-1, mip-1, mip-2, mcp-1, and keratinocyte - derived chemoattractant (kc) levels in uti (/) mice when compared to vehicle challenge (p<0.01). in wt mice, lps treatment significantly enhanced the expression of il-1, mip-1, mip-2, mcp-1, and kc when compared to vehicle challenge. in the presence of lps, the lung expression of mcp-1 was significantly greater in uti (/) mice than in wt mice (p<0.05). these results suggest that uti protects against systemic inflammation induced by intraperitoneal administration of lps, at least partly, through the inhibition of proinflamatory cytokine production / release. in another series of studies, the role of uti in another type of sirs, acute lung inflammation induced by pulmonary exposure to lps, was examined. in brief, uti (/) and wt mice were intratracheally treated with vehicle or lps, and sacrificed 24 h later. in both genotypes, lps treatment induced significant increases in the numbers of total cells and neutrophils in bronchoalveolar lavage (bal) fluid as compared with vehicle treatment (p<0.01). lps treatment caused greater and significant increases in the numbers of bal total cells (p<0.05) and neutrophils (p<0.01) in uti (/) mice than in wt mice. also, lps treatment increased the lung water content in both genotypes of mice (p<0.05) when compared to vehicle treatment. uti (/) mice, however, showed a significantly greater increase in lung water content when compared to wt mice (p<0.05) following lps treatment. lung specimens stained with hematoxylin and eosin 24 h after intratracheal instillation showed that in the presence of lps, wt mice showed moderate infiltration of neutrophils. in uti (/) mice, lps treatment led to a marked recruitment of neutrophils and interstitial edema. vehicle administration alone caused no histological changes in either wt or uti (/) mice. lps treatment induced significant elevation of the protein levels of il-1, mip-1, mcp-1, and kc in lung homogenates when compared to vehicle treatment in both genotypes (p<0.01). in the presence of lps, the local expression of mcp-1 and kc was significantly higher in uti (/) mice than in wt mice (p<0.05 for kc, p<0.01 for mcp-1). furthermore, immunohistochemical examination showed that in the presence of lps, immunoreactive 8-hydroxy-2'-deoxyguanosine was detected in the lungs of both strains of mice, but the staining was more prominent in uti (/) mice than in wt mice. on the other hand, immunoreactive nitrotyrosine was strongly detected only in uti (/) mice challenged with lps. quantitative gene expression analyses of lung homogenates obtained 4 h after intratracheal challenge showed that compared to vehicle treatment, lps treatment resulted in significant elevation of gene expression for inducible nitric oxide synthase (inos) in both genotypes of mice (p<0.05). in the presence of lps, the local expression of inos was higher in uti (/) mice than in wt mice. these results suggest that uti is also protective against acute lung inflammation induced by intratracheal administration of lps, at least in part, via the local suppression of proinflammatory cytokines and antioxidation. in conclusion, uti protects against sirs pathophysiology and subsequent organ damage induced by lps in mice, at least partly, via the modulation of the proinflammatory cytokine il-1, as well as chemokines such as mip-2, mcp-1, and kc (fig. 1). these in vivo results provide direct and novel molecular evidence for the rescue therapeutic potential of uti against systemic inflammatory responses syndrome such as dic, acute lung injury, and multiple organ dysfunction syndrome. | urinary trypsin inhibitor (uti), a serine protease inhibitor, has been widely used in japan as a drug for patients with acute inflammatory disorders such as disseminated intravascular coagulation (dic), shock, and pancreatitis. recent in vitro studies have demonstrated that serine protease inhibitors may have anti - inflammatory properties beyond their inhibition of neutrophil elastase at the site of inflammation. however, the therapeutic effects of uti in vivo remain unclear. in this review, we introduce the roles of uti in the experimental systemic inflammatory response induced by both intraperitoneal and intratracheal administration of lipopolysaccharide using uti deficient and wild - type mice. our experiments suggest that uti can protect against systemic inflammatory response and subsequent organ injury induced by bacterial endotoxin, at least partly, through the inhibition of proinflammatory cytokine and chemokine expression. uti may therefore present an attractive rescue therapeutic option for systemic inflammatory response syndromes such as dic, acute lung injury, and multiple organ dysfunction. |
lumbar foraminal stenosis (lfs) is a condition seen in degenerative lumbar spinal disorders in which a nerve root or spinal nerve is entrapped in a narrowed lumbar foramen. there is a dorsal root ganglion that functions as a pain receptor at this site making this condition refractory and likely to cause severe lower limb pain10). however, macnab14) suitably referred to this region as the hidden zone and despite major strides in imaging technology today, this site is still often overlooked, making it a factor that can adversely impact surgical success rates. nerve decompression sites differ in intra - spinal lesions and foraminal stenosis, and it has been reported that many cases of failed back surgery syndrome are caused by inappropriate treatment of foraminal stenosis4). double - crush lesion where the l4/5 level is compressed by an intraspinal canal lesion and the l5/s1 level is compressed by a lateral lesion so that the nerve is compressed at 2 points (medial and lateral), hence the name. however, traditional imaging studies do not allow the clinician to differentially diagnose whether the compressing lesion is inside or outside the spinal canal, or if a double - crush lesion is responsible. the japanese orthopaedic association back pain evaluation questionnaire (joabpeq)6) provides specific, yet multidimensional outcome measures for patients with low back pain (lbp), including dysfunction and disabilities caused by the disease, as well as resulting psychosocial problems. the purpose of this study was to investigate the use of the joabpeq to diagnose lfs in symptomatic patients. our findings from using the joabpeq to study clinical symptoms in detail to determine the scale s usefulness in diagnosing lfs are presented below. thirteen cases (mean age, 72 years) of lfs and 30 cases (mean age, 73 years) of lscs involving one intervetebral space were enrolled as subjects from among the 143 patients (mean age, 66.8 years) who underwent lumbar surgery between april 2013 and october 2015 at our institution. lfs neuropathy was level l3 in 2 cases, l4 in 2 cases and l5 in 9 cases. lscs was level l1/2 in 1 case, l2/3 in 2 cases, l3/4 in 5 cases, and l4/5 in 22 cases. lfs was diagnosed by microendoscopic intrapedicular partial pediculotomy in 3 cases15), and posterior lumbar inter - body fusion in 10 cases. lscs was diagnosed in all patients through lumbar spinous process - splitting laminectomy17). before surgery, lfs was diagnosed comprehensively based on clinical symptoms, physical findings, plain x - rays, computed tomography (ct), magnetic resonance imaging (mri), and 3-dementional - mri (3d - mri). foraminal stenosis was defined as : abnormalities such as nerve indentation, swelling, and running transversely in their course through the foramen on 3d - mri. ultimately, nerve roots were blocked selectively to diagnose damaged nerve roots based on function. if the visual analog scale (vas 100-mm method) of the lower limbs was alleviated by 20 mm or less at 30 minutes after nerve root block, the diagnosis was considered positive. patient exclusion criteria were as follows : (1) those with residual lower limb pain, (2) those who had previous lumbar spinal surgery, (3) those who had multiple levels of lumbar canal stenosis, (4) those who had myelopathy, and (5) those who had spinal tumor, infectious disease, or spinal trauma. clinical symptoms were evaluated using the vas score for lbp and leg pain ranging from 100mm(extreme amount of pain) to 0 mm (no pain), the japanese orthopedic association (joa ; 029 points) scoring system, and the roland - morris disability questionnaire (rdq ; 024 points). the normal joa score is 29 points, based on 3 subjective symptoms (9 points), 3 clinical signs including straight - leg raising (6 points), and 7 activities of daily living (14 points). the normal rdq is zero points with the total number of items checked from a minimum of 0 to a maximum of 24. the joabpeq includes 25 questions based on rdqs and short form 36 (sf-36). for q1 - 1 through q4 - 1 and q5 - 1, a score of 1 was considered positive for symptoms, while a 2 or q4 - 3, and q5 - 2 through q5 - 7, a score of 1 or 2 was considered positive for symptoms, and 3 to 5 were negative (table 1). scores are calculated based on the answers to questions in 5 domains : pain - related disorders, lumbar spine dysfunction, gait disturbance, social life dysfunction, and psychological disorders. the score for each domain was calculated according to the official guidelines and ranged from 0 to 100 points, which is deemed proportional to the patient s clinical condition. all human and animal studies have been approved by the chiba university and shimoshizu national hospital and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. 5.0 (sas institute inc., cary, nc, usa). for each clinical symptom, differences between both groups were evaluated using an unpaired t - test. for each joabpeq item vas scores (lbp) were lfs : 735.17 mm, lscs : 604.67 mm (p=0.105) ; vas scores (leg pain) were lfs : 805.37 mm, lscs : 655.25 mm(p=0.090) ; joa scores were lfs : 150.69, lscs : 190.54 (p=0.00047) ; rdq scores were lfs : 131.30, and lscs : 110.99 (p=0.157). joa scores were significantly lower (p<0.001) in lfs compared to lscs (fig. 1). categories in joabpeq include pain - related disorders lfs : 387.48, lscs : 576.51 (p=0.087), lumbar spine dysfunction lfs : 446.86, lscs : 674.22 (p=0.026), gait disturbance lfs : 286.34, lscs : 415.01 (p=0.082), social life disturbance lfs : 325.86, lscs : 473.28 (p=0.009), and psychological disorders lfs : 355.81, lscs : 433.33 (p=0.199). lfs showed significantly lower scores in lumbar dysfunction (p<0.05), and social life disturbance (p<0.01) compared to lscs (fig. specifically, in pain - related disorders : (1) have difficulty sleeping : lfs, 53.8% ; lscs, 16.6%(p=0.0125) ; in lumbar spine dysfunction : (2) have difficulty standing up from a chair : lfs, 53.8% ; lscs, 6.6%(p=0.0004) ; (3) have difficulty turning over : lfs, 76.9% ; lscs, 40%(p=0.0261) ; (4) have difficulty putting on socks or stockings : lfs, 76.9% ; lscs, 26.6% (p=0.0021) ; in gait disturbances : (5) have difficulty walking more than 15 minutes : lfs, 61.5% ; lscs, 26.6%(p=0.0298) ; in social life disturbance : (6) do not do routine housework : lfs, 38.4% ; lscs, 0% (p=0.0003) ; and in psychological disorders : (7) are not in decent health : lfs, 69.2% ; lscs, 30.0%(p=0.0166). there were 7 question items and incidence was consistently higher in lfs than lscs (table 2). appropriately named the hidden zone by macnab14), lfs is often overlooked, accounts for approximately 60% of failed back surgery syndromes, and plays a major role in lowering surgical success rates4). diagnostic imaging of lumbar spinal canal stenosis involves a comprehensive review of x - rays, ct, and mri7,12,16), together with functional diagnosis through selective nerve root imaging and infiltration8). conventional mri reportedly produces false positives in 30% to 40% of lfs cases and this is therefore a difficult condition to diagnose. recently, 3d - ct, mr myelography13), 3d - mri2,20), and diffusion tensor imaging5) have been reported to be diagnostically useful. nerve root damage in lfs is most common in the l5 nerve root, accounting for 75% of cases10). there are no useful diagnostic methods to differentiate between possible causes of l5 nerve damage that could be medial stenotic lesions in the l4/5 level, lateral lesions in the l5/s1 level, or double - crush lesions. compared to medial lesions, distal latency is delayed in lateral lesions allowing for a differential diagnosis between the two. however, this is an invasive test and non - invasive diagnostic methods are virtually nonexistent1,9). due to drg involvement in patients with symptomatic lfs, they have generally been recognized as demonstrating more severe symptoms than patients with lscs3,10,18,19). in this study we investigated clinical symptoms specific to patients with lfs symptoms. joa scores were significantly lower in lfs compared to lscs and lumbar spine dysfunction and social life disturbance in joabpeq - measured functionality was also significantly lower in those with lfs. the rdq and oswestry disability index are used as specific scales of low - back - pain associated quality of life, while sf-36 and euroqol are widely used around the world as comprehensive measures of health. joabpeq is a patient - based assessment of treatment results that includes both the scientific and psychological aspects4). an excel file can be shared from the joa website, allowing for automatic assessment of individual patient severity. statistically significant differences were noted in 7 of the joabpeq domains, namely difficulties in : (1) sleeping, (2) standing up from a chair, (3) turning over, (4) putting on socks, (5) walking for 15 minutes, (6) doing household chores, and (7) remaining in decent health. in previous reports, yamada.19) found that pain when recumbent, pain on sitting, the bonnet test, and the freiberg test were specific symptoms of lfs. watanabe.18) reported that incidences of kemp test, intermittent claudication, leg pain in a sitting position, and leg pain at night were high among lfs patients. baba.3) reported that all patients suffered from leg pain caused by nerve root involvement, and the incidence of kemp sign (84.6%), intermittent claudication (84.6%), and leg pain at rest (61.5%) were all high. they assigned an integer score to each identified risk factor as follows : pain when recumbent, 9 points ; positive freiberg test result, 5 points ; positive bonnet test result, 3 points ; and pain on sitting, 3 points. for each patient, all applicable risk score values were added for a total risk score which ranged from 0 to 20. receiver operating characteristic (roc) curve analysis demonstrated cutoff value was 5 points, and the area under the roc curve was 0.87435, with a sensitivity of 75.5%, and specificity of 82.3%. in this study, a high incidence of difficulties were reported in lfs patients such as in sleeping (53.8%), getting up from a chair (53.8%), turning over in bed (76.9%), putting on socks (76.9%) and intermittent claudication such as resting state pain and inability to walk 15 minutes or more (61.5%), findings which do not differ from previously published reports. our study suggests that if resting state pain and intermittent claudication are noted on the joabpeq, an established and widely available patient based outcome scale, further diagnosis and treatment should be considered with potential lfs in mind. by using existing and established assessment methods, it may be possible to diagnose lfs easily in a general practice setting. further studies are needed to investigate whether our findings remain valid in a large population. second, joabpeq only assesses lumbar pain and there are no questions related to lower limb pain. jones.11) reported that there was a significant improvement in lbp in patients with lscs undergoing spinal decompression. in this study, vas score (leg pain) decreased from lfs : 805.37 mm and lscs : 655.25 mm to lfs : 165.70 mm (p<0.001) and lscs : 133.11 mm (p<0.001) after decompressed surgery. vas score (lbp) decreased from lfs : 735.17 mm and lscs : 604.67 mm to lfs : 247.04 mm (p<0.001) and lscs : 163.30 mm(p<0.001) after decompressed surgery. not only leg pain but also lbp significantly improved by decompressed surgery in both group (fig. there are a number of possible explanations for this phenomenon such as improvement of posture, distressed facet joint, and improved nutrient supply to ischemic nerves11). finally, in this study we looked at spinal stenosis in lsf patients, but no comparisons were made with lumbar disc herniation, so further investigations will be necessary. compared to lscs, those with lfs had significantly lower joa scores and both the lumbar spine dysfunction and social life disturbance scores on the joabpeq scale were also significantly lower. joabpeq scores showed a significantly higher incidence of difficulties in : sleeping, getting up from a chair, turning over, and putting on socks together such as resting state pain with a higher incidence of intermittent claudication in those with lfs. results suggest that of the items in the joabpeq, if pain during rest or intermittent claudication is noted, lfs should be kept in mind as a cause during subsequent diagnosis and treatment. | objectiveit is important to develop an easy means of diagnosing lumbar foraminal stenosis (lfs) in a general practice setting. we investigated the use of the japanese orthopaedic association back pain evaluation questionnaire (joabpeq) to diagnose lfs in symptomatic patients.methodssubjects included 13 cases (mean age, 72 years) with lfs, and 30 cases (mean age, 73 years) with lumbar spinal canal stenosis (lscs) involving one intervertebral disc. the visual analogue scale score for low back pain and leg pain, the joabpeq were evaluated.resultsthose with lfs had a significantly lower joa score (p<0.001), while joabpeq scores (p<0.05) for lumbar dysfunction and social functioning impairment (p<0.01) were both significantly lower than the scores in lscs. the following joabpeq questionnaire items (lfs vs. lscs, p - value) for difficulties in : sleeping (53.8% vs. 16.6%, p<0.05), getting up from a chair (53.8% vs. 6.6%, p<0.001), turning over (76.9% vs. 40%, p<0.05), and putting on socks (76.9% vs. 26.6%, p<0.01) such as pain during rest, and signs of intermittent claudication more than 15 minutes (61.5% vs. 26.6%, p<0.05) were all significantly more common with lfs than lscs.conclusionresults suggest that of the items in the joabpeq, if pain during rest or intermittent claudication is noted, lfs should be kept in mind as a cause during subsequent diagnosis and treatment. lfs may be easily diagnosed from lscs using this established patient - based assessment method. |
allergic conjunctivitis is a complex disorder that significantly contributes to the burden of misery and economic impact resulting from environmental allergies, especially in those patients who are either untreated or ineffectively treated.1 allergic conjunctivitis is reported to affect approximately 15% to 20% of the us population.2 some suggest that as much as 30% of the us population is affected by seasonal allergy symptoms with as many as 70% to 80% of these people having ocular symptoms.3 recent reports suggest that ocular allergy is both under - diagnosed and under - treated ; therefore, the actual global impact may be underrepresented.4 seasonal and perennial allergic rhinitis and conjunctivitis cause disruption in daily activities, which is reflected by diminished quality of life measures.1,5 managing allergy effectively requires adequate relief of symptoms and prevention of future symptoms. with this approach, patients have reported enhanced quality of life.1,6 the ocular manifestations of seasonal and perennial allergy occur as a result of mast cell degranulation and subsequent allergic inflammation in sensitized individuals. during this cascade, allergens bind and cross - link allergen - specific ige antibodies on conjunctival mast cell surfaces, initiating degranulation and the release of histamine. once released, histamine binds to histamine receptors on the conjunctival surface, causing itching, redness, and swelling.7 recent evidence suggests that histamine - stimulated conjunctival epithelial cells may upregulate the allergic inflammatory cascade.8,9 mast cells additionally release both preformed and synthesized mediators that contribute to the allergic response.10,11 the comparatively large surface area of the conjunctiva, a robust vascular supply, and a dense concentration of mast cells make allergic conjunctivitis a particularly vexing form of allergy for affected patients. as such, appropriate diagnosis and effective treatment can have a positive impact on patient s quality of life and disease management. in addition, meeting a patient s perceived needs is paramount for effective allergy management, particularly with ocular allergy, due to the eyes habitual but necessary exposure to the environment. the newest class of topical anti - allergy medication for allergic conjunctivitis is the dual - action agent, which combines strong antihistaminic activity (providing rapid relief) with mast - cell stabilizing properties (responsible for prolonged relief).12 five dual - action agents have earned approval from the us food and drug administration (fda) for the treatment of allergic conjunctivitis : epinastine 0.05% (elestat ; allergan, inc., irvine, ca, usa), ketotifen 0.025% (zaditor ; novartis ophthalmics, duluth, ga, usa), azelastine 0.05% (optivar ; medpointe pharmaceuticals, somerset, nj, usa), olopatadine 0.1% (patanol ; alcon laboratories, inc., fort worth, tx, usa), and olopatadine 0.2% (pataday ; alcon laboratories, inc., fort worth, tx, usa). despite similar classification, there is evidence that significant differences exist between the various agents. specifically, olopatadine 0.1%, the first dual - action agent approved by the fda, has demonstrated superior efficacy compared to the other dual - action agents in numerous clinical studies.1315 olopatadine also demonstrates minimal surface activity on mast cell and corneal epithelial cell membranes, which is dose - dependent and potentially clinically relevant.16 as the first agent in its class, olopatadine 0.1% has become the agent by which all other agents in this class are judged. olopatadine 0.2% was recently introduced in an attempt to improve on the qualities of the 0.1% formulation. it has demonstrated patient acceptance and clinical efficacy comparable or superior to olopatadine 0.1%.17 like other dual - action agents, olopatadine 0.2% is approved for the treatment of itching associated with allergic conjunctivitis.18 however, it is the only dual - action agent to be indicated for once daily (qd) dosing,18 providing a potential advantage over twice daily (bid) medications.1922 once daily dosing provides increased convenience and possibly even improved patient adherence to treatment. with more convenient qd dosing, it is likely that olopatadine 0.2% will supplant its lower concentration predecessor.23 olopatadine 0.2% has demonstrated superior comfort and efficacy compared to the dual - action agent epinastine 0.05%.24 no controlled clinical studies have been published directly comparing olopatadine 0.2% and azelastine 0.05% ; however, olopatadine 0.1% has demonstrated superior clinical efficacy compared to azelastine 0.05% in a comparison using the controlled conjunctival antigen challenge (cac) model.15 to examine the relative clinical characteristics of olopatadine 0.2% and azelastine 0.05%, this report explores the relationship among clinical efficacy, perceived comfort and therapeutic satisfaction of these 2 agents. specifically, selected results from 2 independent, prospective, patient - reported outcome studies focusing on allergic conjunctivitis management using either or both olopatadine 0.2% and azelastine 0.05% are examined to gain insight into this clinically important paradigm.25,26 the pace (pataday allergic conjunctivitis evaluation) study was a multi - center, prospective, open - label, single - arm study conducted at 10 allergy, ophthalmology, and optometry practices throughout the us during the spring of 2008 that examined adult patients with allergic conjunctivitis. the purpose of this study was to evaluate patient perceptions of olopatadine 0.2% and previous bid anti - allergy medication (olopatadine 0.1%, azelastine 0.05%, ketotifen 0.025%, or epinastine 0.05% used within the last 6 months) for the treatment of allergic conjunctivitis. patients 18 years or older with active signs and/or symptoms of allergic conjunctivitis (as assessed by the investigator) who had been treated in the last 6 months with a prescription, topical, ocular, anti - allergy, bid medication were included in this study. exclusion criteria were any serious ocular or other medical condition that could result in a patient s inability to safely complete the study ; hypersensitivity or other contraindication to the use of the study medication or its components ; known history of recurrent corneal erosion syndrome ; ocular trauma in either eye within 3 months prior to visit 1 ; any ocular surgical intervention within 6 months prior to visit 1 or anticipation of ocular surgery during the study ; presumed or actual ocular infection or history of ocular herpes in either eye ; and any significant illness that could be expected to interfere with the study, including autoimmune disease, psoriasis, eczema, rosacea, severe cardiovascular disease, poorly controlled hypertension, poorly controlled diabetes, history of status asthmaticus, or history of moderate to severe allergic asthma. on day 1 (visit 1), patients completed an allergy questionnaire to evaluate their previous bid medication and investigators completed a medical history. the questionnaires asked patients to rate their perceptions of efficacy for their anti - allergy medication (previous bid medication was rated at day 1 and olopatadine 0.2% at day 7) with respect to ocular itching, redness, tearing, and swelling using the 5 descriptors : very / somewhat effective, undecided, and very / somewhat ineffective. patients were also asked to rate their satisfaction with their anti - allergy medication with respect to 3 parameters (drop comfort, speed of relief, and overall satisfaction) using the descriptors : very / somewhat satisfied, undecided, and very / somewhat dissatisfied. within - patient changes from baseline to follow - up in the global score were tested using paired t test. between - group comparisons were carried out using pearson s chi square test, or fisher s exact test when sample sizes < 30. statistical analysis was performed in sas (pc version 9.1.2, sas institute, cary, nc, usa) by an independent biostatistician. this was a single - center, prospective, double - masked, placebo- and contralaterally controlled cac study of patients with a documented history of allergic conjunctivitis conducted by ophthalmic research associates, inc. the purpose of this cac study was to determine the comfort of qd olopatadine 0.2% relative to currently available bid anti - allergy medications. patients 18 years or older must have manifested a positive allergen challenge reaction (ie, itching and redness) in each eye at both visit 1 (screening visit) and visit 2 (confirmatory visit), manifested a positive skin test reaction within the past 24 months to the allergen reportedly causing the allergic conjunctivitis, and had a best - corrected logmar visual acuity score of 0.60 or better in each eye. exclusion criteria included any allergy or contraindication to the use of any study medication, active ocular infection, any ocular or medical condition that could affect study parameters, signs or symptoms of allergic conjunctivitis (greater than 1 + redness or any itching) in either eye at the start of any visit, dry eye syndrome requiring daily use of artificial tear substitute, history of ocular surgery within the past 3 months, use of an investigational drug or device within 30 days before visit 1 or during the study period, and use of any medications (ie, h1-antagonist antihistamines, mast cell stabilizers, corticosteroids) within 72 hours before visit 1 or anytime during the study that could interfere with the study parameters. this study followed a standardized cac protocol.27 after identifying and confirming the proper dosage of a known allergen at visits 1 and 2, patients were randomized by treatment and by eye to receive 1 of 4 study medications (olopatadine 0.2%, olopatadine 0.1%, azelastine 0.05%, or ketotifen 0.025%) in 1 eye. all patients received placebo (tears naturale ii, alcon laboratories, inc., fort worth, tx, usa) in the contralateral eye. this report presents data only from those eyes treated with olopatadine 0.2%, azelastine 0.05%, or placebo. at visit 3, investigators instilled study medication (anti - allergy medication in one eye and placebo in the other) 5 minutes prior to and 30 minutes after cac. patients assessed drop comfort using an 11-point scale (0 = very comfortable to 10 = very uncomfortable) immediately, 30 seconds, 1 minute, and 2 minutes after second instillation. differences in mean drop comfort scores between treatment groups were assessed using a paired t test at each time point. safety variables assessed were corrected distance visual acuity, slit lamp biomicroscopy, and all adverse events (reported, elicited, and observed). the pace (pataday allergic conjunctivitis evaluation) study was a multi - center, prospective, open - label, single - arm study conducted at 10 allergy, ophthalmology, and optometry practices throughout the us during the spring of 2008 that examined adult patients with allergic conjunctivitis. the purpose of this study was to evaluate patient perceptions of olopatadine 0.2% and previous bid anti - allergy medication (olopatadine 0.1%, azelastine 0.05%, ketotifen 0.025%, or epinastine 0.05% used within the last 6 months) for the treatment of allergic conjunctivitis. patients 18 years or older with active signs and/or symptoms of allergic conjunctivitis (as assessed by the investigator) who had been treated in the last 6 months with a prescription, topical, ocular, anti - allergy, bid medication were included in this study. exclusion criteria were any serious ocular or other medical condition that could result in a patient s inability to safely complete the study ; hypersensitivity or other contraindication to the use of the study medication or its components ; known history of recurrent corneal erosion syndrome ; ocular trauma in either eye within 3 months prior to visit 1 ; any ocular surgical intervention within 6 months prior to visit 1 or anticipation of ocular surgery during the study ; presumed or actual ocular infection or history of ocular herpes in either eye ; and any significant illness that could be expected to interfere with the study, including autoimmune disease, psoriasis, eczema, rosacea, severe cardiovascular disease, poorly controlled hypertension, poorly controlled diabetes, history of status asthmaticus, or history of moderate to severe allergic asthma. on day 1 (visit 1), patients completed an allergy questionnaire to evaluate their previous bid medication and investigators completed a medical history. the questionnaires asked patients to rate their perceptions of efficacy for their anti - allergy medication (previous bid medication was rated at day 1 and olopatadine 0.2% at day 7) with respect to ocular itching, redness, tearing, and swelling using the 5 descriptors : very / somewhat effective, undecided, and very / somewhat ineffective. patients were also asked to rate their satisfaction with their anti - allergy medication with respect to 3 parameters (drop comfort, speed of relief, and overall satisfaction) using the descriptors : very / somewhat satisfied, undecided, and very / somewhat dissatisfied. within - patient changes from baseline to follow - up in the global score were tested using paired t test. between - group comparisons were carried out using pearson s chi square test, or fisher s exact test when sample sizes < 30. statistical analysis was performed in sas (pc version 9.1.2, sas institute, cary, nc, usa) by an independent biostatistician. this was a single - center, prospective, double - masked, placebo- and contralaterally controlled cac study of patients with a documented history of allergic conjunctivitis conducted by ophthalmic research associates, inc. the purpose of this cac study was to determine the comfort of qd olopatadine 0.2% relative to currently available bid anti - allergy medications. patients 18 years or older must have manifested a positive allergen challenge reaction (ie, itching and redness) in each eye at both visit 1 (screening visit) and visit 2 (confirmatory visit), manifested a positive skin test reaction within the past 24 months to the allergen reportedly causing the allergic conjunctivitis, and had a best - corrected logmar visual acuity score of 0.60 or better in each eye. exclusion criteria included any allergy or contraindication to the use of any study medication, active ocular infection, any ocular or medical condition that could affect study parameters, signs or symptoms of allergic conjunctivitis (greater than 1 + redness or any itching) in either eye at the start of any visit, dry eye syndrome requiring daily use of artificial tear substitute, history of ocular surgery within the past 3 months, use of an investigational drug or device within 30 days before visit 1 or during the study period, and use of any medications (ie, h1-antagonist antihistamines, mast cell stabilizers, corticosteroids) within 72 hours before visit 1 or anytime during the study that could interfere with the study parameters. this study followed a standardized cac protocol.27 after identifying and confirming the proper dosage of a known allergen at visits 1 and 2, patients were randomized by treatment and by eye to receive 1 of 4 study medications (olopatadine 0.2%, olopatadine 0.1%, azelastine 0.05%, or ketotifen 0.025%) in 1 eye. all patients received placebo (tears naturale ii, alcon laboratories, inc., fort worth, tx, usa) in the contralateral eye. this report presents data only from those eyes treated with olopatadine 0.2%, azelastine 0.05%, or placebo. at visit 3, investigators instilled study medication (anti - allergy medication in one eye and placebo in the other) 5 minutes prior to and 30 minutes after cac. patients assessed drop comfort using an 11-point scale (0 = very comfortable to 10 = very uncomfortable) immediately, 30 seconds, 1 minute, and 2 minutes after second instillation. differences in mean drop comfort scores between treatment groups were assessed using a paired t test at each time point. safety variables assessed were corrected distance visual acuity, slit lamp biomicroscopy, and all adverse events (reported, elicited, and observed). a total of 125 patients was enrolled in the pace study.25 this report presents data from the 49 patients with a history of using the bid medication azelastine 0.05%. forty - eight patients (98%) completed the questionnaire at both day 1 and day 7 ; one patient was lost to follow - up. the average age of the 49 patients with a history of azelastine 0.05% use was 56.6 years (range, 2685 ; table 1). approximately three - quarters of the patients were female ; 51% were white and 24% were hispanic. similar percentages of patients rated olopatadine 0.2% and azelastine 0.05% as somewhat effective for both itching and redness, but more patients reported that olopatadine was very effective compared with azelastine 0.05% (46% vs 20% for itching, 42% vs 17% for redness ; figure 1). while patients reported similar overall favorable results (somewhat to very effective) between the 2 medications for tearing and swelling, more of these patients reported that olopatadine 0.2% was very effective compared with azelastine 0.05% (25% vs 9% for tearing, 47% vs 8% for swelling ; figure 2). the difference between medications in relief of swelling was statistically significant (p = 0.0404) ; a trend toward superiority for olopatadine was reported for both itching (p = 0.0691) and redness (p = 0.0715). approximately 3 to 4 times as many patients rated themselves very satisfied with current olopatadine 0.2% use compared with past azelastine 0.05% use on 3 different parameters (figure 3) : drop comfort (75% vs 25%, p < 0.0001), speed of relief (60% vs 20%, p = 0.0004), and overall satisfaction (70% vs 16%, p 5 adverse events in 3 patients were reported during olopatadine 0.2% treatment : adverse taste (n = 2), dry eye (n = 1), post - nasal drip (n = 1), and dilated pupil (n = 1). all adverse events resolved without treatment. thirty - six patients were enrolled in the cac study;26 data from 17 eyes included in the olopatadine 0.2% (n = 8) and azelastine 0.05% (n = 9) groups and all 36 contralateral eyes included in the placebo groups are reported here. upon instillation, olopatadine 0.2% was rated significantly more comfortable than azelastine 0.05% (mean comfort score, 2.8 vs 7.6, p = 0.0223) and was indistinguishable from that of placebo (mean comfort score, 2.8 ; figure 4). patients using olopatadine 0.2% also reported significantly better comfort scores than patients using azelastine 0.05% at both 30 seconds (p = 0.0479) and 1 minute (p = 0.0240) ; although olopatadine 0.2% was numerically better at 2 minutes, this did not reach statistical significance (p = 0.2984). a total of 125 patients was enrolled in the pace study.25 this report presents data from the 49 patients with a history of using the bid medication azelastine 0.05%. forty - eight patients (98%) completed the questionnaire at both day 1 and day 7 ; one patient was lost to follow - up. the average age of the 49 patients with a history of azelastine 0.05% use was 56.6 years (range, 2685 ; table 1). approximately three - quarters of the patients were female ; 51% were white and 24% were hispanic. similar percentages of patients rated olopatadine 0.2% and azelastine 0.05% as somewhat effective for both itching and redness, but more patients reported that olopatadine was very effective compared with azelastine 0.05% (46% vs 20% for itching, 42% vs 17% for redness ; figure 1). while patients reported similar overall favorable results (somewhat to very effective) between the 2 medications for tearing and swelling, more of these patients reported that olopatadine 0.2% was very effective compared with azelastine 0.05% (25% vs 9% for tearing, 47% vs 8% for swelling ; figure 2). the difference between medications in relief of swelling was statistically significant (p = 0.0404) ; a trend toward superiority for olopatadine was reported for both itching (p = 0.0691) and redness (p = 0.0715). approximately 3 to 4 times as many patients rated themselves very satisfied with current olopatadine 0.2% use compared with past azelastine 0.05% use on 3 different parameters (figure 3) : drop comfort (75% vs 25%, p < 0.0001), speed of relief (60% vs 20%, p = 0.0004), and overall satisfaction (70% vs 16%, p 5 adverse events in 3 patients were reported during olopatadine 0.2% treatment : adverse taste (n = 2), dry eye (n = 1), post - nasal drip (n = 1), and dilated pupil (n = 1). all adverse events resolved without treatment. thirty - six patients were enrolled in the cac study;26 data from 17 eyes included in the olopatadine 0.2% (n = 8) and azelastine 0.05% (n = 9) groups and all 36 contralateral eyes included in the placebo groups are reported here. upon instillation, olopatadine 0.2% was rated significantly more comfortable than azelastine 0.05% (mean comfort score, 2.8 vs 7.6, p = 0.0223) and was indistinguishable from that of placebo (mean comfort score, 2.8 ; figure 4). patients using olopatadine 0.2% also reported significantly better comfort scores than patients using azelastine 0.05% at both 30 seconds (p = 0.0479) and 1 minute (p = 0.0240) ; although olopatadine 0.2% was numerically better at 2 minutes, this did not reach statistical significance (p = 0.2984). ocular allergy is often overlooked but represents a significant component of the burden of allergic disease for many patients.4 the eye is an organ critical for survival yet is directly exposed to the environment and offending allergens. as such, managing ocular allergy can pose a significant therapeutic challenge. previous studies have shown superior targeting and greater effectiveness of topical treatment compared with systemic therapies in managing ocular allergies.28 this analysis compares 2 currently available dual - action topical ocular anti - allergy agents in an attempt to better understand what constitutes therapeutic success from the patient s perspective. results from these 2 independent studies reinforce the importance of comfort and perceived clinical efficacy in overall patient satisfaction with therapy. olopatadine 0.2% demonstrates superior tolerability compared to azelastine 0.05% in both studies, with significantly greater comfort upon instillation. in addition, patients from the pace study consistently rated olopatadine 0.2% more favorably in the efficacy endpoints of itching, redness, tearing, and swelling, although swelling was the only efficacy parameter to reach statistical significance. greater efficacy and tolerability likely play a role in the increased satisfaction with olopatadine 0.2% of patients with allergic conjunctivitis.29 although olopatadine 0.1%, the original bid formulation, had been previously judged to be significantly more comfortable than azelastine 0.05% in a randomized, double - masked, crossover study of 91 patients with allergic conjunctivitis,30 the increased concentration of olopatadine 0.2% makes tolerability a valid question. however, the current studies demonstrate that the comfort of this compound relative to azelastine is maintained even at the higher concentration, showing 3 times as many patients who were very satisfied with olopatadine s drop comfort compared with azelastine 0.05% (75% vs 25%, pace study). these results are supported by results from a patient perception study in which patients who had used both olopatadine 0.1% and 0.2% rated them equally comfortable.17 the reasons for the superior comfort of olopatadine 0.2% compared with azelastine 0.05% have not been clearly defined, but they may be at least partly attributed to ph differences ; azelastine 0.05% has a more acidic ph, ranging from 5.0 to 6.5,21 whereas olopatadine 0.2% has a physiologic ph of approximately 7.18 another possible explanation for the greater comfort of olopatadine may arise from the differing effects of these drugs on cell membrane integrity ; unlike azelastine and other dual - action agents, olopatadine does not perturb the membranes of ocular surface epithelial cells, the damage of which may explain the stinging and burning that can be encountered upon instillation of dual - action agents.16 the patient - reported results from the pace study, showing a clear advantage of current olopatadine 0.2% use over past azelastine 0.05% use for swelling and a numeric trend toward superiority for itching and redness, are consistent with the investigator - reported efficacy results from the well - controlled cac study published by spangler and colleagues.15 this study established the clinical superiority of the 0.1% formulation of olopatadine over azelastine. increasing the concentration of olopatadine in the 0.2% formulation would not be expected to have a negative impact on efficacy. the conclusions from the pace study are limited by its nonrandomized, open - label, single - arm design. because patients may have used their bid medications as much as 6 months prior to taking the day 1 allergy questionnaire, patients had to rely on memory, which may have introduced bias. medication (olopatadine 0.2%) to be superior to the previous medication (azelastine 0.05%), despite being provided with no information regarding the efficacy of olopatadine 0.2%. although the cac study had a randomized, double - masked, controlled trial design, its small size limits its conclusions as well. these confounding variables should be considered when deciding which medications to use in the clinical setting. allergic conjunctivitis is an under - diagnosed and often suboptimally treated component of allergic disease. although currently available ophthalmic medications have facilitated the management of this common malady, clinically significant differences among these agents have been reported in the literature.1315 effective therapy requires in - depth understanding of patient perceptions and responses to these therapeutic agents. this exploration of data from 2 independent studies provides insight into the therapeutic dynamic for successfully managing ocular allergy by comparing 2 agents of the same class that have different clinical performance and patient - perceived qualities. among the patients in these 2 studies, olopatadine 0.2% dosed once daily proved more comfortable and was perceived as clinically more efficacious than azelastine 0.05%. patient perception is critically important in ensuring sustained compliance and overall satisfaction with both the treatment and the provider. | purpose : results from 2 patient - reported outcome studies of allergic conjunctivitis sufferers who used olopatadine 0.2% and azelastine 0.05% are analyzed.methods:the pace (pataday allergic conjunctivitis evaluation) multi - center, prospective, open - label study examined patient perceptions of olopatadine 0.2% once daily (qd) and previous twice daily (bid) allergic conjunctivitis medications via questionnaire in allergic conjunctivitis sufferers who had previously used bid medication and then initiated olopatadine. a second conjunctival antigen challenge (cac) study evaluated comfort of 4 allergic conjunctivitis medications.results:forty-nine patients from the pace study (n = 125) with prior azelastine use were examined. significantly more patients rated themselves very satisfied with current olopatadine use compared with past azelastine use on drop comfort (p < 0.0001), speed of relief (p = 0.0004), and overall satisfaction (70% vs 16%, p < 0.0001). significantly more patients reported olopatadine very effective against swelling compared with azelastine (47% vs 8%, p = 0.0404). in the cac study (n = 36), data from olopatadine (n = 8), azelastine (n = 9) and placebo (n = 36) groups were reported. olopatadine was rated significantly more comfortable than azelastine upon instillation (p = 0.0223), at 30 seconds (p = 0.0479), and at 1 minute after instillation (p = 0.0240).conclusion : in the reported studies, olopatadine 0.2% qd was more comfortable than azelastine 0.05% and preferred by patients with allergic conjunctivitis by a ratio of 4:1. |
a 43-year - old female patient was diagnosed with osteonecrosis of the right femoral head and therefore was treated with core decompression and replacement of tantalum trabecular metal system (zimmer, parsippany, nj, usa) about 5 months ago in soonchunhyang university cheonan hospital. the patient was in a lateral decubitus position ; trabecular metal prostheses were removed via a skin incision made by the posterolateral approach. subsequently, the posterior joint capsule was incised in a ' t ' shape and then the piriformis muscle, superior musculus gemellus, internal obturator muscle, and inferior musculus gemellus were confirmed and desquamated from the trochanteric insertion site. desquamated short external rotator muscles were then sutured, followed by drilling of the trochanteric insertion site to restore the short external rotator muscles via the tendon - bone attachment, which was sutured by passing a surgical thread through the drilled holes. excessive tendon - bone attachment was prevented by checking the hip movement range before and after suturing. a cementless prosthesis with a ceramicceramic articular surface was used as an artificial prosthesis. a 48-mm sph delta acetabular cup (lima corporate, udine, italy), a 32-mm biolox delta articular surface liner (ceramtec, plochingen, germany), and a c2 stem (lima corporate) were used. postoperative radiographs indicated that the leg length was increased by approximately 5.0 mm in comparison with that before surgery (fig. 1). no significant complications (such as postoperative trauma or infection) were noted, while the strength of the lower extremity was found to be medical research council (mrc) grade v. the lower extremity touch sensory was also in the normal range. three days after surgery, the patient complained of numbness and reduced muscular strength in the right lower limb. neurologic examination revealed that knee joint extension, ankle joint flexion, and hallux flexus were mrc grade iv, whereas the ankle joint extensor and hallux extensor were grade i. in addition, sensory examination showed a reduction in pain in the dorsum and foot plantar. a medical history review showed that the patient complained of pain at the surgical site when changing posture to the left in her bed in the evening 2 days after surgery. the patient did not report any other problems other than slight pain following surgery, which is considered normal. the authors suspected peroneal nerve palsy because 1) the patient used an abduction pillow immediately after surgery, 2) sudden symptoms were reported when the patient complained of nerve palsy symptoms, and 3) there was a possibility of peroneal nerve compression around the fibular neck as the leg with palsy symptoms was slightly externally rotated. initially, we expected that the symptoms would be alleviated by conservative treatment ; the patient was discharged from the hospital 2 weeks after surgery. however, there was no symptom improvement during a 4-week follow - up, and electromyography could not be performed due to extreme pain 2 and 4 months after surgery. electromyography performed 6 and 10 months after surgery indicated 1) right common peroneal nerve lesion approximately at knee level and 2) sciatic nerve lesion (in its dominant common peroneal part) at above - knee level. these lesions were incomplete in nature and were accompanied by axonal degeneration and regeneration. according to neurologic examinations, after a complete explanation of nerve exploration to the patient, preoperative magnetic resonance imaging (mri) was performed. on mri images, the course of sciatic nerve was compressed by architecture, agglutinated on ruptured short external rotator muscles and their surrounding soft tissues in comparison with the normal side (fig. we observed that sciatic nerves were penetrating the piriformis muscle, whereas portion of the piriformis muscle and other short external rotator muscles were ruptured from the trochanteric insertion site, shrunken and constricted ; the cross sections of these muscles were slightly rolled up and adhered to the surrounding tissues. in addition, ruptured surfaces were compressing the course of the sciatic nerve (fig. short external rotator muscles, including the part of the piriformis muscle that were compressing the nerves, were excised to perform nerve decompression (fig. ten months after surgery, the strength of the ankle joint extensor and hallux extensor was grade iii and a reduction in pain sensation was observed. after 13 months, the strength was shown to be grade iv, which was a significant improvement. nerve palsy after tha is uncommon but may result in critical complications ; multiple studies have addressed this rare complication, and its cause(s), prognosis, and treatments are being extensively investigated. the incidence rate of nerve palsy after primary tha ranges between 0.09% and 3.7% but is slightly higher in patients with total hip revision, ranging from 2.0% to 7.6%2348). the incidence rate of sciatic nerve palsy after primary tha is even lower, ranging between 0.17% and 1%124). patient - related risk factors include 1) the affected area being on the left side, 2) female gender, 3) developmental hip dislocation or hip dysplasia. surgery - related risk factors include 1) some surgical approaches ; 2) total hip revision ; 3) excessive nerve tension because of excessive leg lengthening ; 4) direct trauma ; 5) postoperative pressure by a hematoma ; 6) improper traction when using traction tools, located improper sites ; 7) cement leakage and thermal damage ; 8) secondary reactions by wear debris ; and 9) constriction by a trochanteric wire or suture23459). nevertheless, the causes of more than 50% of nerve palsy cases remain unknown468). in our case, leg lengthening was unlikely to be the cause, because 1) leg lengthening was not significant (approximately 5.0 mm), 2) no palsy symptoms were observed until 3 days after surgery, and 3) symptoms were noticeably improved after nerve exploration. we were unable to find any evidence for direct pressure caused by a hematoma at the surgery site, such as foreign sensation or swelling. nerve exploration also did not indicate direct compression by a hematoma. the presence or absence of a hematoma, which may potentially form right after the rupture of short external rotator muscles, was not certain as mri and computed tomography were not performed immediately after the occurrence of nerve palsy. according to hurd), more than half of the cases of unexplained palsy might be due to sciatic nerve palsy caused by pressure between femoral attachment sites of the ischial tuberosity and gluteus maximus, as suggested by assessment using mri. in our case, mri images showed architecture attached to the short external rotator muscles (rather than compression of the sciatic nerves), which modified the course of the nerves, thus indirectly causing compression. sosna.7) reported a case of sciatic nerve palsy caused by compression of anatomically malformed sciatic nerves by the piriformis muscle that underwent tenotomy after primary tha. our case is somewhat similar to these observations, because nerve exploration indicated that part of sciatic nerves were penetrating the piriformis muscle ; in addition, some of the ruptured piriform muscle and shrunken short external rotator muscles were directly compressing the sciatic nerves. in general, nerve palsy does not have a favorable prognosis1234). it was reported that approximately 35% of nerve palsy cases become permanent, while 36% of palsy patients with complete motor paralysis recovered after 21.1 months in average13). in the study by oldenburg and mller2), in another study with a total of 3,126 patients, schmalzried.4) found that prognosis of nerve recovery may be associated with the degree of nerve damage ; therefore, it is difficult to expect satisfactory recovery for patients with serious paresthesia. further, sciatic nerve palsy tends to be found in peroneal components rather than in tibial component and is more serious in most cases234). kyriacou.9) demonstrated that a significant reduction in pain may be achieved by nerve exploration and neurolysis in patients with sciatic nerve palsy after tha. furthermore, there was no significant association between the timing of sciatic nerve exploration and the degree of pain reduction ; therefore, it would be still beneficial to perform exploration until up to 40 months after surgery. unwin and scott10) suggested that the presence of pain in the affected nerves might be the most important factor to consider in order to determine whether nerve exploration is necessary in patients with acute palsy. if a patient has nerve palsy accompanied by pain, nerve exploration is recommended. weber.8) reported that peroneal nerve palsy after tha might be due to direct damage of the sciatic nerves at surgical sites rather than at the knee level. similarly, schmalzried.4) reported that tha - related nerve injuries are observed around the hip. in the present case, exploration was delayed because of somewhat inconsistent results of electromyographic analyses that were performed 6 and 10 months after tha ; in these analyses, lesions of the peroneal nerves and sciatic nerves were shown, respectively. soon after surgery, the patient did not complain of any noticeable pain other than general pain at the surgical site ; furthermore, nerve palsy was initially found around the knee, potentially owing to the patient 's leg posture and the use of an abduction pillow, which prompted us to implement conservative treatment. although surgical treatment was successful in improving symptoms in this case, we were unable to perform nerve exploration and neurolysis at an early stage due to the 12-month conservative treatment, which failed to achieve favorable clinical outcomes. on the basis of this experience, the authors would like to suggest the necessity of a careful follow - up after tha regarding sciatic nerve palsy potentially caused by ruptured short external rotator muscles. | although the incidence of sciatic nerve palsy following total hip arthroplasty is low, this complication can cause devastating permanent nerve palsy. the authors experienced a case of sciatic nerve palsy caused by ruptured and contracted external rotator muscles following total hip arthroplasty in a patient suffering from osteonecrosis of the femoral head. we report this unusual case of sciatic nerve palsy with a review of the literature. |
chronic infection with hepatitis c virus (hcv) is a global health problem affecting a significant proportion of world s population. the world health organization estimated that in 1999 there were 170 million hcv carriers worldwide, with 34 million new cases appearing each year. a 48-week course of peg - ifn- with rbv is the recommended treatment for patients with hcv involves, but many patients will not be cured by it [35 ]. it also has side effects that prevent some patients from completing therapy. for these reasons, identification of the determinants of responsiveness to the peg - ifn- with rbv treatment is a matter of high priority. recent genome - wide association studies performed in order to identify human genetic contributions to anti - hcv treatment response have indicated that genetic polymorphisms near the il28b gene are associated with responses to hcv treatment [711 ]. tanaka. reported that, within a japanese population of patients with hcv genotype 1, those with minor alleles (tg and gg) of snp rs8099917 were more strongly associated with null virological response (nvr) than were those with major alleles (tt) (p = 2.65 10). they also reported a logistic regression model based on snp, age, gender, re - treatment, platelet count, aminotransferase level, fibrosis stage, and hcv - rna level indicated that rs8099917 is the most significant factor for nvr. ge and his colleagues reported not only that a genetic polymorphism near the il28b gene, encoding interferon--3 (ifn--3), is associated with an approximately twofold change in responsiveness to treatment, both among african - american patients (p = 2.06 10) and among patients of european ancestry (p = 1.06 10) but also that a polymorphism on chromosome 19, rs12979860, is strongly associated with sustained virologic (svr) in all patient groups (p = 1.37 10) [810 ]. they also noted that their regression model showed that the cc genotype is associated with a more substantial difference in responsiveness rate than was any of the other known baseline predictors included in the model. reported an association to svr within the gene region encoding interleukin il28b (rs8099917 combined p = 9.25 10, or = 1.98, 95% ci = 1.572.52) and indicated that host genetics may be useful for predicting drug responsiveness. the recent reports made clear that genetic polymorphisms near the il28b gene are associated with the responses of patients with hcv genotype 1 to the recommended drug treatment and indicated that predicting responsiveness to the treatment is an urgent necessity. although the virological responses of patients with hcv genotype 1 to peg - ifn- with rbv have been reported to be strongly associated with genetic polymorphisms, there is no report of responsiveness being predicted from the polymorphisms. it is quite important to know whether or not a new patient with hcv genotype 1 will be cured by peg - ifn- with rbv before beginning the treatment. predicting patient s responsiveness from the related snps would help to reduce side effects and treatment costs. the method we propose for predicting responsiveness uses decision tree learning based on the genome - wide snps. decision tree learning is a method that uses inductive inference to approximate a target function that will produce discrete values. it is generally best suited to problems in which instances are represented by attribute - value pairs and the target function has discrete output values. a decision tree classifies each example into a class corresponding to one of the output values. individual snps and their alleles (major, hetero, and minor genotypes) in the genome - wide association study (gwas) were used as attributes. 142 japanese hcv genotype 1 patients (64 with virologic responses (vrs) and 78 with null virologic responses (nvrs)) were used as training instances. to carry out the supervised learning for their classification, we partitioned the training instances into two data sets : training data for growing the decision tree, and testing data for pruning the decision tree. the classification processes were carried out in two phases : one for growing it and the other for pruning it. the sliq / sprint algorithm uses a two - branch (yes / no) approach, so for two combinations we selected the three types of branches listed in table 1. the previous analyses of genome - wide drug responses for hcv genotype 1 patients indicated that specific snps are closely associated with vrs / nvrs [711 ]. to analyze snp contributions to the drug effects, one first needs to calculate an evaluation function (such as the gini diversity index (gdi)) for three types of branches of individual snps and determine the maximum value of that function. each snp has three gdis for individual alleles, and each gdi can be computed in the following way : (1)gdi=1l=1kp(cl)2m=1jam1l=1kp(cml)2am = nmn (m=1,2,...,j), m=1jam=1where k is the number of classes (in this case k = 2, vr and nvr), p(cl) is the probability of l class for each snp in the instances, j is a number of branches (j = 2 in the sliq / sprint algorithm), p(cml) is the probability of l class in the branch m, nm is a number of the instances for the branch m, and n is the total number of the snp instances (in the first case all instances are used, that is, n = 142). a node is introduced for partitioning the instances in the decision tree, and in this article an snp in the instances is used as a node. the processes for growing the decision tree are, in outline, as follows:(1)the decision tree starts as a single node representing the hcv patient instances.(2)if the instances are all of the same class, the node becomes a leaf and is labeled with that class.(3)otherwise, the attribute that will best separate the instances into individual classes is selected by calculating the gdi for each attribute. the node is labeled with this attribute.(4)two branches are created for the node attribute, and the instances are partitioned accordingly.(5)the same processes are carried out recursively to form a decision tree for the instances at each partition.(6)the recursive partitioning stops only when one of the following conditions is met:all instances for a given node belong to the same class, orthere are no remaining attributes on which the instances may be further partitioned. if the instances are all of the same class, the node becomes a leaf and is labeled with that class. otherwise, the attribute that will best separate the instances into individual classes is selected by calculating the gdi for each attribute. two branches are created for the node attribute, and the instances are partitioned accordingly. the same processes are carried out recursively to form a decision tree for the instances at each partition. the recursive partitioning stops only when one of the following conditions is met:all instances for a given node belong to the same class, orthere are no remaining attributes on which the instances may be further partitioned. all instances for a given node belong to the same class, or there are no remaining attributes on which the instances may be further partitioned. working backward from the bottom of the tree, the subtree starting at each non - terminal if removing a subtree improves the error (misclassification) rate on the testing data, the subtree is removed. this process continues until no further improvement is made. after completing the growth and pruning of the decision tree, that is, the individual leaves are labeled with the most common classes (vr and nvr) in the instances. in addition, vr and nvr classes are assembled as the total vrs and nvrs for predicting vr and nvr ratios. the vr ratio predicted from the decision tree learning based on the snp information is given by(2)pvr = dvrivrwhere dvr is the total number of vrs in the predicted vr class and ivr is the number of vr instances. the nvr ratio predicted from the decision tree learning is given by(3)pnvr = dnvrinvrwhere dnvr is the total number of nvrs in the predicted nvr class and invr is the number of nvr instances. the total number of vrs and nvrs predicted from the decision tree learning is based on eqs. (3) and (4) and is given by(4)pvr+nvr = dvr+dnvraiwhere ai is the total number of instances (all the hcv patients). individual snps and their alleles (major, hetero, and minor genotypes) in the genome - wide association study (gwas) were used as attributes. 142 japanese hcv genotype 1 patients (64 with virologic responses (vrs) and 78 with null virologic responses (nvrs)) were used as training instances. to carry out the supervised learning for their classification, we partitioned the training instances into two data sets : training data for growing the decision tree, and testing data for pruning the decision tree. the classification processes were carried out in two phases : one for growing it and the other for pruning it. the sliq / sprint algorithm uses a two - branch (yes / no) approach, so for two combinations we selected the three types of branches listed in table 1. the previous analyses of genome - wide drug responses for hcv genotype 1 patients indicated that specific snps are closely associated with vrs / nvrs [711 ]. to analyze snp contributions to the drug effects, one first needs to calculate an evaluation function (such as the gini diversity index (gdi)) for three types of branches of individual snps and determine the maximum value of that function. each snp has three gdis for individual alleles, and each gdi can be computed in the following way : (1)gdi=1l=1kp(cl)2m=1jam1l=1kp(cml)2am = nmn (m=1,2,...,j), m=1jam=1where k is the number of classes (in this case k = 2, vr and nvr), p(cl) is the probability of l class for each snp in the instances, j is a number of branches (j = 2 in the sliq / sprint algorithm), p(cml) is the probability of l class in the branch m, nm is a number of the instances for the branch m, and n is the total number of the snp instances (in the first case all instances are used, that is, n = 142). a node is introduced for partitioning the instances in the decision tree, and in this article an snp in the instances is used as a node. the processes for growing the decision tree are, in outline, as follows:(1)the decision tree starts as a single node representing the hcv patient instances.(2)if the instances are all of the same class, the node becomes a leaf and is labeled with that class.(3)otherwise, the attribute that will best separate the instances into individual classes is selected by calculating the gdi for each attribute. the node is labeled with this attribute.(4)two branches are created for the node attribute, and the instances are partitioned accordingly.(5)the same processes are carried out recursively to form a decision tree for the instances at each partition.(6)the recursive partitioning stops only when one of the following conditions is met:all instances for a given node belong to the same class, orthere are no remaining attributes on which the instances may be further partitioned. if the instances are all of the same class, the node becomes a leaf and is labeled with that class. otherwise, the attribute that will best separate the instances into individual classes is selected by calculating the gdi for each attribute. two branches are created for the node attribute, and the instances are partitioned accordingly. the same processes are carried out recursively to form a decision tree for the instances at each partition. the recursive partitioning stops only when one of the following conditions is met:all instances for a given node belong to the same class, orthere are no remaining attributes on which the instances may be further partitioned. all instances for a given node belong to the same class, or there are no remaining attributes on which the instances may be further partitioned. working backward from the bottom of the tree, the subtree starting at each non - terminal node is examined. if removing a subtree improves the error (misclassification) rate on the testing data, the subtree is removed. after completing the growth and pruning of the decision tree, majority voting is carried out for individual leaves. that is, the individual leaves are labeled with the most common classes (vr and nvr) in the instances. in addition, vr and nvr classes are assembled as the total vrs and nvrs for predicting vr and nvr ratios. the vr ratio predicted from the decision tree learning based on the snp information is given by(2)pvr = dvrivrwhere dvr is the total number of vrs in the predicted vr class and ivr is the number of vr instances. the nvr ratio predicted from the decision tree learning is given by(3)pnvr = dnvrinvrwhere dnvr is the total number of nvrs in the predicted nvr class and invr is the number of nvr instances. the total number of vrs and nvrs predicted from the decision tree learning is based on eqs. (3) and (4) and is given by(4)pvr+nvr = dvr+dnvraiwhere ai is the total number of instances (all the hcv patients). one hundred and forty - two japanese patients with hcv (78 nvr and 64 vr) receiving peg - ifn-/rbv treatment were analyzed by using the snps evaluated in a previous gwas study. although a total of 621,220 snps were used for the genome - wide association analysis, approximately 500 of those with the lowest p - values calculated by using a test for allele frequencies were selected for the decision tree learning. the node with the largest gdi was selected as the root node of the decision tree. in the first case, that is, 142 japanese hcv patients were divided into two branches based on rs8099917. 1, where one sees that the yes and no branches from the root node b1 were, respectively, het + mm and mm. the numbers of instances in the two branches divided by rs8099917 were, respectively, 65 in the yes branch and 77 in the no branch. as the most instances in the yes branch node were nvrs (59 of 65, or 90.8%), this node was labeled as the leaf node. from the descendent node on the no branch, a new descendant of the node was obtained using the processes described in section 2.2. after the decision tree was grown, it was pruned as described in section 2.3. the decision tree learning was carried out for the 142 japanese hcv patients with the 500 lowest - p snps, and the predicted vr and nvr ratios were calculated using eqs. (2) and (3):pvr=48 + 764=5564=0.859pnvr=59 + 1478=7378=0.936the predicted total number of vrs and nvrs was also calculated using eq. (4):pvr+nvr=55 + 73142=0.901before the decision tree learning by the snp information, we knew only that 64 of the japanese hcv patients were vr for the drug response and 78 were nvr. therefore, all we can calculate is that the vr and nvr response rates for those patients are respectively 0.451 (64/142) and 0.549 (78/142). after the decision tree learning, however, we can predict that the vr and nvr drug responses are respectively 0.859 and 0.936 and can predict that the drug response rate for the total 142 samples is 0.901 ((55 + 73)/142). this means that if a new hcv genotype 1 patient wants to know whether or not he / she is vr / nvr for the drug treatment, he / she can predict his / her vr / nvr ratio by checking his / her snp information in the decision tree shown in fig., suppose a new male hcv patient having rs8099917, rs4906195, and rs3816768 alleles that are, respectively,, it is predicted that he will be vr for the recommended hcv drug treatment. on the other hand,, it is predicted that she will mostly likely be nvr for the recommended hcv drug treatment. it is important to increase the prediction ratio in the decision tree learning. the higher the prediction ratio, the better the drug treatment response for a new hcv patient is predicted. in the decision tree model we therefore also generated decision tree models by using root nodes based on the snps having the 30 highest gdis. as a result, we got two decision trees that can predict ratios higher than that of the first decision tree. they are shown in figs. 2 and 3. the results of prediction ratios (probabilities predicted) for these decision tree models and the individual snp attributes used in the models are listed in table 2. this is 2.8 percentage points higher than that of model 1. as the root node plays an important role, we examined what chromosome the root node belongs to in each model. the chromosomes with individual root nodes in the models 1, 2, and 3 were, respectively, 19, 1, and 8 (table 2). although snps near the il28b gene on chromosome 19 were recently reported to be the most significant factors, the root nodes in models 2 and 3 are on other chromosomes. other snps used in the models 2 and 3 are also on other chromosomes (table 2). these results therefore imply that there may be significant snps other than those near the il28b gene on chromosome 19. although that the virological responses of hcv patients treated with peg - ifn- and rbv have been reported to be strongly associated with genetic polymorphisms, there is no report of what responses can be predicted from the polymorphisms. if the responsiveness of hcv patients to a drug treatment could be predicted from information about related snps, side effects and treatment cost could be greatly reduced. furthermore, because the results of the proposed method implied that snps other than those in the il28b region are strongly related to the prediction of the drug response, the relations between those snps and the drug response should be examined experimentally. | highlights we modeled drug responses using decision tree learning based on snps in a genome - wide association study. we can predict the drug responses of a new patient with hcv genotype 1. responsiveness to pegylated interferon (peg - ifn-) plus rebavirin (rbv) treatment was predicted. we can predict with 93% probability whether a new patient with hcv genotype 1 will be helped by drug treatment. |
opioids have been increasing in usage and play an important role in every aspect of modern anesthesia. among opioids, fentanyl, alfentanil, sufentanil, and remifentanil are commonly chosen for analgesia, sedation, hemodynamic stability, as well as attenuation of stress response during anesthesia. however, the administration of opioids has sometimes been found to induce unanticipated pain sensitivity changes, such as opioid - induced hyperalgesia (oih) or tolerance. hyperalgesia is defined as enhanced pain response to a noxious stimulus, in this case induced by opiate use. although still being debated, the presence of oih would be a clinical challenge not only in chronic cancer pain management, but also perioperative pain. in addition to oih, administration of opioids may also tolerance, defined as a decreased response to the drug 's analgesic effects over time, followed by loss of analgesic efficacy. although oih is frequently conflated with opioid tolerance in the literature as the clinical features are similar, in fact they are different phenomena ; increasing opioid dose aggravates pain in oih, whereas tolerance does not. thus, although the mechanisms underlying these two phenomena are likely distinct, they are clearly related and on the same continuum of pain sensitization processes. the prevalence of oih and tolerance related with opioids remains unknown, however, these states appear to occur have increased in frequency with the growing use of remifentanil. moreover, the clinical significance of occurrence of oih or tolerance after perioperative use of opioid is been still under debate. the aim of this review is to present a brief overview of oih in the setting of surgical anesthesia. an understanding of current knowledge of potential oh mechanisms underlying oih as well as the clinical implication should be helpful to clinical anesthesiologists in helping to plan better perioperative pain control strategies. andrews first reported reduced pain thresholds after morphine administration in opioid addicts in 1943. similarly, tilson. first demonstrated that abrupt cessation of opioids induced decreased pain thresholds in rats and showed that this enhanced pain sensitivity was highly correlated with the administered dose of opioid. consistent with these animal results, clinical investigators demonstrated the occurrence of oih after intraoperative remifentanil infusion, characterized by increased pain, combined with increased consumption of postoperative opioid, which in turn, resulted in decreased opioid efficacy. moreover, significant pain reduction was observed after detoxification from high dose opioids which was observed in surgical patients also supports the existence of perioperative oih. although there have also been numerous experimental studies in human and animals on oih or opioid tolerance, the differentiation between them has been indistinct. both oih and tolerance are more evident in patients receiving a high rather that low intraoperative opioid doses. pharmacologically, tolerance is characterized by a loss of drug potency, likely by means of a desensitization of the antinociceptive pathways to opioids, while oih is characterized by increased pain sensitivity and involves sensitization of pronociceptive pathways, both phenomena resulting in increased dose requirements. in despite of these clear differences in definition and mechanism, it is very complicated to differentiate them in clinically because the symptoms of both are somewhat relieved by increased doses of opioid. quantitative sensory testing (qst) has been shown to be the most accurate means of differentiating oih and tolerance, but the complexity of time - consuming process of qst limits its wide spread use. the neurobiology of oih is complex and several mechanisms for oih have been proposed. to date, activation of central glutaminergic pathways, mainly via n - methyl d - aspartate (nmda) receptor, have been regarded as a key pronociceptive mechanism for inducing oih. in an early study, mao. proposed that an increase in responsiveness of the nmda receptor contributes to the development of opioid tolerance and hyperalgesia, as evidenced by his finding that the nmda antagonist mk-801 prevented the development of oih in rats. these concepts also supported by the finding that ketamine, a clinically - used nmda receptor antagonist, reduced fentanyl - induced hyperalgesia. descending spinal facilitation mediated via changes in activity of on- and off- cells within the rostro - ventral medulla (rvm) involving nmda system comprises another suggested mechanism to explain oih. these neurons, which project to the spinal cord and display changes in activity in response to noxious stimuli facilitate or inhibit nociceptive transmission respectively. administration of -opioid receptor (mor) ligands changes the circuit into the off - cell state, whereas the presence of a prolonged noxious stimulus changes it into an on - cell state. globally, oih may partly result from an unbalanced activity of the off- and on - cells underlying the apparent development of tolerance. in support of this theory,. demonstrated that injection of lidocaine into the rvm or bilateral lesions of the dorsolateral funiculus blocked opioid - induced hyperalgesia and restored antinociceptive morphine potency. the opioid receptor family, part of the large g - protein - coupled receptor (gpcr) family, consists of 4 different distinct receptors :, (dor / oprd), (kor / oprk1) and opioid receptor - like (orl1/oprl) receptor, all of which are present in nuclei of the pain modulation circuit. in contrast, kor agonists act presynaptically and orl1 agonists act postsynaptically to inhibit both on- and off - cells in the rvm. when morphine is administered systemically or into the periaqueductal grey matter, the on - cells become silent and the off - cells fire continuously. in this off - cell state, this inhibition is reversed by inactivation of the rvm or selective inhibition of off - cell firing. in the off - cell activated state, microinjection of either an orl1 or a kor agonist will inhibit off - cells and has anti - analgesic action. adrenergic and opioid receptors both belong to the gpcr family, couple to analogous signal transduction pathways, and affect the nociceptive system. various biochemical studies have proposed the existence of gpcr dimerization, which may facilitate transport of receptors to the cell surface and g protein coupling and activation. a heterodimer formation between mor and -adrenoceptor units (2ar) enhances mor signaling in response to morphine but severely decreases the opioid response following the simultaneous addition of morphine and 2ar agonist. vilardaga. proposed a model in which morphine binding to the mor rapidly changes conformation of the activated 2ar, and this transconformational change permits direct inactivation of a gi protein. the direct conformational switching of one receptor by the other that enables inhibition of receptor activation is likely a means of rapidly preventing overstimulation of signaling pathways and may contribute to oih. some neuropeptides, which oppose anti - opioid peptides has been investigated and shown convincing results. the administration of neuropeptide cholecystokinin, the neuropeptide ff, and orphanin fq / nociception have all demonstrated anti - hyperalgesic effect. as a possible third mechanism, spinal dynorphin, an endogenous opioid ligand, may also play an important role in the development of oih. increased concentration dynorphin in spinal cord and primary afferents after noxious stimuli stimulates the release of calcitonin gene - related peptide and thus increase stimulus - evoked spinal excitation. based on the fact that there exists individual difference in the occurrence of oih, there may well be a genetic predisposition of oih among the patients.. found that polymorphisms of the catechol - o - methyl transferase gene were more prevalent in patients demonstrating oih and pain sensitization. finally, beyond the physiological mechanisms discussed here, psychological factors including anxiety and catastrophizing about pain could be modulating factors in the development of oih. clrier proposed a model of neuroadaptative changes linking oih. before the first exposure to opioid, an initial equilibrium is associated with a low level balance between opioid - dependent analgesic systems (pain inhibitory) and nmda - dependent pronociceptive systems (pain excitatory). repeated opioid administrations induces a gradual decrease in the nociceptive threshold (pronociceptive systems sensitization) leading to hyperalgesic state. this progressively shifts the unchanged analgesic response, giving the impression of less analgesia (apparent tolerance). after withdrawal of opioid, counter - adaptation of opioid - dependent analgesic systems is built by changes in the endogenous opioidergic system, and thus a new equilibrium between opioid - dependent analgesic systems and nmda - dependent pronociceptive systems is established. this new, reset equilibrium (allostasis) balance leads to long - term pain vulnerability. oih has been studied mainly after opioid - based anesthesia and during postoperative analgesia. for several decades, importantly, a meta - analysis of studies demonstrated that while oih is consistently present in patients given remifentanil, it 's occurrence with fentanyl administration has not been established. thus, this review deals mainly with literature reports of clinical manifestation and modulatory factors associated with remifentanil infusion : dose, infusion duration, speed of withdrawal, and other combined anesthetic drugs. oih manifests itself by increased sensitivity to painful stimulation which extends to throughout the entire body from the site of preexisting pain. thus, oih exacerbates preexisting painful conditions and therefore can further progress a painful disease state. through the literature, high doses of remifentanil have been regarded as an important factor of oih : most trials show that oih is most likely to occur at infusion rates of > 0.32 ug / kg / min. this dose dependency in inducing oih has been demonstrated both in animal and human study. in rats, a decrease in both thermal threshold and mechanical thresholds are directly proportional to the administered dose of remifentanil. similarly, in surgical patients, postoperative pain scores and cumulative morphine consumption have been shown to be enhanced more in patients receiving high dose remifentanil. a potential mechanism underlying this dose dependence was demonstrated in vitro in a patch clamp single - cell electrophysiologic study which demonstrated cumulative dose, duration of administration, and modality of withdrawal could all influence the extent of oih. bolus only or shorter infusion of remifentanils led to long - term potentiation but with lower incidence. moreover, tapered withdrawal more than 30 minutes after 1 hr infusion prevented long - term potentiation compared with abrupt withdrawal. co - administration of n2o or propofol with remifentanil decreases oih development and consumption of analgesics. all of these factors could be an auxiliary method to minimize the pronociceptive effects of opioids. to date, possible treatment pharmacologic regimens for oih include partial mor agonist (buprenorphine) nmda receptor antagonists (ketamine and dextromethorphan), cyclooxygenase (cox) inhibitors (nonsteroidal anti - inflammatory drugs), and 2 receptor agonists. buprenorhine, a partial mor agonist and - and - receptor antagonist, has a unique property : its antihyperalgesic effects lasted longer than its analgesic effects (2.6 times and 1.9 times for i.v. and s.c. this effect may be mediated through the blockade of -receptors, as agonists at this receptor are known to promote hyperalgesia mediated by descending facilitation. methadone also has an antihyperalgesic action, and has the potential to be widely used in the clinical setting to reduce oih. spinal nmda receptors appear to contribute to the development and maintenance of oih. numerous animal and human studies these studies have shown beneficial effects of supplementation of opioid treatment with ketamine : higher pain thresholds and lessened hyperalgesia in animals and better pain control scores and less use of postoperative morphine in humans. another nmda receptor antagonist, dextromethorphan, has not been widely studied as a means of modulation of oih, likely due to its lack of antihyperalgesic action. prostaglandins, including pge2, can stimulate glutamate release in the spinal cord resulting in activation of nmda receptors. thus, coadministration of the cox inhibitors parecoxib and ketorolac significantly decreased the area of pinprick hyperalgesia during remifentanil, however, pretreatment prior to remifentanil infusion was without effect.. further study of this promising treatment is needed to guide cox inhibitor use in preventing oih. several studies have provided biochemical evidence for the physical association of 2ar with mors and have identified that functional mor-2ar complexes can form in brain and spinal cord neurons. although the significance and function of such a receptor complex are not fully understood, the effect of 2ar antagonist on oih could be accomplished by interactions with this heterodimer complex. clinical and laboratory observations also indicate that 2-adrenoceptor agonists may deter the development of oih as well as alleviate the symptoms of opioid - withdrawal. for example, coadministration of clonidine with morphine in rats wherein oih had been induced normalized both mechanical and thermal thresholds to baseline sensitivities. clinically, a case report presented the experience of administering dexmedetomidine, an 2ar agonist, during opioid dose reduction in patients with oih, allowed normalization of nociceptive and antinociceptive responses. when compared to the chronic pain management setting, oih is less well recognized and the clinical implications less well understood in perioperative settings, despite the fact that there multiple articles have been published establishing this side effect of perioperative opioid administration. several clinical trials of oih have reported increased postoperative pain and morphine consumption. however, those changes are typically controlled acutely without severe side effects by increased morphine consumption. anesthesiologist should however improve their armamentarium in dealing with oih, using some modulatory or pharmacologic approaches to oih which should be helpful both in terms of eliciting fewer and less severe acute side effects but also limiting the lasting consequences of oih. clinical anesthesiologists need to better understand oih and its implications for pain control and opioid usage during the perioperative period. although the clinical implication of oih is not fully established, we need to understand that oih could be a starting point of pain sensitization and pain chronicification. therefore, anesthesiologists should endeavor to prevent or treat oih through modulatory or pharmacologic means based on and understanding of the likely mechanisms underlying oih and these treatment means. | pain is difficult to investigate and difficult to treat, in part, because of problems in quantification and assessment. the use of opioids, combined with classic anesthetics to maintain hemodynamic stability by controlling responses to intraoperative painful events has gained significant popularity in the anesthetic field. however, several side effects profiles concerning perioperative use of opioid have been published. over the past two decades, many concerns have arisen with respect to opioid - induced hyperalgesia (oih), which is the paradoxical effect wherein opioid usage may decrease pain thresholds and increase atypical pain unrelated to the original, preexisting pain. this brief review focuses on the evidence, mechanisms, and modulatory and pharmacologic management of oih in order to elaborate on the clinical implication of oih. |
salmonellosis due to nontyphoidal salmonella (nts) infection is a global public health concern, particularly in salmonella endemic low and middle income countries (lmics). treatment is critical for persons with severe disease, particularly children and immune compromised people. treatment with an appropriate antibiotic can shorten the duration of symptoms, significantly reduce severity of disease and the risk of transmission, and prevent potentially lethal complications. emergence of resistance to first - line therapy like ampicillin, chloramphenicol, and cotrimoxazole including ciprofloxacin among salmonella spp. during the last decades has complicated the situation [1, 2 ]. for treatment of salmonella infection resistant to these drugs, extended - spectrum cephalosporins (escs) are considered as an alternative therapeutic choice. with the increased use of -lactam antibiotics to treat enteric infection, salmonella spp. had acquired resistant to third generation cephalosporin antibiotics in different parts of the world and had been associated with clinical treatment failure [3, 4 ]. extended - spectrum beta - lactamases (esbl) are usually encoded by large plasmids (100 kb) that are transferable from strain to strain and between bacterial species [57 ]. resistance to escs is mediated primarily by production of class a esbls, which can hydrolyze oxyimino cephalosporins but are not active against cephamycins and carbapenems. the plasmid - encoded ctx - m type esbls production was initially identified in 1983 in germany. the ctx - m family enzymes, which confer high levels of resistance to escs, have similar substrate specificities and inhibitor profiles to tem. the ctx - m type esbls have been reported to be found worldwide in different members of enterobacteriaceae isolated from human and other animal sources. earlier we have reported the emergence of blactx - m and blatem type esbl producer s. typhi in one - year - old child with recurrent high - grade fever. in recent years there have been several reports indicating the emergence of resistance to -lactam antibiotics among salmonella species. here, we present blatem gene mediated esbl production among salmonella spp. isolated from stool specimen of patients with diarrhea in an urban setting of bangladesh. as part of a microbiological analysis of stool sample received at dhaka treatment centre of international centre for diarrheal diseases research, bangladesh (icddr, b) during 20052013, we have analyzed 128,312 fecal specimen following standard microbiological method to identify salmonella spp. the putative extended - spectrum beta - lactamase (esbl) producing strains were tested by the double - disc synergy method and detection of -lactamase genes (blactx - m, blatem, blashv, and blaoxa genes) was performed by pcr as describe elsewhere. overall, the annual incidence of salmonella infection showed a decreasing trend and the proportion came down significantly from 2% in 2005 to 1.0% in 2013 (p < 0.001). (2120, [1.7% ]), nontyphoidal salmonella (nts) isolates were more frequently isolated than typhoidal salmonella (ts) (72.8% versus 27.2%, p < 0.001). demographic information showed that salmonellae were isolated from patients of all age groups with a maiden age of 4.04 years. of the total salmonella positive patients, 51.2% (n = 1086) were children aged less than five years. among the ts serogroups, s. typhi was predominant (404, [65.1% ]) followed by s. paratyphi b (139, [22.4% ]) and s. paratyphi a (78, [12.6% ]). of the nts isolates, serogroups c1 strains were more frequently isolated (560, [37.4% ]), followed by b (203, [13.5% ]), c2 (203, [13.5% ]), e (127, [8.5% ]), d (94, [6.3% ]), g (79, [5.3% ]), nontypeable salmonella (54, [3.6% ]), s. typhimurium (2, [0.1% ]), and a (1, [0.06% ]) serogroup. the yearly distribution of salmonella spp. showed distinct seasonality with higher isolation during may to october in each year. temporal shift was noted in the prevalence of serogroups, seasonality, gender distribution, and resistance pattern between ts and nts isolates. around 40% of the salmonella isolates showed resistance to nalidixic acid (na) followed by 36% to ampicillin (amp), 20% to cotrimoxazole (sxt), 4% to ciprofloxacin (cip), 13% to chloramphenicol (c), and 4% to ceftriaxone (cro). resistance to 3 antibiotic classes was more common among ts strains than nts counterpart (36.6% versus 19.8%, p < 0.001). among the representative salmonella isolates with unusual resistant phenotype (ampccipcronasxt), first identified in 2011, blatem gene was detected in 88% (7/8) of the strains (figure 1). interestingly, one s. typhi was positive for both blatem and blactx - m genes which is a very rare phenomenon. in our earlier report, we noticed that this same phenomenon was observed in one s. typhi isolated from blood of a typhoid patient. these findings suggest that multi - esbl producer strains are circulating in bangladesh. however, to the best of our knowledge, this is the first report of esbl production among salmonella isolated from stool specimen of diarrheal patients in urban dhaka, bangladesh. detailed molecular characterization including sequencing is necessary for further geno- and subtyping of these strains. the study finding reflects the higher prevalence of mar salmonella spp. among children aged < 5 years and blatem gene mediated esbl production among salmonella spp. isolated from stool sample of diarrheal patient in urban bangladesh. therefore, it is important to establish a surveillance program to understand actual disease burden due to salmonella as well as promote specific and actual line of therapy for salmonellosis. | salmonellosis, an acute invasive enteric infection, is endemic in bangladesh. we analyzed 128,312 stool samples of diarrheal patients to identify salmonella spp. during 20052013. a total of 2120 (1.7%) salmonella spp. were isolated and the prevalence of salmonella spp. decreased significantly over time (21%, p < 0.001). among the typhoidal salmonella (ts) serogroups, s. typhi was predominant (404, [65.1% ]) followed by s. paratyphi b (139, [22.4% ]) and s. paratyphi a (78, [12.6% ]). of the nontyphoidal salmonella (nts) isolates, the serogroup c1 (560, [37% ]) was predominant followed by b (379, [25% ]), c2 (203, [14% ]), e (127, [9% ]), and d (94, [6% ]). most of the resistance was found towards nalidixic acid (40%), ampicillin (36%), cotrimoxazole (20%), chloramphenicol (13%), ciprofloxacin (4%), and ceftriaxone (4%). interestingly, 32% of the isolates showed reduced susceptibility to cip. multiantibiotic resistance (mar, 3 drugs) was more common among ts than nts strains (p < 0.001). among the representative ceftriaxone - resistant isolates, blatem gene was detected among 88% (7/8) of the strains, whereas only one strain of s. typhi was positive for both blatem and blactx - m genes. the study reflects higher prevalence of mar salmonella spp. and is the first to report the blatem gene mediated esbl production among salmonellae in bangladesh. emergence of mar salmonella spp. in particular esbl strains should be considered a public health concern. |
basal cell carcinoma (bcc) is defined by the world health organization committee on the histological typing of skin tumors as a locally invasive, slowly spreading tumor which rarely metastasize, arising in the epidermis or hair follicles and in which, in particular, the peripheral cells usually simulate the basal cells of the epidermis. it is usually observed in older patients, especially in those frequently and intensively exposed to ultraviolet radiation during their lives. thus, bcc is often observed in head and neck areas, especially the eyelid and nose. bcc may be treated by surgery, cryotherapy, radiotherapy, and curettage and electrodessication. other less frequently used treatment modalities include the topical application of 5-fluorouracil (5-fu) ointment, laser treatment, and systemic chemotherapy. to achieve a favorable outcome, it is important to recognize the histological subtypes, identify the anatomic locations that can increase the risk of spread, and understand the limitations of all available treatment modalities. if surgical defects are repaired, it is necessary to carefully plan the reconstruction after the tumor margins have been cleared. this paper discusses the histopathology, clinical presentation, and management of bcc of the head and neck. the role of sunlight as a causative factor in cutaneous carcinoma is further reflected in its geographic distribution. individuals with more darkly pigmented skin have a lower rate of bcc, being rare in those of african descent. embryonic fusion planes the regions of mesenchymal migration and fusion of the five primordial facial processes during the 5th to 10th weeks of human development have been implicated in the pathogenesis of basal cell carcinoma. newman and leffell reported that basal cell carcinoma was more than four times more likely to occur on the embryonic fusion plane than on other regions of the midface. this may reflect a higher rate of sun exposure of males because of employment patterns. a population - based incidence study in minnesota gave annual incidence figures for males and females of 175 and 124 per 100,000, respectively. however, the incidence in women is increasing because of changing fashions in clothing and time spent outdoors due to recreation patterns or specific occupations. it has been suggested that the incidence of persons affected by bcc is likely to substantially underestimate the true incidence of this cancer. this is due to the fact that bccs are not routinely registered because of their high frequency and low mortality. in addition, occurrence of multiple primary tumors within individuals synchronously or at different times is common in bcc patients. australian surveys demonstrated that the incidence of people treated for new primary bccs was 1.5% in 10 years and that over 700 persons per 100,000 person years were affected by multiple bccs. bcc is more frequent in the elderly, and the incidence of bcc increases with age. more than 90% of bccs are detected in patients aged 60 and older [1013 ]. the actively growing tissue is at the periphery of the lesion, with cellular apoptosis and resultant ulceration in the central region. to treat these lesions, it is important to eradicate the farthest marginal areas because these tend to have the most aggressively behaving cells. growth may continue for months or even years, gradually invading and destroying bone as well as soft tissues. there is a predilection for invasion along tissue planes, the periosteum, and nerves. a common theory states that the embryonic fusion planes, such as the nasolabial fold, are more susceptible to tumor growth. dermoscopy is a noninvasive technique that is known to increase the diagnostic accuracy of benign versus malignant pigmented skin lesions [1419 ]. recently proposed a simple dermoscopic model for the diagnosis of pigmented bcc, based on the absence of a pigment network and the presence of at least one of six positive morphologic features. positive dermoscopic features include ulceration, multiple blue - gray globules, leaflike areas, and telangiectasia. furthermore, large blue - gray ovoid nests have been defined as pigmented ovoid or elongated areas, larger than globules, and not intimately connected to the pigmented tumor body. spoke wheel areas are an additional parameter appearing as well - circumscribed radial projections, usually tan, but also blue or gray, meeting at an often darker (dark brown, black, or blue) central axis (figure 1). dermoscopy is frequently able to differentiate between bcc and other pigmented skin lesions, such as malignant melanoma and seborrheic keratosis. bcc is characterized by large nuclei that are oval and composed mostly of cellular matrix, with little cytoplasm. there is a higher nucleus - to - cytoplasm ratio in malignant compared with normal cells. tumor masses are surrounded by a peripheral cell layer in which the nuclei form a palisade or picket fence - type arrangement. tumors can be classified as nodular, superficial, morpheaform, infiltrating, metatypic, and fibroepithelioma of pinkus. nodular bcc is the most frequent form of bcc, accounting for 75% of all cases, being superficial or ulcerated and often visualized on actinic damaged skin. further, around 90% of nodular bcc lesions are found on the head and neck (figure 2). superficial bcc appears as a plaque or as an erythematous squamous plaque, often found on the trunk and extremities, although 40% still occurs on the head and neck (figure 3). morpheaform bcc accounts for approximately 6% of all bcc, but 95% of these will be located on the head and neck [22, 23 ]. it tends to be more aggressive, sometimes infiltrating deeper in muscles or fat tissue (figure 4). there are no sites of predilection, and these lesions rarely bleed or get ulcerated. metatypic bcc shows clinical signs of bcc as well as squamous cell carcinoma (scc). this subtype tends to be more aggressive than the other subtypes, and it could grow and extend as scc does, with a marked presence of metastasis. fibroepithelioma of pinkus often appears in the lumbar and resembles a fibroepithelial polypus or seborrheic keratosis [24, 25 ]. common treatments for bcc of the head and neck include methods such as mohs micrographic surgery, surgical excision, liquid nitrogen cryosurgery, and curettage and electrodessication. other less frequently employed treatment modalities include the topical application of 5-fluorouracil (5-fu) ointment, laser treatment, radiotherapy, and systemic chemotherapy. the choice of treatment should be determined based on the histological type of lesion, cost, its size and location, patient age, medical condition of the patient, treatment availability, and the patient 's wishes. the aims of any therapy for the treatment of a bcc are to ensure complete removal, the preservation of function, and a good cosmetic outcome. mohs micrographic surgery is well established as the standard of care in many cases of bcc and squamous cell carcinomas. in 1941, frederick mohs described a surgical technique he had developed for the staged removal of skin cancer using in situ fixation of cutaneous tissue using a paste containing zinc chloride. in 1953, he used frozen section without chemical fixation to excise a recurrent tumor on the eyelid. they noted less pain, discomfort, and anxiety in patients treated with this technique compared with those treated with chemical fixation [27, 28 ]. a detailed map of the tumor site was made to record the positive margins and to direct the next excision. despite advances in techniques, the basic principles have remained the same in that histologically examined tissue directing further resection until all margins are clear of tumor. first, mohs micrographic surgery is the most effective method of eradicating bcc, with a five - year cure rate of 99 percent [3032 ]. is of paramount importance, especially around the eyes, nose, ears, mouth, and genitalia. finally, compared with other surgical techniques involving postoperative repair, the cost of mohs micrographic surgery is similar to that of simple excision in the office with permanent section postoperative margin control. mohs micrographic surgery is less expensive than excisions with intraoperative margin control with frozen sections performed in a private office or in an outpatient surgical facility. treatment requires total excision of the lesion. surgical excision facilitates pathologic assessment of the tissue [34, 35 ]. the surgical specimen should be oriented for the pathologist so that the margins can be examined, allowing the surgeon to verify residual tumor presence or complete excision. the margins will depend on the size of the lesions, anatomic location, clinical features, ulceration, and apparent depth of penetration. it has been common practice to employ a 5 mm margin for excision around bcc. some authors suggested that surgical margins of less than 5 mm might be adequate for noninvasive small bcc of the head and neck. wolf demonstrated that margins of 4 mm were adequate in 95% of nonmorpheaform bcc less than 2 cm in diameter when treated by standard excision. in addition, lalloo and sood reported that a clinical excision margin of 2 mm was adequate for the treatment of simple, well - demarcated bccs arising in the head and neck except for recurrent or morpheaform tumors. while these margins are adequate for a small bcc whose histologic subtype is such that the tumor does not warrant marked lateral or deep excision, it is not an acceptable margin for large tumors or lesions exhibiting a morpheaform histology. silverman. analyzed 588 primary and 135 recurrent bccs treated by surgical excision. primary - treated tumors had a cumulative 5-year recurrence rate of 4.8%, whereas recurrent tumors recurred at a rate of 11.6%, showing a statistically significant difference. the recurrence rate is higher for head and neck tumors, with that of the ear being the highest. however, dubin and kopf reported that bcc recurrence rates increased with an expanding lesion size. lesions smaller than 2 mm did not recur, lesions 6 to 10 mm showed a recurrence rate of 8.8%, and lesions larger than 30 mm recurred 23.1% of the time. as described above, bcc often originates in the skin of the nose, eyelid, or ear. surgeons should be familiar with the structures of these lesions and also with the reconstruction methods following the surgical resection of deep penetrating lesions. numerous reconstruction methods have been devised and utilized according to the characteristics of the defect. although skin grafting is a simple option, it is not a suitable reconstruction method for most defects of the nose, because it is difficult to obtain a good texture and color match. a local flap is a more favorable reconstruction method for the lower portion of the nose, where the skin is thick and dense with sebaceous glands. however, if a defect of the nasal tip or the ala is superficial and too large to cover with a local flap, a full - thickness skin graft can be used, especially when the skin is relatively thin and sebaceous glands are sparse. some local flaps often used for reconstruction of the nose are listed as follows. (1) nasolabial flapthe superiorly based nasolabial flap is useful for defects of the nasal sidewall, ala, and tip, while the inferiorly based nasolabial flap is useful for defects of the upper and lower lip, nasal floor, and columella. the blood supply to this flap is excellent due to perforating branches of the facial artery. the color and texture are excellent matches, while the donor site scar is acceptable in the nasolabial sulcus. using a template defect, the medial incision for the flap follows the nasolabial sulcus, and the lateral incision is placed no higher than the level of the inferior defect margin. the flap is elevated in the subcutaneous plane, and the plane goes deeper as it proceeds superiorly (figure 5(b)). the flap is rotated counterclockwise on the right side and transferred to the defect (figure 5(c)). the superiorly based nasolabial flap is useful for defects of the nasal sidewall, ala, and tip, while the inferiorly based nasolabial flap is useful for defects of the upper and lower lip, nasal floor, and columella. the blood supply to this flap is excellent due to perforating branches of the facial artery. the color and texture are excellent matches, while the donor site scar is acceptable in the nasolabial sulcus. using a template defect, the medial incision for the flap follows the nasolabial sulcus, and the lateral incision is placed no higher than the level of the inferior defect margin. the flap is elevated in the subcutaneous plane, and the plane goes deeper as it proceeds superiorly (figure 5(b)). the flap is rotated counterclockwise on the right side and transferred to the defect (figure 5(c)). (2) subcutaneous v - y flapsliding, subcutaneous v - y flaps for the reconstruction of nasal defects have been gaining in popularity, especially in nasal dorsum reconstruction. the flaps have also been used in ala nasi reconstructions, for defects generally limited to less than 1.5 cm in diameter and not involving the rim. the advantages of having similar tissue in the same operative field, with an excellent blood supply, make the v - y flap a common choice for nasal reconstruction. once all margins are known to be clear after tumor excision, the v - y flap is dissected out and moved inferiorly on a subcutaneous pedicle to repair the defect. however, this flap has limitations, particularly in instances involving the inferior margins of the nose near the anterior nares. some notching along the alar rim may occur and, in younger individuals, would probably be severe. for repair of the nostril rim, this flap may not be effective. the higher the defect is located on the nostril away from the rim, the easier the reconstruction is and the more favorable the result is. sliding, subcutaneous v - y flaps for the reconstruction of nasal defects have been gaining in popularity, especially in nasal dorsum reconstruction. the flaps have also been used in ala nasi reconstructions, for defects generally limited to less than 1.5 cm in diameter and not involving the rim. the advantages of having similar tissue in the same operative field, with an excellent blood supply, make the v - y flap a common choice for nasal reconstruction. once all margins are known to be clear after tumor excision, the v - y flap is dissected out and moved inferiorly on a subcutaneous pedicle to repair the defect. however, this flap has limitations, particularly in instances involving the inferior margins of the nose near the anterior nares. some notching along the alar rim may occur and, in younger individuals the higher the defect is located on the nostril away from the rim, the easier the reconstruction is and the more favorable the result is. (3) bilobed flap the bilobed nasal flap is a useful and time - honored technique for reconstructing defects of the nose, especially defects of the lower third of the nose [41, 43 ]. the bilobed flap is appropriate for partial - thickness losses of less than 1.5 cm of the lateral aspect of the nose, ala, and tip area. this flap is essentially a rotation flap divided into two transposition flaps, with an excellent blood supply from angular and supraorbital arteries. it recruits skin from the middorsum and sidewall.the two flaps have a common base and typically form an arc of no more than 90110 to avoid tension development on wound closure (figure 6(a)). the angle between the defect and first lobe is equal to that between the first and second lobe. the size of the first lobe equals that of the defect, and the second lobe is 2/3 the size of the first lobe. the primary flap closes the tumor defect, and the secondary flap is used to close the donor site (figure 6(b)). the donor site of the second lobe is primarily closed (figure 6(c)). the bilobed nasal flap is a useful and time - honored technique for reconstructing defects of the nose, especially defects of the lower third of the nose [41, 43 ]. the bilobed flap is appropriate for partial - thickness losses of less than 1.5 cm of the lateral aspect of the nose, ala, and tip area. this flap is essentially a rotation flap divided into two transposition flaps, with an excellent blood supply from angular and supraorbital arteries. the two flaps have a common base and typically form an arc of no more than 90110 to avoid tension development on wound closure (figure 6(a)). the angle between the defect and first lobe is equal to that between the first and second lobe. the size of the first lobe equals that of the defect, and the second lobe is 2/3 the size of the first lobe. the primary flap closes the tumor defect, and the secondary flap is used to close the donor site (figure 6(b)). the donor site of the second lobe is primarily closed (figure 6(c)). (4) midline forehead skin flap (seagull flap)the midline forehead skin flap can serve as a cover for any nasal reconstruction from severe tip and ala loss to a total nasal defect. using this flap, esthetic and functional reconstruction can be achieved by creating a nose that blends well with the face. its vertical axis is placed over the midline of the forehead, and the wings are designed to lie in natural transverse creases. the body of the seagull lies along the bridge, the wings curl at the ala and turn into the nostril sills, and the seagull head and neck creates the tip and columella. the midline forehead skin flap can serve as a cover for any nasal reconstruction from severe tip and ala loss to a total nasal defect. using this flap, esthetic and functional reconstruction its vertical axis is placed over the midline of the forehead, and the wings are designed to lie in natural transverse creases. the body of the seagull lies along the bridge, the wings curl at the ala and turn into the nostril sills, and the seagull head and neck creates the tip and columella. bcc accounts for 90 to 95% of malignant eyelid tumors [4547 ]. regarding periocular bcc, lower eyelid lesions are the most common, accounting for up to two - thirds of cases, followed by the upper eyelid, medial canthus, and lateral canthus [4547 ]. although small partial - thickness eyelid defects may be closed by simple suture, reconstruction of the lower eyelid after surgical excision is quite challenging. salomon. reported that local flaps or full - thickness skin grafts should be recommended in cases of small- and medium - sized lower eyelid skin defects. they also reported that the bilobed flap seemed to be the most appropriate among numerous possible regional flaps for small- and medium - sized lesions. the flap is based superiorly, so that it can easily be rotated to the lower eyelid position. larger defects of the medial canthus and adjacent eyelids may be covered with midline forehead transposition flaps. full - thickness palpebral defects ranging from one quarter to one - half of the lower eyelid may be repaired easily with the use of a cheek rotation flap. in such cases, orbital exenteration and/or resection of the paranasal sinuses may be required [4749 ]. in a study of invasive bcc, leibovitch. suggested that medial canthus bcc posed a higher risk of orbital invasion. a wedge excision of the auricle with a margin is often used for auricular bcc. a variety of local flaps for external ear and conchal reconstruction have been described, and full - thickness skin grafts (ftsg) have been used as well. dessy. reported that their first choice for the skin graft donor site is usually the contralateral postauricular area. after bcc removal from the external auditory canal, closure of the skin defect of the external auditory canal may not be needed if the underlying bone of the external ear canal is intact. to cover the exposed bone of the external auditory canal (the skin defect), a skin graft from the postauricular area may be a possibility. based either inferiorly or superiorly, the entire conchal skin and subcutaneous tissue can be elevated and transposed across the meatus of the external auditory canal. curettage and electrodesiccation (ce) comprise one of the most frequently used treatment modalities for bcc. the gross tumor is removed with a curet, and the base is desiccated with a cautery. the disadvantages are that, without biopsy and specimen orientation, histological control is poor or absent, and hypertrophic scars and hypopigmentation may occur. it is generally accepted that when effectively treated by ce experts, cure rates of more than 95% can be expected for appropriately selected bcc. the types of bcc that should not be treated by ce include large, infiltrating, morpheaform, and recurrent tumors. silverman. demonstrated that larger lesions, the diameter, and high - risk anatomic sites were independent factors affecting the recurrence rate (rr). in their study, bcc treated with ce at low - risk sites (neck, extremities) had a cumulative 5-year rr of 3.3% for lesions of any diameter. at medium - risk sites (scalp, forehead, auricular, and malar), bccs with diameters of less than 10 mm led to a 5-year rr of 5.3%, whereas those of 10 mm or larger had a higher rr of 22.7%. at high risk sites (nose, nasal labial groove, canthi, and ear), bccs of less than 6 mm in diameter led to a 5-year rr of 4.5%, whereas those of 6 mm or greater were associated with a 5-year rr of 17.6%. reported that all primary bcc patients with tumor of facial sites exhibited a 5-year cumulative rr of 1.2%, with 3 recurrences (nose, eyelid, and preauricular region) in 256 patients. cryotherapy is destructive modality that has been used in the treatment of bcc [54, 55 ]. two freeze - thaw cycles with a tissue temperature of 50c are required to destroy bcc. kuflik and grage reported 99% cure rates in 628 patients followed for 5 years. august suggested that cryotherapy should be avoided for the scalp and nasolabial fold sites because of the high rate of recurrence of the tumor. ceilley and del rosso also mentioned that aggressive cryotherapy may induce tumor recurrence because of concealment of the tumor by a fibrous scar. reported a case of a nodular bcc with a skip lesion on the nose, near the nasolabial fold, after repeated cryotherapy. radiotherapy can yield a high cure rate for bcc, and adjunctive radiotherapy can improve local regional control in cancer with adverse features such as the presence of perineural spread, extensive skeletal muscle infiltration, bone or cartilage invasion, and positive nodal / extranodal spread. lauritzen. reported that the cure rate with radiotherapy was 92.7% at 5 years in a series of 500 bcc patients. seegenschmiedt. reported that complete remission was achieved in 99% of patients by 3 months after treatment, in 127 bcc lesions of the head and neck region. swanson. reported that radiation therapy for bcc of the medial canthus resulted in a 100% control rate for positive margins and a 92% control rate for gross disease. regarding disadvantage of radiotherapy, radiotherapy may cause common cutaneous side effects such as acute and chronic radiation dermatitis [64, 65 ]. good initial cosmetic results can deteriorate with time, such that skin may show poikiloderma. it is desirable to avoid radiation therapy in young patients because of the late effects of irradiation. the treated area is exposed to monochromatic light after local or systemic administration of a chemical photosensitizer, such as methyl aminolevulinate. the photosensitizer absorbs light energy and then interacts with reactive oxidative species or directly with cellular substrates, resulting in cell death via apoptosis or necrosis. good treatment results of pdt were reported in superficial and nodular bccs with response rates of 85%92% in superficial bcc and 73%91% in nodular bcc [6874 ]. although longer followup studies are required, reported data indicate the potential of pdt as a noninvasive treatment alternative for superficial and nodular bccs. mutations in hedgehog pathway genes, primarily genes encoding patched homologue 1 (ptch1) and smoothened homologue (smo), occur in bcc. von hoff. assessed the safety and pharmacokinetics of gdc-0449 (vismodegib), a small - molecule inhibitor of smo, and responses of metastatic or locally advanced basal cell carcinoma to the drug. they reported that 18 of 33 bcc patients had a response to gdc-0449 and that only one grade 4 adverse event occurred during continuous daily administration of gdc-0449 for up to 19 months. because of its low toxicity and specificity for the hedgehog pathway, this drug has potential advantages compared with conventional chemotherapy and may also be used in combination treatments. nevoid basal carcinoma syndrome, also referred to as gorlin - goltz syndrome or basal cell carcinoma syndrome, is a rare autosomal dominant disease showing a genetic predisposition characterized by multiple bcc [7881 ]. patients with bcc syndrome show multiple abnormalities, none of which are unique to this syndrome [80, 81 ]. the three abnormalities traditionally considered to be the most characteristic of the syndrome are bcc, pits on the palm and sole, and cysts of the jaw. palmoplantar pits are small defects in the stratum corneum and may be pink or, if dirt has accumulated, dark in color. jaw cysts are often the first detectable abnormalities, and they may be asymptomatic and, therefore, diagnosed only radiologically. however, they also may erode enough bone to cause pain, swelling, and loss of teeth. a minority of bccs demonstrate aggressive behavior and involve the craniofacial bones in nevoid bcc syndrome. tabuchi. reported a nonfamilial case of nevoid bcc syndrome with a bcc of the eyelid invading the ethmoid sinus. because the individual abnormalities are not unique to bcc syndrome patients, it is possible to clinically diagnose bcc syndrome only when multiple, typical defects are present. the severity of abnormalities may differ markedly among members of a single family, and diagnosis certainly may be difficult in individuals. generally, the diagnosis is suggested in a patient with bcc arising at an unexpectedly early age and in unexpectedly large numbers. the gene for bcc syndrome has been mapped to chromosome 9q22.3-q31 [82, 83 ]. two researchers have independently demonstrated that bcc syndrome is caused by mutations of the patched1 (ptch1) gene [82, 83 ]. bcc is more common than all other cancers, and the most frequently seen malignancy by most doctors regardless of their specialty. we have to recognize bcc and its different histologic subtypes, as well as areas in which these might occur. mohs micrographic surgery is the standard treatment for cases of bcc on the head and neck. radiation therapy is also used in the treatment of primary bcc or in cases where postsurgical margins are positive for cancer. thus, doctors in all specialties need to become more aware of bcc, and accurate and early diagnoses need to be made by them. | basal cell carcinoma (bcc) is a malignant neoplasm derived from nonkeratinizing cells that originate from the basal layer of the epidermis and is the most frequent type of skin cancer in humans, with cumulative exposure to ultraviolet radiation as an important risk factor. bcc occurs most frequently at sun - exposed sites, with the head and neck being common areas. tumors can be classified as nodular, superficial, morpheaform, infiltrating, metatypic, and fibroepithelioma of pinkus. several treatment options such as surgical excision and nonsurgical procedures are available. the choice of treatment should be determined based on the histological subtype of a lesion, cost, its size and location, patient age, medical condition of the patient, treatment availability, and the patient 's wishes. the aim of any therapy selected for bcc treatment involving the head and neck is to ensure complete removal, the preservation of function, and a good cosmetic outcome. |
twenty to forty - five percent of patients with advanced cancer suffer from moderate to severe pain that requires treatment with opioids [13 ]. pharmacokinetic aspects of morphine and metabolites have been extensively studied and it has been shown that there are large inter - individual differences in serum concentrations of morphine and metabolites, even after dose correction. serum concentrations of morphine and metabolites are related to morphine dose and route of administration [4, 5 ]. while renal function has no influence on the serum concentrations of morphine, the levels of metabolites increase with decreasing renal function. the clinical efficacy of oral oxycodone is similar to that of morphine, but oxycodone is more potent with an equianalgesic ratio of 1/1.52 [6, 7 ]. this may partly be explained by the fact that the oral bioavailability of oxycodone ranges from 6087% [8, 9 ], which is higher than the 2040% for morphine [1012 ]. clinical pharmacokinetic studies on oxycodone and its major metabolites in patients are scarce [1316 ], especially for cancer pain patients. oxycodone is extensively metabolised in the liver, mainly via cyp3a4 to the inactive metabolite noroxycodone (47% of the dose), by 6-keto reduction to the most likely inactive metabolites, - and -oxycodol (8% of the dose), and via cyp2d6 to the active metabolite oxymorphone (11% of the dose), which is mainly found in a conjugated form in plasma. a third, possibly active metabolite, noroxymorphone, is formed from noroxycodone via cyp2d6, but also to a lesser degree from oxymorphone via cyp3a4 (fig. 1). the major metabolic pathway (bold arrows) of oxycodone is the formation of noroxycodone via cyp3a4 enzymes. the minor metabolic pathways (narrow arrows) are formation of oxymorphone via cyp2d6 enzymes, and 6-keto reduction to - and -oxycodol. the major metabolic pathway (bold arrows) of oxycodone is the formation of noroxycodone via cyp3a4 enzymes. the minor metabolic pathways (narrow arrows) are formation of oxymorphone via cyp2d6 enzymes, and 6-keto reduction to - and -oxycodol. oxymorphone is further metabolised to noroxymorphone via cyp3a4 enzymes cyp3a4 is involved in the metabolism of about 50% of all drugs, and does not seem to be under polymorphic regulation [18, 19 ]. inhibition and induction of cyp3a4 cyp3a4 activity is subject to a sex difference as higher activity is reported in women than in men [22, 23 ]. furthermore, a 50-fold inter - individual difference in cyp3a4 expression has been reported, and diseases such as cancer can down - regulate the expression [25, 26 ]. cyp2d6 is polymorphically regulated and expressed in four different phenotypes [27, 28 ]. cyp2d6 is not inducible, but it may be inhibited by several drugs [2931 ]. thus, an individual s phenotype can change (phenocopying) with co - administration of cyp2d6 inhibitory drugs [32, 33 ]. a recent study by samer. has shown that oxycodone and noroxycodone auc increased, and oxymorphone auc decreased, after blocking the cyp2d6 metabolic pathways of oxycodone with quinidine in healthy volunteers. also found decreased noroxycodone cmax and increased oxymorphone auc after blocking the cyp3a4 metabolic pathway with ketoconazole. moreover, samer s study indicated that oxymorphone, despite its modest levels in serum, may contribute to oxycodone analgesia in humans. the metabolites of oxycodone are excreted through the kidneys in free or conjugated form [9, 35 ]. peak plasma concentrations of oxycodone and noroxycodone are higher in patients with mild - to - moderate hepatic dysfunction or mild - to - severe renal dysfunction, and oxymorphone concentrations are lower in patients with impaired hepatic function [3638 ]. bmi (body mass index) is also potentially important to drug disposition and metabolism. several studies have described pharmacokinetic differences for drugs in obese compared from non - obese subjects. however, insufficient data for morphine on this topic, and no such data on oxycodone, exist. the present study is part of the european pharmacogenetic opioid study (epos). of the 2,294 cancer patients included in epos, 442 used oral oxycodone for their cancer pain. the secondary aim was to explore factors that influence the most important metabolic ratios, noroxycodone / oxycodone and oxymorphone / oxycodone. based on the above considerations, it was expected that the serum concentrations of oxycodone was influenced by dose, time since last dose to blood sample, sex, age and bmi ; the ratios noroxycodone or oxymorphone to oxycodone by age, cyp3a4 inhibitors or inducers, or cyp2d6 inhibitors, number of co - medications and gfr, and the former also by sex. moreover, a large inter - individual variability in serum concentrations and ratios was expected. this multicentre study was performed according to the guidelines of the helsinki declaration and was approved by the relevant research ethics committee of each study centre. before entering the study, participating patients gave written informed consent. patients included in epos were aged 18 years or more, had a verified malignant disease, and were treated with regular oral, subcutaneous, transdermal or intravenous opioids (morphine, methadone, fentanyl, hydromorphone, buprenorphine, ketobemidone or oxycodone) for their cancer pain for a minimum duration of 3 days. exclusion criteria were patients who were not capable of speaking the language used at the study centre. patients treated with oral oxycodone were eligible for the present study. at the time of inclusion age, sex, the patients functional status was assessed by the karnofsky performance status. all medications and dosages including opioids for the previous 24 h, duration of opioid treatment, use of rescue opioids in last 24 h, route of opioid administration, and the time interval between last opioid administration and blood sampling (see below) were recorded. medications were categorised as to whether they were cyp3a4 inducers or inhibitors, or a cyp2d6 inhibitor. the web - based table of flockhart, for instance, lists glucocorticoids as cyp3a4 inducers, while this group of drugs is not listed by wilkinson. body mass index (bmi) was calculated using the international system of units, bmi = weight (kg)/height (m). renal function was expressed as calculated glomerular filtration rate (gfr)/1.73 m body surface [44, 45 ]. blood samples were obtained shortly before drug administration of the patients scheduled oral opioid medication (trough value). for practical reasons blood samples from out - patients (n = 68) were taken at the time of examination. blood samples for opioid analyses in serum were collected in tubes with no additives and left at ambient temperatures for 3060 min before centrifugation at 2,500g (approximately 3,000 rpm) for 10 min. a pre - study formal sample size calculation was not performed since this was an explorative subgroup study within the larger epos study. however, the sample size is larger than green s recommendations (104 + k independent variables), and large enough to detect a medium effect according to miles and shevlin. median oxycodone and metabolites serum concentrations and ratios were calculated from the hospitalised patients (336 slow release, 35 immediate release) independent of time since last scheduled dose to blood sample and opioid used as rescue medication. spearman rank correlations were used to explore the association between patient demographic variables and serum concentrations. to protect against the risk for type i error due to multiple testing, only variables with p values less than 0.01 were considered statistically significant, and intended to be included into the multiple regression analyses. however, variables which did not meet the p 0.1. because data covering all variables for all the patients were not available (as seen in table 4 with the different n for the three regression analyses), the backward stepwise linear regressions were done manually. p values 0.05 (two - sided) were considered statistically significant in the final model. time since starting opioids, time since last oxycodone (scheduled or rescue) dose before blood sample, number of medications taken in last 24 h, oxycodone total (scheduled and rescue) daily dose, systemic steroids, glomerular filtration rate (gfr), albumin serum concentrations (all p 18 from purelab ultra (elga, bergman, norway). formic acid (analytical grade) was obtained from sds (ratstatt, germany). the liquid chromatography system used was an agilent 1100 series hplc system fitted with a g1311a quaternary pump, a g1322a degasser and a g1313a autosampler, all from agilent technologies (matriks, norway). column heating was performed with a universal - thermostat column oven from mikrolab (aarhus, denmark). the zorbax sb - c18 (2.1 150 mm, 5 m) column and the zorbax sb - c8 pre - column (4.6 12.5 mm, 5 m) were purchased from agilent technologies. the ms - ms system consisted of an api 4000 qtrap and api 5000 triple quadruple mass spectrometer from sciex instruments (applied biosystems, streetville, on, canada). edwards and smith s protein precipitation method, with the following modifications, was applied to extract oxycodone and its metabolites oxycodone, noroxycodone, oxymorphone and noroxymorphone from 0.2 ml serum. after addition of internal standards (oxycodone - d6, noroxycodone - d3 and oxymorphone - d3, 20 l of a 50 ng / ml solution) to the sample, proteins were precipitated by adding acetonitrile (0.9 ml). the sample was then whirl - mixed and left in the fridge for 3060 min. the sample was centrifuged at 12,000g for 10 min, supernatant was then frozen at 80c and evaporated in a vacuum concentrator (maxi dry lyo, heto holten a / s, aller, denmark) until freeze dried (23 h). a solution of 50 l containing 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid was added, the sample whirlmixed and centrifuged at 12,000g for 10 min. an lc - ms / ms method validated in accordance with dadgar and shah was used for identification and quantitative analysis of drug and metabolites in the extracted samples. analytical separation was performed with a column oven at 40c using a zorbax sb - c18 column with a zorbax sb - c8 pre - column at a flow rate of 0.2 ml / min and a gradient elution. the gradient was initiated with 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid (5.0 min), and then with a stepwise increase to 100% (5.20 min) acetonitrile with 0.1% formic acid. after 7.0 min the acetonitrile concentration was decreased to 20% (7.2 min) and was kept constant until end of the run (14.0 min). injection volume was 10 l. the ms / ms system was operated with multiple ion monitoring (mrm), with the following ions monitored : 316.1 241.1 for oxycodone, 302.1 227.2 for noroxycodone and oxymorphone, and 288.0 213.0 for noroxymorphone. the standard curve ranges were oxycodone 0.3216 nm (0.1500 ng / ml), oxymorphone 0.07166 nm (0.0250 ng / ml), noroxycodone 0.173,314 nm (0.051,000 ng / ml) and noroxymorphone 0.17696 nm (0.05200 ng / ml). the calibration curves were obtained from linear or quadratic regression with 1/x or 1/x weighing of the analyte - to - internal - standard ratios of peak area versus the respective analyte concentration. coefficients of variation (intra- and inter - day) for each analyte were 16.5 and 8.3% (0.32 nm), 4.4 and 4.0% (1.6 nm), 6.0 and 3.9% (31.7 nm), 4.5 and 3.7% (634.2 nm), and 5.5 and 4.8% (1,268.4 nm) for oxycodone ; 8.8 and 6.7% (0.8 nm), 5.8 and 3.4% (3.6 nm), 4.4 and 3.9% (76.2 nm), 10.8 and 5.6% (1,325.6 nm), and 6.5 and 5.8% (2,651.2 nm) for noroxycodone ; 10.0 and 7.5% (0.2 nm), 9.7 and 7.1% (0.3 nm), 8.9 and 4.4% (3.3 nm), 6.0 and 3.6% (66.4 nm), and 7.0 and 3.8% (132.7 nm) for oxymorphone ; and 10.0 and 7.1% (0.7 nm), 9.2 and 5.7% (0.9 nm), 6.9 and 6.5 (12.2 nm), 6.4 and 5.5% (208.8 nm), and 14.8 and 7.7% (471.7 nm) for noroxymorphone. the limits of quantification were 0.32 nm (0.1 ng / ml) for oxycodone, 0.07 nm (0.02 ng / ml) for oxymorphone, and 0.17 nm (0.05 ng / ml) for noroxycodone and noroxymorphone. this multicentre study was performed according to the guidelines of the helsinki declaration and was approved by the relevant research ethics committee of each study centre. before entering the study, participating patients gave written informed consent. patients included in epos were aged 18 years or more, had a verified malignant disease, and were treated with regular oral, subcutaneous, transdermal or intravenous opioids (morphine, methadone, fentanyl, hydromorphone, buprenorphine, ketobemidone or oxycodone) for their cancer pain for a minimum duration of 3 days. exclusion criteria were patients who were not capable of speaking the language used at the study centre. at the time of inclusion age, sex, weight, height, ethnicity and cancer diagnosis were registered. the patients functional status was assessed by the karnofsky performance status. all medications and dosages including opioids for the previous 24 h, duration of opioid treatment, use of rescue opioids in last 24 h, route of opioid administration, and the time interval between last opioid administration and blood sampling (see below) were recorded. medications were categorised as to whether they were cyp3a4 inducers or inhibitors, or a cyp2d6 inhibitor. the web - based table of flockhart, for instance, lists glucocorticoids as cyp3a4 inducers, while this group of drugs is not listed by wilkinson. body mass index (bmi) was calculated using the international system of units, bmi = weight (kg)/height (m). renal function was expressed as calculated glomerular filtration rate (gfr)/1.73 m body surface [44, 45 ]. blood samples were obtained shortly before drug administration of the patients scheduled oral opioid medication (trough value). for practical reasons blood samples from out - patients (blood samples for opioid analyses in serum were collected in tubes with no additives and left at ambient temperatures for 3060 min before centrifugation at 2,500g (approximately 3,000 rpm) for 10 min. a pre - study formal sample size calculation was not performed since this was an explorative subgroup study within the larger epos study. however, the sample size is larger than green s recommendations (104 + k independent variables), and large enough to detect a medium effect according to miles and shevlin. median oxycodone and metabolites serum concentrations and ratios were calculated from the hospitalised patients (336 slow release, 35 immediate release) independent of time since last scheduled dose to blood sample and opioid used as rescue medication. spearman rank correlations were used to explore the association between patient demographic variables and serum concentrations. to protect against the risk for type i error due to multiple testing, only variables with p values less than 0.01 were considered statistically significant, and intended to be included into the multiple regression analyses. however, variables which did not meet the p 0.1. because data covering all variables for all the patients were not available (as seen in table 4 with the different n for the three regression analyses), the backward stepwise linear regressions were done manually. p values 0.05 (two - sided) were considered statistically significant in the final model., time since last oxycodone (scheduled or rescue) dose before blood sample, number of medications taken in last 24 h, oxycodone total (scheduled and rescue) daily dose, use of cyp3a4 inducer, use of cyp3a4 inhibitor, systemic steroids, glomerular filtration rate (gfr), albumin serum concentrations (all p 18 from purelab ultra (elga, bergman, norway). formic acid (analytical grade) was obtained from sds (ratstatt, germany). the liquid chromatography system used was an agilent 1100 series hplc system fitted with a g1311a quaternary pump, a g1322a degasser and a g1313a autosampler, all from agilent technologies (matriks, norway). column heating was performed with a universal - thermostat column oven from mikrolab (aarhus, denmark). the zorbax sb - c18 (2.1 150 mm, 5 m) column and the zorbax sb - c8 pre - column (4.6 12.5 mm, 5 m) were purchased from agilent technologies. the ms - ms system consisted of an api 4000 qtrap and api 5000 triple quadruple mass spectrometer from sciex instruments (applied biosystems, streetville, on, canada). edwards and smith s protein precipitation method, with the following modifications, was applied to extract oxycodone and its metabolites oxycodone, noroxycodone, oxymorphone and noroxymorphone from 0.2 ml serum. after addition of internal standards (oxycodone - d6, noroxycodone - d3 and oxymorphone - d3, 20 l of a 50 ng / ml solution) to the sample, proteins were precipitated by adding acetonitrile (0.9 ml). the sample was then whirl - mixed and left in the fridge for 3060 min. the sample was centrifuged at 12,000g for 10 min, supernatant was then frozen at 80c and evaporated in a vacuum concentrator (maxi dry lyo, heto holten a / s, aller, denmark) until freeze dried (23 h). a solution of 50 l containing 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid was added, the sample whirlmixed and centrifuged at 12,000g for 10 min. an lc - ms / ms method validated in accordance with dadgar and shah was used for identification and quantitative analysis of drug and metabolites in the extracted samples. analytical separation was performed with a column oven at 40c using a zorbax sb - c18 column with a zorbax sb - c8 pre - column at a flow rate of 0.2 ml / min and a gradient elution. the gradient was initiated with 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid (5.0 min), and then with a stepwise increase to 100% (5.20 min) acetonitrile with 0.1% formic acid. after 7.0 min the acetonitrile concentration was decreased to 20% (7.2 min) and was kept constant until end of the run (14.0 min). the ms / ms system was operated with multiple ion monitoring (mrm), with the following ions monitored : 316.1 241.1 for oxycodone, 302.1 227.2 for noroxycodone and oxymorphone, and 288.0 213.0 for noroxymorphone. the standard curve ranges were oxycodone 0.3216 nm (0.1500 ng / ml), oxymorphone 0.07166 nm (0.0250 ng / ml), noroxycodone 0.173,314 nm (0.051,000 ng / ml) and noroxymorphone 0.17696 nm (0.05200 ng / ml). the calibration curves were obtained from linear or quadratic regression with 1/x or 1/x weighing of the analyte - to - internal - standard ratios of peak area versus the respective analyte concentration. coefficients of variation (intra- and inter - day) for each analyte were 16.5 and 8.3% (0.32 nm), 4.4 and 4.0% (1.6 nm), 6.0 and 3.9% (31.7 nm), 4.5 and 3.7% (634.2 nm), and 5.5 and 4.8% (1,268.4 nm) for oxycodone ; 8.8 and 6.7% (0.8 nm), 5.8 and 3.4% (3.6 nm), 4.4 and 3.9% (76.2 nm), 10.8 and 5.6% (1,325.6 nm), and 6.5 and 5.8% (2,651.2 nm) for noroxycodone ; 10.0 and 7.5% (0.2 nm), 9.7 and 7.1% (0.3 nm), 8.9 and 4.4% (3.3 nm), 6.0 and 3.6% (66.4 nm), and 7.0 and 3.8% (132.7 nm) for oxymorphone ; and 10.0 and 7.1% (0.7 nm), 9.2 and 5.7% (0.9 nm), 6.9 and 6.5 (12.2 nm), 6.4 and 5.5% (208.8 nm), and 14.8 and 7.7% (471.7 nm) for noroxymorphone. the limits of quantification were 0.32 nm (0.1 ng / ml) for oxycodone, 0.07 nm (0.02 ng / ml) for oxymorphone, and 0.17 nm (0.05 ng / ml) for noroxycodone and noroxymorphone. oxycodone, oxycodone - d6, noroxycodone - hcl, noroxycodone - d3 hcl, oxymorphone, oxymorphone - d3 and noroxymorphone hcl were obtained from cerillant (round rock, tx, usa). acetonitrile (hplc grade) was obtained from labscan and de - ionised water > 18 from purelab ultra (elga, bergman, norway). formic acid (analytical grade) was obtained from sds (ratstatt, germany). the liquid chromatography system used was an agilent 1100 series hplc system fitted with a g1311a quaternary pump, a g1322a degasser and a g1313a autosampler, all from agilent technologies (matriks, norway). column heating was performed with a universal - thermostat column oven from mikrolab (aarhus, denmark). the zorbax sb - c18 (2.1 150 mm, 5 m) column and the zorbax sb - c8 pre - column (4.6 12.5 mm, 5 m) were purchased from agilent technologies. the ms - ms system consisted of an api 4000 qtrap and api 5000 triple quadruple mass spectrometer from sciex instruments (applied biosystems, streetville, on, canada). edwards and smith s protein precipitation method, with the following modifications, was applied to extract oxycodone and its metabolites oxycodone, noroxycodone, oxymorphone and noroxymorphone from 0.2 ml serum. after addition of internal standards (oxycodone - d6, noroxycodone - d3 and oxymorphone - d3, 20 l of a 50 ng / ml solution) to the sample, proteins were precipitated by adding acetonitrile (0.9 ml). the sample was then whirl - mixed and left in the fridge for 3060 min. the sample was centrifuged at 12,000g for 10 min, supernatant was then frozen at 80c and evaporated in a vacuum concentrator (maxi dry lyo, heto holten a / s, aller, denmark) until freeze dried (23 h). a solution of 50 l containing 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid was added, the sample whirlmixed and centrifuged at 12,000g for 10 min. an lc - ms / ms method validated in accordance with dadgar and shah was used for identification and quantitative analysis of drug and metabolites in the extracted samples. analytical separation was performed with a column oven at 40c using a zorbax sb - c18 column with a zorbax sb - c8 pre - column at a flow rate of 0.2 ml / min and a gradient elution. the gradient was initiated with 20% acetonitrile with 0.1% formic acid and 80% water with 1.0% formic acid (5.0 min), and then with a stepwise increase to 100% (5.20 min) acetonitrile with 0.1% formic acid. after 7.0 min the acetonitrile concentration was decreased to 20% (7.2 min) and was kept constant until end of the run (14.0 min). the ms / ms system was operated with multiple ion monitoring (mrm), with the following ions monitored : 316.1 241.1 for oxycodone, 302.1 227.2 for noroxycodone and oxymorphone, and 288.0 213.0 for noroxymorphone. the standard curve ranges were oxycodone 0.3216 nm (0.1500 ng / ml), oxymorphone 0.07166 nm (0.0250 ng / ml), noroxycodone 0.173,314 nm (0.051,000 ng / ml) and noroxymorphone 0.17696 nm (0.05200 ng / ml). the calibration curves were obtained from linear or quadratic regression with 1/x or 1/x weighing of the analyte - to - internal - standard ratios of peak area versus the respective analyte concentration. coefficients of variation (intra- and inter - day) for each analyte were 16.5 and 8.3% (0.32 nm), 4.4 and 4.0% (1.6 nm), 6.0 and 3.9% (31.7 nm), 4.5 and 3.7% (634.2 nm), and 5.5 and 4.8% (1,268.4 nm) for oxycodone ; 8.8 and 6.7% (0.8 nm), 5.8 and 3.4% (3.6 nm), 4.4 and 3.9% (76.2 nm), 10.8 and 5.6% (1,325.6 nm), and 6.5 and 5.8% (2,651.2 nm) for noroxycodone ; 10.0 and 7.5% (0.2 nm), 9.7 and 7.1% (0.3 nm), 8.9 and 4.4% (3.3 nm), 6.0 and 3.6% (66.4 nm), and 7.0 and 3.8% (132.7 nm) for oxymorphone ; and 10.0 and 7.1% (0.7 nm), 9.2 and 5.7% (0.9 nm), 6.9 and 6.5 (12.2 nm), 6.4 and 5.5% (208.8 nm), and 14.8 and 7.7% (471.7 nm) for noroxymorphone. the limits of quantification were 0.32 nm (0.1 ng / ml) for oxycodone, 0.07 nm (0.02 ng / ml) for oxymorphone, and 0.17 nm (0.05 ng / ml) for noroxycodone and noroxymorphone. the epos included 2,294 cancer patients from 17 centres in 11 european countries, with 461 patients (98% caucasians) treated with oxycodone. twenty - two patients were excluded ; 17 because they were treated with intravenous (5) or subcutaneous (12), oxycodone, 2 because of lack of a blood sample and 3 because neither oxycodone nor metabolites could be detected in serum. thus, 439 patients using oral oxycodone (394 slow release, 48 immediate release) were included for analysis (fig. 2). the european pharmacogenetic opioid study included 2,294 cancer patients of whom 461 were treated with oxycodone. four hundred and forty - four used oral oxycodone, 439 were included in the analyses study flow sheet. the european pharmacogenetic opioid study included 2,294 cancer patients of whom 461 were treated with oxycodone. four hundred and forty - four used oral oxycodone, 439 were included in the analyses the patients demographic data are shown in table 1. thirteen percent (n = 59) of the participants were suffering from renal disease / dysfunction (gfr < 60 ml / min per 1.73 m body surface), while 58% (n = 253) had albumin serum concentrations below the normal range (3555 forty - four percent of the patients used rescue opioid doses (90% used oxycodone). one - hundred and sixty - nine patients used oral immediate release, 6 subcutaneous and 1 intravenous oxycodone as rescue medication. table 1demographics and characteristics of the 439 patients included in the analysesdemographic / characteristicstatisticmen / women247/192age (years)63 (1891)bmi (kg / m)24 (1441)height (cm)171 (148199)karnofsky performance status score (%) 70 (2090)time since cancer diagnosis (months)18 (0286)time since start opioids (months)1 (097)time since last oxycodone dose before blood sample (hours)10 (0.117)number of medications in addition to oxycodone6 (017)glomerular filtration rate (gfr) (ml / min/1.73 m)96 (24261)serum albumin (g / l)33 (1091)oxycodone rescue medication (yes / no)176/263 oral immediate release oxycodone169 subcutanous oxycodone6 intravenous oxycodone1 other than oxycodone19cancer diagnosis gastrointestinal (inclusive pancreas, liver)19.8 prostate17.5 lung (inclusive mesothelioma)16.6 breast14.8 female reproductive organs7.5 haematological6.6 other urological6.4 head and neck2.5 skin2.1 sarcoma2.0 other cancer diagnoses5.7 more than one diagnosis4.3 unknown origin2.7metastases (yes / no)359/55 bone49.8 liver22.2 lung19.0 cns5.2 other33.3 more than one47.2pain category somatic pain56.9 mixed pain27.8 visceral pain11.6 neuropathic pain3.6numbermedian (minimum to maximum)percentage (%) demographics and characteristics of the 439 patients included in the analyses median (minimum to maximum) median time from last opioid dose to blood sample was 10 (0.117) h. virtually all patients (99%) used other drugs in addition to opioids, and 12 used herbal medications. patients had taken a median of 6 non - opioid drugs during the previous 24 h. the most frequently co - administered classes of drugs were laxatives (57%), histamine 2 receptor antagonists (57%), paracetamol (53%), systemic steroids (48%) and antiemetics (37%). thirty - five patients used one or more medications known to inhibit cyp2d6 such as haloperidol (28), fluoxetine (2) or paroxetine (1), hydroxyzine (2), doxepine (1), chlorpromazine (1) and amidarone (1). other cyp3a4 inhibitors used were clarithromycin (2), verapamil (3), nelfinavir (1), itraconazole (1) and diltiazem (1). three patients used the cyp3a4 inducer carbamazepine and one used phenobarbital [18, 43 ]. two - hundred and twelve used systemic steroids that according to flockhart are cyp3a4 inducers. the median oxycodone rescue dose was 20 (range 5360) mg, and median oxycodone total daily dose (sum of scheduled and rescue) was 80 (range 10960) mg. the 95% cis for the mean oxycodone scheduled daily dose were 87109 mg/24 h, 2942 mg/24 h for rescue and 102128 mg/24 h total daily dose (table 2). the resulting serum concentrations of oxycodone and metabolites and their ratios displayed wide ranges, even after dose correction (table 3). oxycodone serum concentration varied from 0 to 1,890 (median 100) nm, while those of noroxycodone, oxymorphone and noroxymorphone varied from 0 to 4,858 (median 106), 027 (median 1.5) and 0509 (median 17) nm respectively. the 95% cis for the uncorrected mean values were 139183 nm for oxycodone, 174245 nm for noroxycodone, 2.33.0 nm for oxymorphone and 2634 nm for noroxymorphone. table 2oxycodone scheduled daily dose, total daily dose of oxycodone rescue medication and total daily oxycodone (scheduled and rescue ; mg/24 h) given as median, 25th and 75th percentile, minimum to maximum values, mean and 95% ci for the hospitalised patientsoxycodone daily dose : median25th percentile75th percentileminimum to maximummean95% cilowhighscheduled6040120107609887109rescue2010405360352942total (scheduled and rescue)804012510960115102128table 3uncorrected serum concentrations (nm) of oxycodone, noroxycodone, oxymorphone, noroxymorphone and dose - corrected (nm 100 mg 24 h / dose 24 h) serum concentrations, and ratios noroxycodone / oxycodone and oxymorphone / oxycodone given as median, 25th and 75th percentiles, minimum to maximum values, mean and 95% ci for mean for the hospitalised patientsserum concentrationsmedian25th percentile75th percentileminimum to maximummean95% cilowhighoxycodone974320101,890161139183oxycodone1449022501,294186170203noroxycodone1014421103,571209174245noroxycodone16110326103,032212188235oxymorphone1.50.73.20252.62.33.0oxymorphone2.21.24.20343.43.03.8noroxymorphone178390509302634noroxymorphone2918440360343138ratio noroxycodone / oxycodone1.10.71.90.124.41.61.41.8ratio oxymorphone / oxycodone0.020.010.030.000320.210.020.020.02uncorrected serum concentrationsdose - corrected serum concentrations oxycodone scheduled daily dose, total daily dose of oxycodone rescue medication and total daily oxycodone (scheduled and rescue ; mg/24 h) given as median, 25th and 75th percentile, minimum to maximum values, mean and 95% ci for the hospitalised patients uncorrected serum concentrations (nm) of oxycodone, noroxycodone, oxymorphone, noroxymorphone and dose - corrected (nm 100 mg 24 h / dose 24 h) serum concentrations, and ratios noroxycodone / oxycodone and oxymorphone / oxycodone given as median, 25th and 75th percentiles, minimum to maximum values, mean and 95% ci for mean for the hospitalised patients uncorrected serum concentrations dose - corrected serum concentrations the noroxycodone / oxycodone and oxymorphone / oxycodone ratios varied from 0.1 to 24.4 (median 1.1) and from 0.00032 to 0.21 (median 0.02) respectively (table 3). the 95% ci for the mean was 1.41.8 nm and 0.0200.025 nm respectively. four patients lacked a noroxycodone / oxycodone ratio, because there was no detectable oxycodone and noroxycodone (n = 2), no detectable oxycodone (n = 1) and no detectable noroxycodone (n = 1). fifteen patients had an oxymorphone / oxycodone ratio of zero, 12 because oxymorphone was not detected, and 3 because oxycodone was not found. these patients were given a fictive low serum concentration value (assay detection limit 0.5) and were included in the distribution (fig. fig. 3a log10-transformed distributions of cyp3a4-dependent noroxycodone / oxycodone ratio and b the cyp2d6-dependent oxymorphone / oxycodone ratio. histograms on the left and p - p plots (expected cumulative probability vs observed cumulative probability) on the right a log10-transformed distributions of cyp3a4-dependent noroxycodone / oxycodone ratio and b the cyp2d6-dependent oxymorphone / oxycodone ratio. histograms on the left and p - p plots (expected cumulative probability vs observed cumulative probability) on the right the epos included 2,294 cancer patients from 17 centres in 11 european countries, with 461 patients (98% caucasians) treated with oxycodone. twenty - two patients were excluded ; 17 because they were treated with intravenous (5) or subcutaneous (12), oxycodone, 2 because of lack of a blood sample and 3 because neither oxycodone nor metabolites could be detected in serum. thus, 439 patients using oral oxycodone (394 slow release, 48 immediate release) were included for analysis (fig. 2). the european pharmacogenetic opioid study included 2,294 cancer patients of whom 461 were treated with oxycodone. four hundred and forty - four used oral oxycodone, 439 were included in the analyses study flow sheet. the european pharmacogenetic opioid study included 2,294 cancer patients of whom 461 were treated with oxycodone. four hundred and forty - four used oral oxycodone, 439 were included in the analyses the patients demographic data are shown in table 1. thirteen percent (n = 59) of the participants were suffering from renal disease / dysfunction (gfr < 60 ml / min per 1.73 m body surface), while 58% (n = 253) had albumin serum concentrations below the normal range (3555 forty - four percent of the patients used rescue opioid doses (90% used oxycodone). one - hundred and sixty - nine patients used oral immediate release, 6 subcutaneous and 1 intravenous oxycodone as rescue medication. table 1demographics and characteristics of the 439 patients included in the analysesdemographic / characteristicstatisticmen / women247/192age (years)63 (1891)bmi (kg / m)24 (1441)height (cm)171 (148199)karnofsky performance status score (%) 70 (2090)time since cancer diagnosis (months)18 (0286)time since start opioids (months)1 (097)time since last oxycodone dose before blood sample (hours)10 (0.117)number of medications in addition to oxycodone6 (017)glomerular filtration rate (gfr) (ml / min/1.73 m)96 (24261)serum albumin (g / l)33 (1091)oxycodone rescue medication (yes / no)176/263 oral immediate release oxycodone169 subcutanous oxycodone6 intravenous oxycodone1 other than oxycodone19cancer diagnosis gastrointestinal (inclusive pancreas, liver)19.8 prostate17.5 lung (inclusive mesothelioma)16.6 breast14.8 female reproductive organs7.5 haematological6.6 other urological6.4 head and neck2.5 skin2.1 sarcoma2.0 other cancer diagnoses5.7 more than one diagnosis4.3 unknown origin2.7metastases (yes / no)359/55 bone49.8 liver22.2 lung19.0 cns5.2 other33.3 more than one47.2pain category somatic pain56.9 mixed pain27.8 visceral pain11.6 neuropathic pain3.6numbermedian (minimum to maximum)percentage (%) demographics and characteristics of the 439 patients included in the analyses median (minimum to maximum) median time from last opioid dose to blood sample was 10 (0.117) h. virtually all patients (99%) used other drugs in addition to opioids, and 12 used herbal medications. patients had taken a median of 6 non - opioid drugs during the previous 24 h. the most frequently co - administered classes of drugs were laxatives (57%), histamine 2 receptor antagonists (57%), paracetamol (53%), systemic steroids (48%) and antiemetics (37%). thirty - five patients used one or more medications known to inhibit cyp2d6 such as haloperidol (28), fluoxetine (2) or paroxetine (1), hydroxyzine (2), doxepine (1), chlorpromazine (1) and amidarone (1). other cyp3a4 inhibitors used were clarithromycin (2), verapamil (3), nelfinavir (1), itraconazole (1) and diltiazem (1). three patients used the cyp3a4 inducer carbamazepine and one used phenobarbital [18, 43 ]. two - hundred and twelve used systemic steroids that according to flockhart are cyp3a4 inducers. the median oxycodone rescue dose was 20 (range 5360) mg, and median oxycodone total daily dose (sum of scheduled and rescue) was 80 (range 10960) mg. the 95% cis for the mean oxycodone scheduled daily dose were 87109 mg/24 h, 2942 mg/24 h for rescue and 102128 mg/24 h total daily dose (table 2). the resulting serum concentrations of oxycodone and metabolites and their ratios displayed wide ranges, even after dose correction (table 3). oxycodone serum concentration varied from 0 to 1,890 (median 100) nm, while those of noroxycodone, oxymorphone and noroxymorphone varied from 0 to 4,858 (median 106), 027 (median 1.5) and 0509 (median 17) nm respectively. the 95% cis for the uncorrected mean values were 139183 nm for oxycodone, 174245 nm for noroxycodone, 2.33.0 nm for oxymorphone and 2634 nm for noroxymorphone. table 2oxycodone scheduled daily dose, total daily dose of oxycodone rescue medication and total daily oxycodone (scheduled and rescue ; mg/24 h) given as median, 25th and 75th percentile, minimum to maximum values, mean and 95% ci for the hospitalised patientsoxycodone daily dose : median25th percentile75th percentileminimum to maximummean95% cilowhighscheduled6040120107609887109rescue2010405360352942total (scheduled and rescue)804012510960115102128table 3uncorrected serum concentrations (nm) of oxycodone, noroxycodone, oxymorphone, noroxymorphone and dose - corrected (nm 100 mg 24 h / dose 24 h) serum concentrations, and ratios noroxycodone / oxycodone and oxymorphone / oxycodone given as median, 25th and 75th percentiles, minimum to maximum values, mean and 95% ci for mean for the hospitalised patientsserum concentrationsmedian25th percentile75th percentileminimum to maximummean95% cilowhighoxycodone974320101,890161139183oxycodone1449022501,294186170203noroxycodone1014421103,571209174245noroxycodone16110326103,032212188235oxymorphone1.50.73.20252.62.33.0oxymorphone2.21.24.20343.43.03.8noroxymorphone178390509302634noroxymorphone2918440360343138ratio noroxycodone / oxycodone1.10.71.90.124.41.61.41.8ratio oxymorphone / oxycodone0.020.010.030.000320.210.020.020.02uncorrected serum concentrationsdose - corrected serum concentrations oxycodone scheduled daily dose, total daily dose of oxycodone rescue medication and total daily oxycodone (scheduled and rescue ; mg/24 h) given as median, 25th and 75th percentile, minimum to maximum values, mean and 95% ci for the hospitalised patients uncorrected serum concentrations (nm) of oxycodone, noroxycodone, oxymorphone, noroxymorphone and dose - corrected (nm 100 mg 24 h / dose 24 h) serum concentrations, and ratios noroxycodone / oxycodone and oxymorphone / oxycodone given as median, 25th and 75th percentiles, minimum to maximum values, mean and 95% ci for mean for the hospitalised patients uncorrected serum concentrations dose - corrected serum concentrations the noroxycodone / oxycodone and oxymorphone / oxycodone ratios varied from 0.1 to 24.4 (median 1.1) and from 0.00032 to 0.21 (median 0.02) respectively (table 3). the 95% ci for the mean was 1.41.8 nm and 0.0200.025 nm respectively. four patients lacked a noroxycodone / oxycodone ratio, because there was no detectable oxycodone and noroxycodone (n = 2), no detectable oxycodone (n = 1) and no detectable noroxycodone (n = 1). fifteen patients had an oxymorphone / oxycodone ratio of zero, 12 because oxymorphone was not detected, and 3 because oxycodone was not found. these patients were given a fictive low serum concentration value (assay detection limit 0.5) and were included in the distribution (fig. fig. 3a log10-transformed distributions of cyp3a4-dependent noroxycodone / oxycodone ratio and b the cyp2d6-dependent oxymorphone / oxycodone ratio. histograms on the left and p - p plots (expected cumulative probability vs observed cumulative probability) on the right a log10-transformed distributions of cyp3a4-dependent noroxycodone / oxycodone ratio and b the cyp2d6-dependent oxymorphone / oxycodone ratio. histograms on the left and p - p plots (expected cumulative probability vs observed cumulative probability) on the right serum concentrations of oxycodone (rs = 0.71), oxymorphone (rs = 0.56), noroxycodone (rs = 0.75) and noroxymorphone (rs = 0.68) correlated (p < 0.001 for all) with oxycodone total daily dosage (fig. 4, shown only for oxycodone). serum concentrations of noroxycodone (rs, females = 0.79, rs, males = 0.76), oxymorphone (rs, females = 0.73, rs, males = 0.66) and noroxymorphone (rs, females = 0.64, rs, males = 0.59) were closely associated (p < 0.001 for all) with oxycodone serum concentrations for both men and women. at a given level of oxycodone, correlations between oxycodone daily dose and the ratios oxymorphone / oxycodone (rs, females = 0.32, rs, males = 0.33) and noroxycodone / oxycodone (rs, females = 0.33, rs, males = 0.13) were low (p < 0.001 for all ; data not shown). moreover, there was no correlation between serum concentration and gfr (data not shown). 4spearman rank correlation (rs) between oxycodone total daily dose (mg/24) and serum concentrations of oxycodone (nm ; rs = 0.71, p < 0.001) for the hospitalised patientsfig. 5a c spearman rank correlations (rs) for men (rs, males) and women (rs, females) between oxycodone and noroxycodone, oxymorphone and noroxymorphone serum concentrations (rs = 0.590.79, p < 0.001) for the hospitalised patients spearman rank correlation (rs) between oxycodone total daily dose (mg/24) and serum concentrations of oxycodone (nm ; rs = 0.71, p < c spearman rank correlations (rs) for men (rs, males) and women (rs, females) between oxycodone and noroxycodone, oxymorphone and noroxymorphone serum concentrations (rs = 0.590.79, p < 0.001) for the hospitalised patients table 4 shows the results from the multiple linear regression analysis with the outcomes oxycodone serum concentrations, the oxymorphone / oxycodone ratio and the noroxycodone / oxycodone ratio, respectively. table 4multiple linear regression models with factors predicting the serum concentrations of oxycodone and the ratios oxymorphone / oxycodone and noroxycodone / oxycodonefactors associated withunstandardised coefficientsstandardised coefficients95% confidence interval for bbstandard errorbetasignificancelower boundupper boundoxycodone (n = 433 ; r = 0.35) oxycodone total daily dose0.0020.00020.4910.0000.0020.002 cyp3a4 inducer0.7860.2330.1310.0011.2420.327 cyp3a4 inhibitor0.2040.1010.0780.0440.0050.403 sex0.1130.0450.0980.0120.0250.201 time from last oxycodone dose to blood sample0.0270.0060.1890.0000.0380.016ratio oxymorphone / oxycodone (n = 438 ; r= 0.05) oxycodone total daily dose0.00040.00010.1360.0040.00070.0001 cyp3a4 inducer0.6070.2340.1220.0100.1471.067 number of medications (excluding opioids) taken in the last 24 h0.0240.0080.1400.0030.0390.008ratio noroxycodone / oxycodone (n = 396 ; r = 0.19) oxycodone total daily dose0.0010.00010.2310.0000.00030.001 time from last oxycodone dose to blood sample0.0120.0040.1470.0020.0040.020 albumin0.0060.0020.1320.0050.0020.010 sex0.1100.0330.1600.0010.1750.045 cyp3a4 inducer0.6020.1830.1530.0010.2420.962 cyp3a4 inhibitor0.2940.0680.1970.0000.4270.160 systemic steroids0.0700.0320.1020.0280.1320.008 bmi0.0090.0040.1090.0190.0170.001 glomerular filtration rate0.0010.00040.1610.0010.0020.0005independent variables in all analyses were age (years), sex, bmi (kg m), karnofsky performance status (%), time from last oxycodone dose to sample (h), oxycodone total daily dose (mg/24 h), time since starting opioids (months), number of concomitant medications in the last 24 h, use of cyp3a4 inhibitor (yes / no), use of cyp3a4 inducer (yes / no), glomerular filtration rate (ml min 1.73 m) and albumin (g l) serum concentrationscoefficients are in log 10 form (e.g. 10)yes = 1, no = 0 (user of systemic steroids yes : 10, no : 10)men = 1, women = 0 (male oxycodone serum concentration : 10, women : 10, noroxycodone / oxycodone ratio men : 10, women : 10)the patients in the cyp3a4 inducer (n = 4) group also used systemic steroids multiple linear regression models with factors predicting the serum concentrations of oxycodone and the ratios oxymorphone / oxycodone and noroxycodone / oxycodone independent variables in all analyses were age (years), sex, bmi (kg m), karnofsky performance status (%), time from last oxycodone dose to sample (h), oxycodone total daily dose (mg/24 h), time since starting opioids (months), number of concomitant medications in the last 24 h, use of cyp3a4 inhibitor (yes / no), use of cyp3a4 inducer (yes / no), glomerular filtration rate (ml min 1.73 m) and albumin (g l) serum concentrations coefficients are in log 10 form (e.g. 10) yes = 1, no = 0 (user of systemic steroids yes : 10, no : 10) men = 1, women = 0 (male oxycodone serum concentration : 10, women : 10, noroxycodone / oxycodone ratio men : 10, women : 10) the patients in the cyp3a4 inducer (n = 4) group also used systemic steroids oxycodone total daily dose, time from last oxycodone dose (scheduled or rescue) to blood sample (p 0.001 for both), cyp3a4 inducer (p = 0.001), cyp3a4 inhibitor (p = 0.044) and sex (p = 0.010) were associated with oxycodone serum concentrations. together these factors explain 35% (r = 0.35) of the observed variation. total daily dose had the largest standardised coefficient, and was therefore the most prominent of all variables. this means that increasing the dose and inhibition of the cyp3a4 metabolic pathway favour increased oxycodone serum concentrations. cyp3a4 inducer and time from last oxycodone dose (scheduled or rescue) to blood sample displayed negative associations. thus, longer time between tablet intake and blood sampling and use of a cyp3a4 inducer as concomitant medication decrease oxycodone serum concentrations. users of cyp3a4 inducer drugs are predicted to have 84% (see notes to table 4 for calculation) lower serum concentrations of oxycodone than those without the cyp3a4 inducer drug. users of cyp3a4 inhibitors are predicted to have 60% higher oxycodone serum concentrations than non - users. the variables oxycodone total daily dose (p = 0.004), number of medications except opioids taken in the last 24 h (p = 0.003) and cyp3a4 inducer (p = 0.010), only explained 5% (r = 0.05) of the observed variation in oxymorphone / oxycodone ratio. the variables total daily dose and number of medications were negatively associated with this ratio. thus, an increase in total daily dose or number of medications decreases the oxymorphone / oxycodone ratio. users of cyp3a4 inducers are predicted to have a three times higher oxymorphone / oxycodone ratio, than non - cyp3a4 users. all variables had a similar impact on the explained variation (see their standardised coefficients, table 4). except for age, karnofsky performance status, time since starting opioids and number of medications, the ratio noroxycodone / oxycodone was associated with all examined variables, with oxycodone total daily dose being the most important. however, these variables together only explained 19% (r = 0.19) of the variation. use of cyp3a4 inhibitor (p = 0.000), bmi (p = 0.019) and glomerular filtration rate (p = 0.002) showed a negative association. men are predicted to have 22% (see notes to table 4 for calculation) lower noroxycodone / oxycodone ratio than women (p = 0.002), and use of a cyp3a4 inhibitor is predicted to give a 49% reduction in ratio compared with non - users of cyp3a4 inhibitors. users of cyp3a4 inducers are predicted to have about 4 times higher noroxycodone / oxycodone ratio. users of systemic steroids are predicted to have 15% lower ratio than those not using steroids. forty - seven of the 439 patients used immediate - release oxycodone every 46 h, while the remaining 392 were using controlled - release oxycodone approximately every 12 h. all the regression analyses were also performed without the immediate release users to determine whether oxycodone formulation affected the data. the outcome of this analysis did not differ from the outcome of the complete analysis. serum concentrations of oxycodone (p = 0.49), oxymorphone (p = 0.12) and the ratio oxymorphone / oxycodone (p = 0.15) did not differ between users and non - users of cyp2d6 inhibitors. the following variables included in the analyses did not explain variability in any of the outcomes : age, karnofsky performance status and time since starting opioids. finally, other variables that were explored, but not included in the multiple regression analyses were ; use of steroids, use of dexamethasone, use of cyp2d6 inhibitor, having liver metastases and time since starting opioids in months. the focus of this study was to examine if common clinically observed factors could predict variation in oxycodone serum concentrations and the metabolite to oxycodone ratios in cancer patients. it was shown that oxycodone total daily dose, use of cyp3a4 inducers / inhibitors, sex and the time from the last oxycodone dose to blood sample explained variations in serum concentrations of oxycodone. it was also observed that the ratios for the serum concentrations of the metabolites noroxycodone and oxymorphone to oxycodone were influenced by the use of cyp3a4 inducer drugs and oxycodone total daily dose. the number of medications except opioids taken in the last 24 h was the only additional factor for the oxymorphone / oxycodone ratio. the noroxycodone / oxycodone ratio was in addition influenced by sex, use of cyp3a4 inhibitor, bmi, gfr, albumin and time from last dose to blood sample. however, the regression models could only explain minor parts of the variability of serum concentration and ratios of oxycodone and metabolites. the wide concentration ranges of oxycodone and metabolite serum concentrations observed were expected, not least due to the wide dose range observed in this sample. these observations agree with the studies that showed large individual differences in serum concentrations even after dose correction for patients receiving morphine [4, 5153 ]. total daily dose correlated highly with the variable that best explained the variability of oxycodone serum concentrations. rescue medication is frequently used by cancer pain patients. in this sample 44% of the patients the use of rescue medication may confound data interpretation ; however, this was corrected for by using the two variables time since last dose and total daily dose in the regression analyses. first, men used higher total daily doses than women (median 80 mg/24 h vs 70 mg/24 h, p = 0.03). it is unlikely that the higher body weight of men accounts for this difference as dosing for cancer pain is not based on weight, but titration to the desired effect. it is more likely that it reflects that men may be less sensitive than women to opioids, and therefore may require higher doses to relieve similar levels of pain [54, 55 ]. there are no published data on sex differences in oxycodone dosage requirements, although studies with morphine have shown that men need at least 3040% more morphine than women for pain relief. this agrees with the finding in this study where men had higher serum concentrations of oxycodone than women (about 30%, as calculated from the regression analysis). since sex and daily dose are independent variables in the analyses, sex as a factor in itself also contributes to the difference in oxycodone serum concentration. this may well be explained by a higher metabolic capacity in women as discussed below. the observation that men are predicted to have a 31% lower noroxycodone / oxycodone ratio than women may fit with higher cyp3a4 activity in women. thus, the higher oxycodone serum concentrations in men may be explained by a lower activity of cyp3a4 compared with women. also, in vivo studies have shown that women seem to exhibit faster clearance of cyp3a4-metabolising drugs [22, 23, 60 ], although some studies have failed to detect this clearance difference [61, 62 ]. a secondary outcome was to assess whether clinical variables can be used to predict the metabolite to parent drug ratios, assuming that this exploration of ratios could shed light on the elimination pathways of oxycodone. it was unexpected that oxycodone total daily dose explained the variability of metabolite to parent drug ratios. we would expect both ratios to remain constant for an individual, assuming that the elimination pathways of oxycodone followed first - order kinetics. thus, this finding may indicate that all processes involved in these ratios might not obey first - order kinetics. oxymorphone is formed from oxycodone mainly by cyp2d6 enzymes and excreted mainly as an oxymorphone-3-glucuronide conjugate. while the distribution of the noroxycodone / oxycodone ratio was normal as expected for a potential phenotypic expression of cyp3a4, this was not obvious for the oxymorphone / oxycodone ratio. both histogram and p - p plot showed indices of multimodal distribution, although no clear bimodality could be seen. one reason for this may be that oxymorphone is further metabolised by udp - glucuronosyl - transferase to oxymorphone-3-glucuronide and variability in this metabolic pathway may influence the distribution of the oxymorphone / oxycodone ratio. also, factors other than genotype may explain the variability of this ratio as discussed below. cyp2d6 is subject to significant inhibition of its activity by a number of other drugs [18, 31 ]. most of the co - administered drugs used in our study are not known to inhibit cyp2d6, as only 8%of the patients (n = 35) were treated with known cyp2d6 inhibitors. moreover, the low number of known cyp2d6 inhibitors observed in this large sample of patients recruited from several centres indicates that the use of these drugs is infrequently indicated in cancer pain patients. however, it remains the case that increasing numbers of co - administered drugs reduce the ratio pf oxymorphone to oxycodone. some of the factors, such as sex, gfr, use of cyp3a4 inducers / inhibitors and time from last oxycodone (scheduled or rescue) dose to blood sample can probably be explained by common pharmacokinetic knowledge. gfr is an independent variable predicting a noroxycodone / oxycodone ratio rise when gfr decreases. this rise may possibly be explained by reduced renal clearance, causing a relative accumulation of noroxycodone, which is mainly excreted through the kidneys. in fact, the auc ratio for noroxycodone / oxycodone was three times higher in renal failure patients compared with subjects with normal renal function after intravenous administration of oxycodone. thus, reduced renal function may change several aspects of the overall pharmacokinetics of oxycodone. two of the factors that influence the noroxycodone / oxycodone ratio, bmi and albumin, are difficult to explain. this could potentially have explained the ratio, although albumin did not contribute to the variability of oxycodone serum concentrations itself. the overall median noroxycodone / oxycodone ratios in this study in cancer patients was in accordance with results from previous studies [9, 35, 39 ]. the oxymorphone / oxycodone ratio was also in line with other studies, and confirms the minor amount of this active metabolite compared with its parent substance in this group. a previous study has claimed that the levels of oxymorphone in the brain relative to the parent drug oxycodone is very low. however, recent experimental studies in humans have documented that oxymorphone contributes to analgesia [64, 65 ]. concomitant medication with cyp3a4 inhibitors, as expected, reduced the cyp3a4-mediated noroxycodone / oxycodone ratio. the use of cyp3a4 inducers on the other hand, influenced all three examined outcomes. the observed changes in the ratios comply with an increased cyp3a4 activity, thus lowering oxycodone concentration and increasing those of noroxycodone. however, except for systemic steroids, cyp3a4 inducers were used by only 4 patients and these 4 alone had a remarkable influence on both ratios and the oxycodone serum concentrations. thus, potential cyp3a4 drug drug interactions, especially with the use of cyp3a4 inducers, are also important to consider when administering oxycodone in cancer patients. we recognise that compared with experimental pharmacokinetic studies in volunteers (healthy or with cancer) this study is subject to a number of confounding factors. however, this multicentre cross - sectional study resulted in a patient population representative of the heterogeneity of cancer patients physicians are faced with in day - to - day clinical work. moreover, this study is unique with its large sample size where clinical characteristics and extensive data on the serum concentrations of oxycodone and its major metabolites are combined. the use of multiple regression analysis made it possible to control for differences between the patients with respect to common clinical variables. thus, despite the heterogeneity of the sample, a number of plausible variables related to the variability of the outcomes were also confirmed in cancer patients. the explained variability of the dependent variables was low in these regression analyses, especially for the ratios, which comply with those of a previous publication. plausible reasons for this are the fact that cancer patients are a very heterogeneous group of patients ; the origin and progression of their cancer differ, their metastatic status differs, their metabolic status differs, and of course there are genetic and perception differences. sex differences related to opioids and metabolism may also be true in a cancer population. drug interactions related to cyp2d6 are probably of little clinical significance ; however, use of cyp3a4 inducers or inhibitors should be carefully monitored, as these might significantly influence the serum concentrations, which may possibly change the effects of oxycodone. pharmacokinetics in special populations, such as patients with renal failure and obesity, should be studied further. finally, the variables, including daily dose, explained one third of the variability of oxycodone serum concentrations and only minor parts of the variability of the ratios in this population. | objectiveoxycodone is widely used for the treatment of cancer pain, but little is known of its pharmacokinetics in cancer pain patients. the aim of this study was to explore the relationships between ordinary patient characteristics and serum concentrations of oxycodone and the ratios noroxycodone or oxymorphone / oxycodone in cancer patients.methodsfour hundred and thirty - nine patients using oral oxycodone for cancer pain were included. the patients characteristics (sex, age, body mass index [bmi ], karnofsky performance status, time since starting opioids, oxycodone total daily dose, time from last oxycodone dose, use of cyp3a4 inducer / inhibitor, use of systemic steroids, number of medications taken in the last 24 h, glomerular filtration rate (gfr) and albumin serum concentrations) influence on oxycodone serum concentrations or metabolite / oxycodone ratios were explored by multiple regression analyses.resultssex, cyp3a4 inducers / inhibitors, total daily dose, and time from last oxycodone dose predicted oxycodone concentrations. cyp3a4 inducers, total daily dose, and number of medications taken in the last 24 h predicted the oxymorphone / oxycodone ratio. total daily dose, time from last dose to blood sample, albumin, sex, cyp3a4 inducers / inhibitors, steroids, bmi and gfr predicted the noroxycodone / oxycodone ratio.conclusionwomen had lower oxycodone serum concentrations than men. cyp3a4 inducers / inhibitors should be used with caution as these are predicted to have a significant impact on oxycodone pharmacokinetics. other characteristics explained only minor parts of the variability of the outcomes. |
osgood - schlatter disease (osd) is a well known condition, characterized by pain over the tibial tubercle with subsequent tubercle prominence. we report an unusual case of simultaneous bilateral tibial tubercle avulsion fracture in a 16 year old boy who was a known case of osd. a 16 year old boy a known case of osd presented to the outpatient department with history of jumping from the school compound wall (two feet height) while playing, followed by severe pain around anterior aspect of both knees and difficulty in walking. he was treated successfully with open reduction and internal fixation with tension band wiring. at the end of 22 months the patient was symptomatically relieved and both the tuberosities were united with the main bone. even though bilateral osgood - schlatter disease (osd) is a well known condition, one should always keep in mind the risk of tibial tubercle avulsion fractures while treating a case of osd. patient should be advised not to involve in strenuous activities till the disease subsides radiologically or till skeletal maturity. osgood - schlatter disease (osd) is a well known clinical condition, characterized by painful tender disabling swelling over the tibial tubercle. in 1903, osgood in the united states and schlatter in germany reported this condition independently for the first time [1, 2 ]. avulsion of the tibial tubercle in an adolescent is rare and is usually produced by sudden violent contraction of the quadriceps muscles. we report a case of simultaneous bilateral tibial tubercle avulsion fracture in a 16 year old boy who was a diagnosed case of osd. a 16 year old boy presented to the outpatient department in november 2009 with the history of painful swelling around the anterior aspect of both proximal tibia. on examination he was diagnosed clinically to have bilateral osd and treated conservatively with nsaids and compression bandage. he was brought to the outpatient department after 2 months in february 2010 with the history of jumping from the school compound wall (two feet height) while playing, followed by severe pain around anterior aspect of both knees and difficulty in walking. after few minutes the knees were swollen and he was unable to bear weight on both the legs and to flex and extend the knees. radiographs of both knee joints ap and lateral views were taken which showed bilateral tibial tubercle avulsion fractures. 1) according to ogden classification right side was type iia and leftside was iib tibial tubercle avulsion fracture. both sides were treated with open reduction and internal fixation with tension band wiring (tbw) (fig. 2 and 3). radiograph of right and left knees respectively showing ogden type iia and iib avulsion fracture of the tibial tubercle. postoperative radiographs of right knee with tbw in situ postoperative radiographs of right knee with tbw in situ fracture was reduced and confirmed clinically and radiologically under image intensifier and fixed with two 2 mm k wires and tension band wiring. the figure - of - eight wire was passed deep to the patellar ligament and through a transosseous tunnel 4 cm distal to the fracture. kirschner wires provided rotational stability, and were inserted through the proximal part of the tubercle and engaged the posterior cortex of the tibia under direct guidance of image intensification. patient was put on cylindrical slab for 2 weeks and gradual mobilization of knee was started. patient was followed up clinically and radiologically at 6 weeks, 12 weeks, and 6 months and 12 months and 22 months (fig. follow up the knee range was 0 to 120 and completely pain free with patient doing all his activities without any functional limitations 22 months follow up radiograph of both knees showing well united tuberosities a painful, tender disabling swelling about the tibial tubercle in adolescence is characteristic of osd. the earliest record of traction epiphysitis is of a middle aged male, dated ninth to tenth century a.d. on the archaeological evidence found in the late saxon buriel ground of st. the disease starts in the second decade of life and usually resolves spontaneously without any sequelae by the time of skeletal maturity. initially, the tibial tubercle is painful following physical activity, may be due to chronic micro trauma to the tibial tubercle secondary to overuse of the quadriceps muscle and gradually becomes prominent and constantly painful. mri will show the patellar tendon attachment more proximally and in a broader area to the tibia (routine mri was not done in our case as clinico - radiological findings were correlating with the diagnosis). clinical conditions like chondromalacia patellae, patellar tendinitis, osteomyelitis of proximal tibia, pes anserinus bursitis and sinding larsen - johanson syndrome should be considered as differential diagnosis and should be ruled out before considering osd. avulsion fractures of the tibial tuberosity are uncommon, comprising only 0.4 to 2.7% of all epiphyseal injuries. the mechanism of avulsion injury usually consists of violent knee flexion against a tightly contracted quadriceps or a violent quadriceps contraction with a fixed foot like in bad landing from a jump. in our patient also our patient was already a diagnosed case of osd on conservative management, but was not complying with our advice to refrain from sports activities. osd has been reported to be a associated risk factor for tibial tuberosity avulsion [11 - 18 ] although scientific correlation is still inconclusive. ogden commented that structural modification in the physeal cartilage, probably change in the columnar cartilage to fibrocartilage may weaken the physis to tensile strain. our case supports the hypothesis that patients with osd have a risk for developing tibial tuberosity avulsion and should be allowed for a adequate period of restricted activities, specially sports related activities. there have been 230 published cases with tibial tuberosity avulsion fractures, but simultaneous bilateral tibial tubercle avulsions are uncommon, with only few cases reported [3 - 5,21 - 25 ] and reports in a case of pre - existing osd are still rare [6,15, 16,17,18 ]. thus our case is among rare bilateral presentations that strengthen the association between pre - existing osd and tibial tubercle avulsion fracture. accepting the association between osd and avulsion fractures, we reviewed the literature with respect to duration between diagnosis of osd and avulsion fractures. inoue too reported one case with pre exiting asymptomatic osd but did not comment on the duration between diagnosis and avulsion. mosier and stanitsk reported another case that presented with avulsion fracture after three weeks of diagnosis of osd. chow reported one case of avulsion fracture with prior diagnosis of osd 6 months back. levi reported one case in 13 year old boy who had history of osd in left knee since one year. thus a time variation from 3 weeks to 1 year has been reported between diagnosis of osd and avulsion fractures, however data is insufficient to make comment on the period after diagnosis of osd, in which activities should be restricted to prevent tibial avulsion fracture. it would be probably safe to have sports restrictions up till the physeal fusion or complete radiological healing of the primary osd. the latter point was not commented on in the reviewed literature and probably a non healed osd will have a higher propensity for avulsion fractures as seen in our case. the original classification system was provided by sir reginald watson - jones which defined three types. type i was an avulsion of a small part of the tibial tubercle, distal to the proximal tibial physis. ryu and debenham described a fracture of the tibial tuberosity that extends posteriorly along the proximal tibial physis creating an avulsion of the entire proximal epiphysis (type iv). in our case both knee had ogden class ii fractures and were treated with k wires and tension band wiring. open reduction of type ii injuries is essential and many methods of fixation for avulsed tibial tubercle are available like tension band wiring, screw fixation with washer, suture anchor fixation, bone staples fixation etc [19,29 - 30 ]. in ogden type iii injuries where there is comminution and intraarticular extension the range of motion may be limited [3, 30 ], however as our case was extraarticular type ii injury the recovery of range of motion was full as reported by other such studies [6, 15,21,30 ]. we report a rare case of bilateral tibial tuberosity avulsion fracture in a case of pre - existing osd. this case shows association between these factors and we can probably suggest to patients to restrict activities till complete radiological healing of the osd to prevent risk of tibial tubercle avulsion. our case was an extraarticular avulsion and open reduction internal fixation results in good clinical and functional results. high strain and sports activities should be restricted till healing of diagnosed osd to prevent risk of tibial tubercle avulsion fractures. in cases with avulsion fracture of tibial tuberosity in adolescent | introduction : osgood - schlatter disease (osd) is a well known condition, characterized by pain over the tibial tubercle with subsequent tubercle prominence. avulsion fracture following osd is a rare complication. we report an unusual case of simultaneous bilateral tibial tubercle avulsion fracture in a 16 year old boy who was a known case of osd.case presentation : a 16 year old boy a known case of osd presented to the outpatient department with history of jumping from the school compound wall (two feet height) while playing, followed by severe pain around anterior aspect of both knees and difficulty in walking. radiographs showed bilateral tibial tubercle avulsion fracture. he was treated successfully with open reduction and internal fixation with tension band wiring. at the end of 22 months the patient was symptomatically relieved and both the tuberosities were united with the main bone.conclusion:even though bilateral osgood - schlatter disease (osd) is a well known condition, one should always keep in mind the risk of tibial tubercle avulsion fractures while treating a case of osd. patient should be advised not to involve in strenuous activities till the disease subsides radiologically or till skeletal maturity. |
the macarthur foundation 's research network on successful midlife development says that midlife is perhaps the most ill - defined of any period in life. until recently it could really be so puzzling that many people are confused and fail to understand this natural event of life. however, it could also be a time when people simultaneously explore their inner lives and restructure their outer lives. the united nations children 's emergency fund reported that the average life span of filipino women is 66 years and of filipino men 62 years. theoretically, then, the midthirties and forties are the middle years of the filipinos. however, developmental books still situate midlifers at the age of 35 - 60. oftentimes, midlife abounds in changing images and myths which include midlife crisis, change of life, the empty nest and many more. it can be an emotionally uncomfortable time which can lead to depression and the need for psychotherapy. on the other hand, johnston contends that midlife is but a developmental stage which is essentially positive and has the specific goal of facilitating the process of becoming a whole person. midlifers who are in the teaching profession may in fact be experiencing anxieties and other negative behaviors arising from the multiplicity of roles they play in their work, family, and community. as workers in the family, they might be taking care of their elderly and dying parents or experiencing their grown - up children leaving home. they take generative roles by being members of community organizations that would take care of the next generations. on top of these, they may also have personal needs to satisfy and dreams to realize. the aforementioned conditions may create role confusions and ambivalence among midlifers leading them to undesirable effects. however conversely, the same conditions may also be their wellspring of vigor, zest, and inspiration ; they energize them, keep them going and make their life meaningful. it was therefore the aim of this study to take another look at midlife by studying the health statuses of the faculty midlifers and analyze their implications to human resources management in education. the participants of this study were the 106 out of 142 permanent filipino full - time faculty members of the different basic education departments and colleges of the university of san jose - recoletos (usj - r), cebu city, philippines, who are in their midlife. this number showed that the sample size of the conducted survey has a margin error of 5% at 95% level of confidence. this study utilized a survey to come up with descriptive - correlation analyses between the profiles of the research participants and their health statuses. this survey includes a three - part test designed to assess the physical health, emotional health and psychological health (attitude toward midlife) of the research participants. it evaluates how well the person is physically and how good he is in taking good care of his body. this inventory has been constructed to find out how a person deals with the day - to - day situations that involve emotions. this instrument has been administered to 300 midlifers to test its internal consistency and factor analysis. this study also utilized multivariate analyses to determine the relationships between the sociodemographic profiles of the research participants and their health statuses. specifically, the pearson - product - moment coefficient of correlation was performed using statistical program for social sciences (spss) is manufactured by ibm corporation to determine their relationships. the data present that majority of the midlifers of the university are females. they composed 63.21% of the overall population. it was also shown that the faculty midlifers of the university are predominantly married (82.07%). the ages of the participants of this study range between 35 and 59 years old. participants were clustered into three groups such as the early bloomers (36 - 40 years), bloomers (41 - 50), and late bloomers (51 - 59). a little more than half (51.89%) of the faculty midlifers it has a standard deviation of 6.86, which means that their ages are moderately dispersed. the means of their year of service is 18.23 years with a standard deviation of 8.27, which means that the research participants are widely dispersed in terms of their years of teaching. practically half (43.40%) of the midlifers of usj - r have rendered 10 - 19 years in the teaching profession. seventeen % (16.98%) are teaching from 0 to 9 years while 11.32% have embraced the profession for 30 - 39 years. with regards to their educational attainment, it was found out that most (37.73%) of the faculty midlifers attained masters (m.a. or m.s.) they are followed by faculty who are taking up doctoral studies (22.64%) and college graduates who are adding m.a. or m.s. some faculty midlifers (16.04%) have reached the peak of educational attainment having earned a doctorate degree. a very few (1.89%) of them are teaching with only a college degree. more than half (64 of 106 or 60.38%) of the research participants have an individual gross monthly income of 28,000-32,999. some of them (15% or 14.15%) get a monthly salary of 23,000-27, 999 while (11% or 10.38%) among them receive a monthly salary of 33,000-37,999. only a few of the faculty midlifers of usj - r are receiving a monthly salary of 38,000 and above. it was likewise illustrated that the combined family income of the most (28 of 106 [26.42% ]) is between 28,000 and 32,999. this is somehow similar to the amount received by the majority of the faculty midlifers as individual monthly income. however, significantly, they are followed by faculty midlifers (23.58%) whose combined family monthly income (cfmi) has reached 68,000 or more. a few of them (16 of 106 [15.09% ]) gets a combined family income between 33,000 and 37,999. sociodemographic characteristics for the sample are summarized in table 1a and b. socio - demographic characteristics of the faculty midlifers 1 socio - demographic characteristics of the faculty midlifers 2 furthermore, it was revealed that a good majority of the research participants (69 of 106 [65.09% ]) are physically fair while a number of them (32 of 106 [30.19% ]) are found out to be physically good. their mean score is 40.19 (fair) with a standard deviation of 7.39 denoting that they are widely dispersed in term of the physical health status. nevertheless, barely 4.72% (5 of 106) of them are also physically in the data showed that a great majority of the participants (87 of 106 [82.08% ]) are okey in terms of their emotional health. only some of them (19 of 106 [17.92% ]) do need to review their reactions to situations that involve their emotions. the faculty midlifers got a mean score of 19.47 (you 're okay) in their emotional health with a standard deviation of 1.87 indicating that they are not widely dispersed in term of their emotional health. finally, it was found out that an overwhelming majority (97 of 106 [91.51% ]) of the faculty midlifers in the study have very positive attitude toward midlife. their mean score is 130.68 (very positive) with a standard deviation of 14.08. none among them have slightly positive, negative, and much more extremely negative attitude toward midlife. health statuses of the faculty midlifers tests on correlations discovered that basically there are no significant relationships between the physical health and the sociodemographic profile of the faculty midlifers except with sex which indicates a significant relationship (r = 0.20, p = 0.04). since their relationship is positive, this implies that the females among them have better physical health status compared to the males. females have a mean physical health status of 41.12 while that of their counterpart is 38.13. however, computation on correlation also tells us that the emotional health status of the faculty midlifers are significantly related to their age positively (r = 0.20, p = 0.04). it means that the higher their age, the higher also is their emotional health status. thus, as their number of teaching increases, their emotional health status likewise improves (r = 0.25, p = 0.01). the two variables, age and years of teaching, usually come together due to the reality that oftentimes as the worker ages, his / her years of service in his / her workplace also increases. lastly, two variables in the sociodemographic profile of the faculty midlifers were found to have significant relationship with their psychological health. their psychological health (attitude toward midlife) is related to their educational attainment and their cfmi. the correlates of the health statuses of the faculty midlifers are presented in table 3. correlates of the health statuses of the faculty midlifers psychological health and educational attainment are highly correlated (r = 0.32, p = 0.001). it suggests that the higher their educational attainment, the more positive is their attitude toward midlife. on the other, a significant positive relationship between psychological and cfmi is also observed (r = 0.24, p = 0.01). table 3 illustrates the correlation between the health statuses and the sociodemographic profile of the faculty midlifers. this finding supports many of the studies on the association between sex and physical health. christie reported what recent researchers have confirmed and what many have already suspected ; that women are not only the fairer sex, but also the healthier sex as well. the studies of and added that nowadays, women outlive men by about 5 - 6 years. with respect to that most essential proof of robustness or the power to stay alive, kirkwood assumed that women are really tougher than men from birth through the extreme old age. his research even found that by the age 85 there are roughly six women to every four men. at age 100, the ratio is more than two to one. moreover, by age 122, the current world record for human longevity the score stands at one - nil in favor of women. it might be that women live longer because they develop healthier habits than men. for instance, women smoke and drink less than men and choose a better diet. they have a secret weapon to use if there are little battles in their bodies. a study done by mcgill university indicated that estrogen gives women an edge when it comes to fighting off infections. that is because estrogen confronts a certain enzyme that often hinders the body 's first line of defense against bacteria and viruses. it is also discovered that high levels of testosterone, which boost male fertility, are quite bad for long - term survival. there is evidence in rodents that cells in a female body do repair damage better than in the body of a male, and that surgical removal of the ovaries eliminates this difference. in addition, castration of men in institutions for the mentally disturbed which was surprisingly a commonplace a number of years ago gave researchers astonishing findings. in one study of several 100 men in an unnamed institution in kansas, the castrated men were found to live on average 14 years longer than their uncastrated fellows. women do live longer and may have fewer problems with certain infectious diseases than men. women 's hearts beat more rapidly than men 's (80 beats / min vs. 72 beats / min), but women have less tendency to develop high blood pressure. however, they have also some drawbacks as women have much higher risk for autoimmune diseases such as lupus and rheumatoid arthritis. on the other, age and years of teaching served as correlates of the faculty midlifers ' emotional health. psychology professor and longevity expert, laura carstensen contends that as people age, they become more emotionally balanced and better able to solve highly emotional problems. her study found out that over the years, the older subjects reported having fewer negative emotions and more positive ones compared with their younger days. urry and gross expounded that people seem to develop better skills for regulating their emotions as they age. for example, older people often have smaller and closer social networks than younger people. this may show that they are choosing to put themselves in pleasant situations with people they like. furthermore, studies have found that older adults pay more attention to positive information than to negative information, which may improve mood. some evidences also suggest that older people are better at predicting how a certain situation will make them feel, which gives them a better chance of choosing enjoyable situations and avoiding unpleasantness. while teenagers and young adults experience more frustrations, anxiety, and disappointment over things such as test scores, career goals, and finding a soulmate, older people typically have made their peace with life accomplishments and failures., they tend to have learned to accept what comes and to regulate their emotions. according to them, several studies also found a gradual average decline in such negative emotions as anger, fear, and anxiety through midlife and beyond. florin dolcos, an assistant in psychiatry and neuroscience, offered a physiological explanation of this phenomenon. he had identified brain patterns that help healthy older people regulate and control emotions better than their younger counterpart. in his study, where participants were shown standardized pictures of emotionally challenging situations, the older participants rated the images as less negative than the younger participants. the brain scan of these older participants also showed increased interaction between the amygdala, a brain region involved in emotion detection, and the anterior cingulate cortex, a brain region involved in emotion control. according to dr. dolcos, these findings indicate that emotional control improves with aging and that it is the interaction between these two regions of the brain that allows healthy seniors to control their emotional response so that they are less affected by upsetting situations. his study further acknowledges results of previous studies that provided evidence that healthy older individuals have a positivity bias which means they can actually manage how much attention they give to negative situations, so they are less upset by them. furthermore, the study of fariselli. showed that emotional intelligence increases slightly with age. in their study, age is found to be slightly predictive of self - awareness and has a strong relationship with self - direction. finally, the educational attainment of the faculty midlifers is highly related to their psychological health. thus, the higher their educational attainment or cfmi, the higher also is their psychological health. finds a significant association between low level of education and psychological distress in both genders. they find out that low level of education is associated with a low sense of mastery, low social support, negative life events, low household income, and unemployment. among those variables considered, sense of mastery emerged to really have a strong mediating influence between the level of education and psychological distress. their sense of mastery could have given them the positive attitude toward their midlife events. in addition, steele. noticed marked inequity in mental health services utilization by educational level. they noted that people with higher education are more likely to avail mental health services. they are more likely to see psychiatrist, family doctor, psychologist, or social worker. this could be the reason why they were able to maintain their psychological health. orpana. noted in their study that low - income respondents were at a significantly higher risk of becoming psychologically distressed. low levels of household income are associated with several lifetime mental disorders and suicide attempts, and a decrease in income is associated with a higher risk for anxiety, substance use, and mood disorders. moreover, studies over the 20 years also indicate a close interaction between factors associated with poverty and mental ill - health. for example, common mental disorders are seen to be about twice as frequent among the poor as among the rich most especially among people experiencing hunger or facing debts. evidence also indicates that depression is 1.5 - 2 times more prevalent among the low - income groups of population. in addition, reporting that one is on the lowest rung of the socioeconomic status ladder, or that children in the household are often hungry, is likewise associated with reporting more depressive symptoms. on the other, those with higher income are much more likely than those with lower incomes to report excellent mental health, and this persists even when other variables such as age, education, gender, and marital status are taken into accounts. at midlife, the faculty members of usj - r can still generally be considered as physically well. it can also be said that female faculty midlifers are better able to maintain a healthy body than their male colleagues. faculty midlifers likewise manifest emotional maturity. their emotional health becomes better as they grow older and as they stay longer in the teaching profession. they maintain an optimistic view on midlife experiences and having a higher educational attainment and higher family income contribute to their level of optimism. thus, if they are well - managed, they can become relevant and better contributors to the attainment of the basic goals and objectives of the educational institution and the educational system in general. | background : between the school years of 2009 - 2012, the turnover record of the university of san jose - recoletos (usj - r), cebu city, philippines showed that permanent faculty members who left the institution were all midlifers. their reasons varied from health issues to greener pasture elsewhere.materials and methods : this study then sought to explore the health statuses of the faculty midlifers of the usj - r. the data were collected through survey conducted among the 106 faculty midlifers of the university. this study applied multivariate analyses to the survey data using pearson - moment of correlation to determine the relationship between the sociodemographic profile of the research participants and their health statuses.results:this research revealed that faculty midlifers are generally well physically. they showed emotional maturity and have positive outlook toward midlife. more so, their health conditions are significantly related with their sex, age, years of teaching, educational attainment, and income.conclusion:at midlife, the faculty members of usj - r can still generally be considered physically well. thus, if they are well - managed, they can become relevant and better contributors to the attainment of the basic goals and objectives of the educational institution and the educational system in general. |
the constellation of findings consisted of a solid ovarian mass, ascites and hydrothorax, which should be considered as a malignant process until proven otherwise. however, in 1937, meigs and cass1) reported the clinical picture of nonmalignant ascites and/or pleural effusion in association with a benign ovarian tumor, with resolution of the ascites and hydrothorax after removal of the ovarian lesion, which thereafter has been called meigs ' syndrome2, 3). when the same clinical features exist, but involve other ovarian or gynecologic tumors, it is referred to as pseudo - meigs ' syndrome4, 5). granulosa - stromal cell tumors, including granulosa cell tumors, thecomas and fibromas, are the most common diseases of sex cord stromal tumors, and may be associated with endometrial hyperplasia and an endometrial carcinoma6). granulosa cell tumors are low - grade, estrogen secreting malignancies, and can be seen in women of all ages. an ovarian mass and an elevated serum ca125 level in a postmenopausal woman generally suggest a malignant process. herein, a case of meigs ' syndrome from an ovarian granulosa cell tumor, with elevated ca125, and the unique principles of its management are reported, with a review of the literature. a 69-year - old korean woman was admitted to our hospital complaining of abdominal fullness and dyspnea., there was dullness to percussion, with decreased breath sounds in the lower half of the right lung field, a huge palpable mass in the lower abdomen, and on a rectovaginal examination a large, smooth solid mass was palpated on the left adnexa. laboratory data on admission were within normal limits. a chest x - ray (figure 1a) and ct scan (figure 1b) showed massive right pleural effusion, and ultrasonography, ct and mri scans revealed a 61012 cm in sized intrapelvic huge solid mass, including cystic components involving both adnexae and the uterus, and a moderate amount of intra - abdominal fluid collection (figure 2, 3). the mri and ct scans showed no evidence of a metastatic disease involving the lymph nodes, bone or abdominal organs. the serum ca125 concentration was 82.49 u / ml (normal, 0~35 u / ml). the serum levels of carcinoembrionic antigen (cea) and carbohydrate antigen 19 - 9 (ca19 - 9) were normal. a cytological examination of both the ascites and pleural fluid showed no signs of malignant cells. serous ascites, a moderately enlarged uterus and a large right ovarian mass were found, but there were no signs of metastatic spread. a total abdominal hysterectomy (tah) was performed, with a bilateral salpingo - oophorectomy (bso), partial omentectomy and pelvic lymph nodes sampling. during the operation, the ovarian tumor ruptured in the pelvic cavity and 2,500 ml of serous ascites were drained during the laparotomy. due to the right pleural effusion, a trocar tube was inserted into the right thorax, with a total of 650 ml of serous fluid collected. the specimens of the left ovary and salpinx, macroscopically, showed a previously ruptured fragile, with numerous grayish brown ovary tissues. the nuclei of this tumor were pale and round and oval or angular and have occasional nuclear grooves (figure 4). the totally removed uterus measured 742.2 cm in size, and showed adenomyosis and senile endometrium. the right ovary and salpinx were grossly clean, but no definitive lesion was found. a cytological examination of the ascites drained during the operation showed no sign of malignant cells. the final diagnosis of the left ovarian mass was that of a granulosa cell tumor. the pathologic stage of the tumor was designated as t1cnomo and ic, according to the tnm stage and to the international federation of gynecologists and obstetricians (figo) staging system, respectively. the hydrothorax and ascites rapidly resolved in the early postoperative period (figure 5), and the trocar tube was removed on the seventh postoperative day. four cycles of postoperative chemotherapy were planned due to the tumor rupturing during the operation. the adjuvant chemotherapy consisted of cap (cyclophosphamide 500 mg / m, adriamycin 50 mg / m and cisplatin 50 mg / m on day 1) regimen. meigs and cass1) described a rare triad of benign, solid ovarian tumor, with the gross appearance of a fibroma (e.g., an actual fibroma, a thecoma, or a granulosa cell tumor), ascites and hydrothorax, where a cure was achieved by completely removing the benign tumor. this syndrome occurs in association with only 1 to 2 percent of fibromas, which in themselves are rare in children2). since the original description by meigs, ascites and hydrothorax have been reported in a variety of other gynecologic conditions, including fibroids7), degenerative ovarian changes, mature cystic teratoma and ovarian - hyperstimulation syndrome, and are usually referred to as " pseudo - meigs ' syndrome ". however, as ryan5) indicated, the discrimination of true meigs ' syndrome is considered fundamentally academic, and does not affect the therapeutic aspects of the problem8). the mechanism of formation of the ascites is unknown, but several theories have been offered to explain the origin of the hydrothorax and ascites in meigs ' and pseudo - meigs ' syndrome. meigs believed it due to leakage from the fibroma, as fibromas are frequently edematous, possibly due to pressure on the lymphatic vessels on the tumor 's surface2). it has also been suggested that the presence of fluid results from cyst formation within the tumor as a result of injury or necrosis. meigs showed the hydrothorax generally developed from movement of the ascitic fluid into the pleural space through congenital defects in the diaphragm. generally, a plausible theory explaining meigs syndrome has been proposed, as follows ; first, the ascites may be caused by transudation of interstitial edema fluid from the tumor. in relation to this explanation, samanth and black9) proposed a discrepancy between the arterial supply to a large mass of tumor tissue, and that it 's venous and lymphatic drainage could lead to stromal edema and transudation. second, it is possible that pressure on the lymphatics in the tumor itself may cause the escape of fluid through the superficial lymphatics situated just beneath the single layered cuboidal epithelium covering the tumor10). third, the production of fluid by the peritoneum, other than around the tumor surface, has recently been suggested as the main factor in the production of ascites8). recently, abramov.11) reported that the findings strongly suggested the involvement of vasoactive growth factor (vegf and fgf) and the inflammatory cytokine il-6 in the pathogenesis of meigs ' syndrome. all 3 factors possess potent vascular permeability - enhancing properties, and have been associated with capillary leakage and the formation of ascites and pleural effusion in other gynecologic abnormalities, such as the ovarian hyperstimulation syndrome12) and ovarian cancer. concentrations of all 3 vasoactive factors were significantly higher in the ascites than in the serum. this suggests local rather than systemic secretion of these factors by the ovarian neoplasm mediates the hyperpermeability of the ovarian or peritoneal vasculature, with the subsequent transudation of fluid into the peritoneal cavity. the etiology of hydrothorax is also unclear, although the transfer of ascitic fluid via the transdiaphragmatic lymphatic channels is the current prevailing theory13, 14). although the highest levels of ca125 are seen in association with a pelvic malignancy, such as ovarian cancer with dissemination, there is no ca125 value 100% specific for ovarian cancer. elevated levels have been noted in various benign processes, such as endometriosis, pelvic inflammatory disease, pregnancy, ascites and benign ovarian tumors9, 15, 16). in our case, the elevated serum ca125 level was strongly suggestive of primary ovarian cancer, with the decline and normalization of the elevated levels after surgery confirming the association of raised ca125 levels with ovarian granulosa - cell tumors. due to the rarity of this disease, meigs ' syndrome caused by an ovarian granulosa cell tumor may be misdiagnosed as severe peritoneal and pleural dissemination. granulosa cell tumors are the most common sex cord - stromal tumors, and may be associated with endometrial hyperplasia and an endometrial carcinoma. in general, they tend to present with a stage i disease and are frequently associated with hormonal effects, such as precocious puberty, amenorrhea, postmenopausal bleeding or virilizing symptoms. the surgical staging of sex cord - stromal tumors is the same as that for epithelial ovarian cancers, with their surgical management based on the tumor stage and age of the patient17). in women who have completed childbearing, the surgery should be more aggressive, including a tah, with a bso and the standard surgical staging. women older than age 40 at diagnosis are more likely to experience a recurrence of granulosa cell tumors, which is why adjuvant therapy is recommended older populations, although definitive evidence for its efficacy in preventing or delaying recurrences is lacking. patients with an advanced stage disease (i.e., stage ii to iv) may benefit from additional therapy, with cisplatin - based combination chemotherapy being the most frequently used treatment18, 19). eighty percent of granulosa cell tumors were diagnosed as stage i, according to figo staging system. the 10 year survival rate after a recurrence was 56.8%, and the mitotic rate, tumor stage and residual tumor disease were associated with a poor prognosis20). in our case, with an age of 69-years and an ic stage, cisplatin - based adjuvant chemotherapy was performed due to the rupturing of the ovarian tumors in pelvic cavity during the operation. in conclusion, an intrapelvic tumor, with ascites, pleural effusion and elevated serum ca125, should suggest a malignant ovarian tumor, but if both the ascites and pleural effusion cytology are negative, the diagnosis and treatment of meigs ' syndrome is required. clinicians should be aware that an ovarian granulosa cell tumor may cause meigs ' syndrome, and that resection of the ovarian lesions can improve the prognosis. | herein, a rare case of ovarian granulosa cell tumor, presenting as meigs ' syndrome, with elevated carbohydrate antigen 125 (ca125), is reported. a 69-year - old woman was admitted for the investigation of abdominal fullness and dyspnea. a preoperative examination revealed a huge pelvic tumor and an abdominopelvic magnetic resonance image (mri) assumed ovarian cancer. a chest computed tomography (ct) scan revealed pleural effusion. a laparotomy confirmed the huge mass to be an ovarian tumor. a total abdominal hysterectomy (tah), with a bilateral salpingo - oophorectomy (bso) and partial omentectomy, was performed. although short - term intrathoracic drainage was required, the hydrothorax and ascites rapidly resolved in the postoperative period. |
incidence and mortality of neonatal - acquired brain damage, especially hypoxic - ischemic brain damage (hibd), have an increasing trend. brain damage of prematurity is the predominant form of the acquired brain damage in neonates who undergo neurological morbidity. administration of the recombinant human - erythropoietin (rh - epo) into extremely preterm infants at neonatal intensive care unit improved neurodevelopmental outcomes. however, the underlying mechanisms that support the neuroprotective effects of the rh - epo following premature brain damage remain unclear. the present study therefore used an animal model to investigate the role of rh - epo in enhanced neurological recovery, which was thought to be related to the promotion of angiogenesis. some in vitro experiments suggest that epo enhances vascular endothelial growth factor (vegf) secretion in neural progenitor cells through the activation of phosphatidylinositol 3 kinase (pi3k)/akt signaling pathway. the neural progenitor cells, treated with the rh - epo, upregulate vascular endothelial growth factor receptor 2 (vegfr2) expression in the cerebral endothelial cells (ecs). in addition, the pi3k / akt signaling also mediates angiogenesis and vegf expression in the ecs. wang. found that the rh - epo increased the mobilization of endothelial progenitor cells (epcs) and subsequent promotion of angiogenesis. based on these studies, we hypothesized that : (1) rh - epo may increase the amount of epcs or ecs in the hypoxia - ischemic (hi) region of the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway by promoting neovascularization ; (2) rh - epo may induce the secretion of vegf in the hi region in the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway and then promote neovascularization through the vegf / vegfr system. to verify our hypothesis, we tested the phospho - akt, cd34, vegfr2 proteins (the surface markers for ecs and epcs), and vegf mrna after administration of the rh - epo. the rh - epo injection was obtained from zhongda hospital affiliated to southeast university (china). antibodies were obtained from the following sources : phospho - akt (ser 473, d9e) xp rabbit mab from cst (usa) ; rat cd34 antibody from r and d (usa) ; and anti - vegf receptor 2 antibody from abcam (uk). pregnant sprague - dawley rats were obtained from nanjing medical university of china and were allowed to deliver. the animals were double housed with food and water supply ad libitum at a temperature- and light - controlled environment (12-h light / dark cycle, daily humidity, and temperature monitoring). all the protocols were approved by the institutional animal care and use committee of southeast university. five - day - old (pd5) postnatal rats underwent permanent ligation of the right common carotid artery (cca). the infant rats were then exposed to hypoxia (94% n2, 6% o2) for 2 h as described by back. all the rat pups were randomized into five groups as follows : (1) control group (i) : without hi, rh - epo, and ly294002 (c19h17 no3 ; a pi3k inhibitor) ; (2) hi group (ii) : underwent permanent ligation of the right cca and exposed to hypoxia, but without rh - epo and ly294002 ; (3) hi + ly294002 group (iii) : underwent permanent ligation of the right cca ; exposed to hypoxia ; and given ly294002, without rh - epo ; (4) hi + rh - epo group (iv) : underwent permanent ligation of the right cca, exposed to hypoxia, and given rh - epo, without ly294002 ; (5) hi + rh - epo + ly294002 group (v) : underwent permanent ligation of the right cca ; exposed to hypoxia ; given rh - epo and ly294002. coronal sections (10 m) from the brains were cut using a freezing microtome (leica cm3050 ; leica instruments, germany). the slides were then baked for 1 h at 50c. after repairing antigen of the slides, the slides the slides were then incubated overnight at 4c with the primary antibody : anti - cd34 (10 g / ml), and then followed by treatment with appropriate secondary antibody for cd34 for 1 h at room temperature. each of the aforementioned steps was followed by 5-min rinses in pbs for three times. the nuclear staining procedures using dapi (abcam, uk) were the same as above. the cd34 protein (red) and nucleus (blue) were examined with an epifluorescence microscope (nikon, japan). three sections from each rat were taken from the right white matter region and the cells in the white matter were counted (per 400) in three areas by two blinded independent observers. protein concentration was determined by the bca protein assay kit (keygen biotech, china). homogenate protein (30 l) was heated for 5 min at 99c and then subjected to 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis gel. after electrophoresis, the proteins were electroblotted on the nitrocellulose (nc) membrane and blocked with 5% skim milk. the immobilized proteins were exposed to commercially available antibodies such as phospho - akt (1:2000), cd34 (0.1 g / ml), and vegfr2 (0.2 g / ml). specific proteins were detected with secondary antibody and visualized by high - sig ecl reagents (tanon, china). western blotting for the glyceraldehyde-3-phosphate dehydrogenase (gapdh, 1:1000 ; abcam, uk) was performed as loading control, and band densities were semi - quantified using image - j software (national institutes of health, bethesda, usa). total rna was extracted from the right brains of rats using trizol (invitrogen, california, usa), and cdna was synthesized using a kit (invitrogen). polymerase chain reaction (pcr) was performed using the sybr green opcr mix kit (invitrogen), and -actin was used as an internal control. the reverse transcription conditions were 10 min at 25c, 30 min at 42c, and 5 min at 85c. the amplification program consisted of activation at 94c for 30 s, followed by 45 amplification cycles, each consisting of 94c for 10 s, 60c for 12 s, and 72c for 30 s. primer pairs used for amplification were as follows : vegf, forward 5-aacgtcactatgcagatcatgc-3, reverse 5-ctccgctctgaacaaggct-3 ; -actin, forward 5-ctgaaccctaaggccaacc-3, and reverse 5-agcgcgtaaccctcatagat-3. data were analyzed using gel - pro32 analyzer (thermo fisher scientific, usa). the relative value of mrna = the absorbance value of objective fragment / the absorbance value of -actin. multiple comparisons were conducted using one - way analysis of variance (anova) followed by student newman a statistically significant difference between means was considered when p 0.05). group ii was higher than group i [p 0.05 ; figure 1b and 1c ]. group ii was a little higher than group i [p 0.05). group ii was higher than group i [p 0.05 ; figure 1b and 1c ]. group ii was a little higher than group i [p < 0.05 ; figure 1b and 1c ]. the trends of the cd34 and vegfr2 proteins in the five groups were consistent with the amount of phospho - akt in the right white matter of premature brains. two days after the whole operation, the trends of cd34 cells count in the five groups after the immunofluorescence experiments were consistent with results for the cd34 protein in the western blot assays [figure 2a and 2b ]. at the same time, 0.05 ; figure 2a and 2b ] and group iii [p < 0.05 ; figure 2a and 2b ]. expressions of cd34 cells and vegf in the right white matter of premature brain 2 days after the whole operation. (a) immunofluorescence staining for cd34 cells (red : cd34 ; blue : nucleus). group iv showing significant difference from other groups (p < 0.05) ; group iii showing significant difference from groups iv, v, and ii (p < 0.05) ; group ii showing significant difference from group i (p < 0.05). gapdh : glyceraldehyde-3-phosphate dehydrogenase ; vegf : vascular endothelial growth factor ; qrt - pcr : quantitative real - time - polymerase chain reaction. the vegf mrna level was assessed by quantitative real - time pcr during hi in the right white matter of premature brains. two days after the whole operation, the vegf mrna level in group iv was significantly higher than in the other four groups [p < 0.05 ; figure 2c ]. group iii was significantly lower than in the other four groups [p < 0.05 ; figure 2c ]. the exogenous rh - epo could have upregulated the expression of vegf through the pi3k / akt signaling pathway during the hi in the white matter of premature brains. group ii was a little higher than group i [p < 0.05 ; figure 2c ] and group iii [p < 0.05 ; figure 2c ]. administration of the rh - epo after ischemia, either peripherally or centrally injected, suggested beneficial effects on brain edema by delaying the neuronal death and resulted in functional improvement of neurons in neonatal animal models. the injection of rh - epo centrally is not a practical approach in clinical settings, and indeed, systemic delivery of the rh - epo has advantages in that it is universally available to the capillary endothelium and thus potentially present everywhere in the brain. in addition, the receptors of epo are extensively expressed in the mature brain, including hippocampus. a study showed that the peak time of administrating epo peripherally was 3 h in the brain tissue of rodents. based on this finding, in our experiments, we administered the rh - epo by intraperitoneal injection before hi (2 h). akt was originally identified by staal in 1987, which was also known as pkb. the akt is the downstream protein for the pi3k and can be activated by growth factors and other extracellular stimulators. ly294002 can inactive the sites (ser473/thr308) of the akt to inhibit its phosphorylation. in this study, the rh - epo increased the number of cd34 cells in the hi zone of rat brain through the pi3k / akt signaling pathway. moreover, the rh - epo induces epcs immigration into the hi zone and then the epcs proliferation and differentiation to form the new blood vessels. this physiological process is called vasculogenesis, whereby the new blood vessels are formed where no preexisting vessels were present. on the other hand, the ecs from the capillaries in the hi zone of brain proliferate and form the new blood vessels. this physiological process is called angiogenesis, which involves the growth and development of new blood vessels from preexisting vessels. the rh - epo may therefore regulate blood vessels growth through two mechanisms : vasculogenesis and angiogenesis. vegfr2 can combine with the vegf and can be activated to induce neovascularization. in this study, the expression of vegfr2 protein and vegf mrna levels were significantly upregulated by the rh - epo through the pi3k / akt pathway in the hi zone of the brain. first, this result indicated that the rh - epo could activate the vegf / vegfr2 signaling pathway through the pi3k / akt pathway. second, the increased expression of vegfr2 could combine with more vegf and then enhance neovascularization of the vegf. third, the vegfr2 is 100% expressed in the mature ecs but not in the epcs. our study has showed that the rh - epo could significantly stimulate the ecs proliferation by the pi3k / akt pathway in the hi brain. in our study, the protein levels of p - akt, cd34, vegfr2, and mrna level of vegf in the hi group were a little higher than in the same group. hence, we hypothesized that these changes were induced by the endogenous epo which was induced by hypoxia. the expressions of p - akt, cd34, vegfr2, and vegf in group v were all between group iii and group iv in our study. there may also be some other signaling pathways which were activated by the rh - epo that could not be inhibited by ly294002. moderate neovascularization is beneficial to the hi zone of premature brain which can provide enough oxygen and energy to the surrounding cells. this information offers hope for the potential neovascularization by these molecules for clinical benefits. in conclusion, our study has demonstrated that the pi3k / akt signaling pathway is one of the essential mechanisms for neovascularization of rh - epo. the rh - epo induces cd34 cell immigration into the hypoxic and ischemic zone of premature rat brain and also upregulates the expression of vegfr2 protein and vegf mrna level through the pi3k / akt signaling pathway. this work was supported by a grant from the national natural science foundation of china (no. this work was supported by a grant from the national natural science foundation of china (no. | background : recombinant human - erythropoietin (rh - epo) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti - inflammation. in the present study, we found that the rh - epo was related to the promotion of neovascularization. our aim was to investigate whether rh - epo augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (pi3k)/protein kinase b (akt) signaling pathway.methods:postnatal day 5 (pd5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. all the rat pups were randomized into five groups as follows : (1) control group ; (2) hypoxia - ischemic (hi) group ; (3) hi + ly294002 group ; (4) hi + rh - epo group ; and (5) hi + rh - epo + ly294002 group. the phospho - akt protein was tested 90 min after the whole operation, and cd34, vascular endothelial growth factor receptor 2 (vegfr2), and vascular endothelial growth factor (vegf) were also tested 2 days after the whole operation.results:in the hypoxic and ischemic zone of the premature rat brain, the rh - epo induced cd34 + cells to immigrate to the hi brain zone (p < 0.05) and also upregulated the vegfr2 protein expression (p < 0.05) and vegf mrna level (p < 0.05) through the pi3k / akt (p < 0.05) signaling pathway when compared with other groups.conclusions:the rh - epo treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the pi3k / akt signaling pathway. besides, the endogenous epo may exist in the hi zone of rat brain and also has neovascularization function through the pi3k / akt signaling pathway. |
nanotechnology was incorporated in an estimated us$82 billion worth of manufactured goods in 2008, according to currall 1 and appeared in over 800 consumer products on the market.2 pre - global financial crisis estimates were that nanotechnology would be in us$2.6 trillion worth of goods by 2014.3 yet despite this rapid uptake and the undoubted major impacts that nanotechnology is going to have on our lives, the general public appear to know very little about it. the risk of course, is obvious, that the public may react to the rapid proliferation of nanotechnology the way they reacted to gm crops, and a large consumer backlash against nanoproducts would be understandable if product developers were not developing products that aligned with public needs or values. however, as has been pointed out by kahan, slovic, braman, gastil and choen, not much more is known about public perception of the risks of nanotechnology than is known about nanotechnology risks themselves.4 the answer in many countries is to try and match rapid product development with increased public understanding and awareness of nanotechnology. but this might be only half the answer, as improved understanding of what the public actually know and think about nanotechnology should also inform public policy development and product development. there are, of course, many advocacy groups who are willing to state what they believe the public feel about nanotechnology, whether that be highly supportive of or highly concerned about the technology. for example, friends of the earth s out of the laboratory and on to our plates, publication of 2008, states, early studies of public opinion show that given the ongoing scientific uncertainty about the safety of manufactured nanomaterials in food additives, ingredients and packaging, people do not want to eat nanofoods.5 (this was repeated morning television program nine am with david and kim, on 18 august 2008). this may or may not be true, but with no reference to the surveys it is hard to know. but the best method for really knowing what the public think is to ask them. the risk of not doing so, or presuming to have a good feel for the public s attitudes, without actually undertaking quality research can be demonstrated, once again, by the gm debate. public sentiments to new technologies can be pivotal in shaping the direction and pace of scientific activity.6 two studies conducted in the usa and the european union clearly showed that the perception that a majority of industry and public policy developers had about what the public thought about gm foods and crops did not align with what the public actually thought.7 another problem is undertaking over - simplified polling and holding it up as a valid explanation of complex attitudes. but as much as the general public tends to be attracted to attitudes that mirror their values, so are advocacy groups attracted to polls that mirror their attitudes. it is no surprise then to find industry groups looking for poll results that demonstrate widespread consumer support for nanotechnology products, and ngos looking for poll results that show consumer concerns about nanotechnology. the reality is that both are a part of the public s attitudes towards nanotechnology, but more in - depth study and analysis of public attitudes needs to be undertaken to better inform policy and product development. nature nanotechnology listed 12 significant survey studies of public perceptions of nanotechnology in february 2009, with the overall conclusion that while literature on public perceptions is becoming more mature and rigorous, there is still a need to understand public attitudes better, as scientists, policymakers and business will therefore be better positioned to anticipate trends that will dictate how the public might react to new scientific developments or products.8 a few of the studies are worth commenting on in a little more detail, for their impact both in what they found or how they were reported. one us study compared the attitudes of the general public with the attitudes of scientists, and interestingly was widely reported as finding that nano - scientists were more concerned about nanotechnology than the general public,9 while what the study actually found was that on some issues the scientists were more concerned, and on some the public were more concerned. across most indicators, the public perceived more benefits than scientists, in relation to topics such as better treatment of diseases, a cleaner environment and a solution of energy problems. however, when risks were addressed the public generally had much higher perception of risks than the scientists, across issues such as loss of privacy, use of the technology by terrorists and loss of jobs. also of note, the most divergent perceptions were for job losses, which had almost a 40% perceived risk by the public and only 5% perceived risk by the scientists. on two issues, however, the nano - scientists had a higher perception of risk. these were more pollution and new health problems.10 a study conducted by the woodrow wilson international center for scholars project on emerging nanotechnologies, addressed the impact of information on attitudes and found that the more information people obtain about nanotechnology, the more concerned they tend to become. however this had to be understood in terms of risk perceptions being largely affect driven, and exposure to information leading to a general polarization along cultural and political lines.11 a follow up study to examine the cultural predispositions towards technology and environmental risks found that general unattributed advocacy tended to polarise beliefs, with significant racial and cultural indicators. they study also found that the gap between people who are generally inclined to credit and those inclined to dismiss claims of environmental risk widens dramatically after exposure to such arguments. biased assimilation was a key determinant of attitude, meaning that people tend to credit and dismiss arguments about nanotechnology in patterns that reflect their cultural predispositions towards risk. in practice this means that people assign greater credibility to the opinions of those whose values they share.12 the department of innovation, industry, science and research s australian office of nanotechnology commissioned studies into public attitudes towards nanotechnology in 2005, 2007 and 2008, detailed in market attitude research services.13 each study, conducted by an independent market research company, and the methodology employed was random representative sampling through telephone polling of over 1,000 people aged 18 years and over, seeking their responses to a broad range of questions, seeking to understand both attitudes to risks and benefits and different applications. the findings of the three studies show that public has very high expectations of nanotechnology, concerns are only moderate, while knowledge and awareness are rather low. but to hold these up as the key findings does not do justice to what can be determined by more detailed analysis, which shows these generalisations are often nuanced by attitudes to specific applications. it is crucial to take public opinion polling to this next level of detail to better understand its implications. interestingly, while discussions of public attitudes towards new technologies need to generally address distinctive demographics differences of attitudes, whether by age, gender or education, breaking down the public into different demographic publics showed very little major differences, so in this instance it is reasonably accurate to refer to the public. in 2008, 86% of the surveyed population stated they were excited or hopeful about nanotechnology, and believed strongly that it would improve the quality of life in australia, would have a positive impact on employment and the economy, and would provide great strides in qualitative comments expressed by people indicated that key areas of science and technology driving the average person s interest were towards particular applications that were seen to have clear benefits. these included : medical treatment advances particularly cervical cancer vaccination;science s role in increasing the understanding and potential threat of climate change and potential solutions offered by renewable energy and biofuel technical developments ; andongoing advances in computer and internet technology. medical treatment advances particularly cervical cancer vaccination ; science s role in increasing the understanding and potential threat of climate change and potential solutions offered by renewable energy and biofuel technical developments ; and ongoing advances in computer and internet technology. as has been stated, while at the simplest level the survey findings could be used to support the claim that the australian public is strongly supportive of nanotechnologies, when attitudes to different applications are examined, this statement is shown to not always hold up. currall stated that examining specific applications as opposed to nanotechnology in general is an important direction for further public attitude studies.14 for some applications, such as specific medical technologies, support rose over the survey period. for instance, support for using nanotechnology to provide machines that can exist in the blood stream to clear arterial clots and cancer cells, rose from 90% in 2005 to 94% in 2008. using nanotechnology in filers that control pollutants from entering the environment had 96% support in 2005 and 95% in 2008, and technology that disassembled and broke down waste and garbage had 91% support in 2005 and 93% in 2008. however application that had lower support included using nanotechnology in protective suits against chemical or biological weapons (77% support in 2005 and 74% support 2008) and stain repellent fabrics (51% support in 2005 and 2008). the lowest levels of support, that rated below 50%, were for miniature surveillance devices (34% in 2008), computers in clothes or goods (31%) and changing nutrients in foods (32%). it is worth noting that attitudes to food showed the only significant change (49% support in 2005 to 32% in 2008), and there were clearly different attitudes towards using nanotechnology in food and in food packaging, with the applications of food packing that monitors condition being supported by 73% of respondents in 2008. the implications of this is that it is important for nanotechnology applications to align with public values such as public good, rather than more commercial ones. an issues in understanding public attitude polling on nanotechnology is whether people know what they are responding to, and qualitative comments indicated that outcomes of the application of the technology were a larger driver of attitudes to nanotechnology than attitudes to the technology itself. there is an adage that not knowing what something is does not stop people having strong attitudes towards it, which tends to be supported by the data. this can be very significant in understanding different response to different polls, and provides insight into attitudes to new technologies may not always be responding to the technology itself, and indicates that public discussions on the technology should focus primarily on its uses rather than how the technology works, to better align with public understandings. this is reinforced by data that while awareness of the term nanotechnology (when prompted) was found to be moderate (rising from 51% in 2005 to 66% awareness in 2008), specific detailed knowledge of what nanotechnology means was still low (at 8%). interestingly, us data shows not dissimilar levels of awareness. a 2008 study undertaken for the woodrow wilson international center for scholars project on emerging nanotechnologies, found that 49% had heard nothing at all about nanotechnology (compared to 38% in australia) and 26% had heard just a little (compared to 34% in australia).15 after testing initial awareness and knowledge of nanotechnology the following definition was provided to respondents in the surveys in an attempt to enable more informed attitudes through providing a common understanding:nanotechnology is science at a very small scale ; and refers to a new array of devices and materials whose key parts are less than 10 nanometers, about 10,000 times smaller than the width of a human hair. working at a scale a million times smaller than a pinhead allows science researchers to tune material properties at the atomic level, making them behave in different ways to create new materials and products.16 nanotechnology is science at a very small scale ; and refers to a new array of devices and materials whose key parts are less than 10 nanometers, about 10,000 times smaller than the width of a human hair. working at a scale a million times smaller than a pinhead allows science researchers to tune material properties at the atomic level, making them behave in different ways to create new materials and products.16 there are many different definitions of nanotechnology available, of course, and it may be that a different definition elicited somewhat different response from this point on in the survey. many public attitude studies seek to define attitudes as a risk - benefit equation, which can provide a useful indicator of the relativeness of these two factors. between 2005 and 2008, the public 's perception of the benefits outweighing the risks increased (from 39% to 53%) and the perception of risks equalling benefits diminished (from 35% to 18%). in 2008 only three per cent perceived there were more risks than benefits, 18% perceived the risks and benefits might be equal and 26% were unsure. again, focussing solely on public perceptions orf risk, or benefit, does not provide the full picture. asked to articulate more detail on their perceived risks and benefits of nanotechnology the majority (64%) surveyed in the study expressed no concerns about nanotechnology. the mild concerns included : nanotechnology regulation and safeguards are not keeping up with the development of nanotechnology;because nanotechnology is so new there might be problems for public safety or worker safety ; andthe complexity of nanotechnology makes it difficult to understand. nanotechnology regulation and safeguards are not keeping up with the development of nanotechnology ; because nanotechnology is so new there might be problems for public safety or worker safety ; and the complexity of nanotechnology makes it difficult to understand. the only high concern expressed (28%) related to the use of nanotechnology in food. the implications of this could be further studies to determine the driver of this attitude and whether it was based more on any response to nanotechnology use or modification of foods. the 2008 us woodrow wilson study sought attitudes towards risks and benefits and then provided a definition of nanotechnology, discussing both risks and benefits and retested risk and benefits. the study found that after hearing information there was a 10 point increase in the proportion of respondents who felt that benefits would outweigh risks (from 20% to 30%) and there was a larger 16% increase from those who thought risks would outweigh benefits (7% to 23%) ; a 13 point increase in the proportion saying risks would be equal (25% to 38%) and large drop in those not sure (48% to 9%).17 this supports the assumption that may poll results are based on initial response that can change when the public is more informed. considering the low levels of awareness of nanotechnology in the public, this also suggests that attitudes are likely to change significantly as people become more informed. of interest to the public debate is the issue of trust, which can underpin the type of information being sought in a the study found that the majority of the australian community gave high levels of continued trust towards scientists (88% trust in 2008) to explain any risks associated with nanotechnology. government agencies and regulators (61% trust) and non - government organisations (ngo s) (at 64% trust) were also expected to provide a role of giving information to the australian community about nanotechnology, and to regulate and monitor the nanotechnology industry. however trust in manufacturers and distributors of consumer products was only 30% and trust in business leaders was 28% as to sources of information on nanotechnology, they were dominated by the media. however they were only rated as being trusted to tell of the risks of nanotechnology by 50%. television, newspapers and radio were cited as reliable sources for information by 80% of respondents. the internet was cited by 55% or respondents, and word of mouth by friends, family and colleagues was cited by 9%. this is also an area for more research, particularly comparing the accuracy of information being obtained from different media. the 2008 survey sought to probe deeper than the two previous surveys, by allowing for qualitative input from a subset of respondents, and new questioning explored issues where respondents could express opinions about different issues relating to nanotechnology. in relation to the adequacy of safety and testing of consumer products which use nanomaterials or are made using nanotechnology, people expressed views that were categorised as it is commonsense to test for any potential risks to consumer safety. specific comments included:i feel ok about this because consumer product testing always seems to be fairly thorough.18(female, aged 4049 years, disability pensioner, perthhopeful about nanotechnology)i have a small amount of concern. but only because it is new technology and the potential exists for unknown factors.19(male, 60 + years, retired, townsvillehopeful about nanotechnology) i feel ok about this because consumer product testing always seems to be fairly thorough.18 (female, aged 4049 years, disability pensioner, perthhopeful about nanotechnology) only because it is new technology and the potential exists for unknown factors.19 (male, 60 + years, retired, townsvillehopeful about nanotechnology) for the impact of nanoparticles entering the environment comments were expressed that any potential health risks should be assessed, and government agencies and manufacturers should keep the public informed. specific comments included:i do nt have any concerns because a lot of particles enter the environment anyway, and it is only when problems occur you need to worry.20(male, aged 5059 years, employed full - time, adelaidehopeful about nanotechnology)i am mildly concerned because i am not sure of the situation, except that it sounds possible that the particles could live in the environment and not break down.21(male, 60 + years, retired, adelaidehopeful about nanotechnology) i do nt have any concerns because a lot of particles enter the environment anyway, and it is only when problems occur you need to worry.20 (male, aged 5059 years, employed full - time, adelaidehopeful about nanotechnology) i am mildly concerned because i am not sure of the situation, except that it sounds possible that the particles could live in the environment and not break down.21 (male, 60 + years, retired, adelaidehopeful about nanotechnology) addressing nanotechnology use being largely self - regulated by those industries using nanotechnology most comments expressed the view that governing body regulation is needed and that specific comments included:i am not concerned at all because although industry has their own interest at heart, if industry does nt self regulate then they know the government will step in.22(female, aged 5059 years, employed part - time, sydneyexcited about nanotechnology)i am mildly concerned because of financial interests interfering with other interests such as public safety and wellbeing of the environment.23(male, 3039 years, employed full - time, adelaideexcited about nanotechnology)i am very concerned because of the motivation and pressure to reduce profits working against self regulation.24(female, 60 + years, retired, melbourne holds neutral view about nanotechnology) i am not concerned at all because although industry has their own interest at heart, if industry does nt self regulate then they know the government will step in.22 (female, aged 5059 years, employed part - time, sydneyexcited about nanotechnology) i am mildly concerned because of financial interests interfering with other interests such as public safety and wellbeing of the environment.23 (male, 3039 years, employed full - time, adelaideexcited about nanotechnology) i am very concerned because of the motivation and pressure to reduce profits working against self regulation.24 (female, 60 + years, retired, melbourne holds neutral view about nanotechnology) on the issue of workers and researchers involved with nanotechnology being potentially exposed to nanoparticles comments expressed were related to the view that any potential health risks need to be addressed by relevant authorities. specific comments included:regulations will have safety nets, therefore, i am not concerned as long as industry does not self regulate.25(male, aged 4049 years, employed full - time, sydneyhopeful about nanotechnology)i am only mildly concerned. health effects always need monitoring to ensure no genetic impacts on offspring and future generations.26(female, 4049 years, employed full - time, brisbanehopeful about nanotechnology) regulations will have safety nets, therefore, i am not concerned as long as industry does not self regulate.25 (male, aged 4049 years, employed full - time, sydneyhopeful about nanotechnology) i am only mildly concerned. health effects always need monitoring to ensure no genetic impacts on offspring and future generations.26 (female, 4049 years, employed full - time, brisbanehopeful about nanotechnology) qualitative comments tended to focus on issues of safety and trust to regulate the technology, and demonstrated that when discussing concerns there were clearly large variations that could not be accurately summarised by simple for and against polling. the qualitative polling also provided insights into some of the values that drove attitudes, as well as factors that would mitigate concerns or increase them. however i should also be pointed out that despite many respondents focussing often on concerns, the market research company mars noted that at the completion of the above intensive qualitative interviews respondents mostly retained their positive view of nanotechnology or strengthened their positive view about nanotechnology. the last question put to respondents was:finally, and again thank you for your thoughts, i just want to touch base with you on your final thoughts about nanotechnology based on our discussion today. overall, what now best describes how you feel about the potential implications of nanotechnology ? finally, and again thank you for your thoughts, i just want to touch base with you on your final thoughts about nanotechnology based on our discussion today. overall, what now best describes how you feel about the potential implications of nanotechnology ? the study found that while 86% had stated that they were excited or hopeful about nanotechnology during the initial questions of the survey, following the intensive follow - up interview most people strengthened positive views held about nanotechnology, with this figure rising to 92%. this might seem, at first glance, contradictory to the finding that the more people learn about new technologies the more concerned they become, which is reinforced the 2008 woodrow wilson study cited earlier, but that may be based on confusing information giving with two - way engagement. while the survey conducted for the australian office of nanotechnology provides interesting tracking data on what the public think about nanotechnology, with the top line findings that perceptions of benefits far outweigh risks, one of the key findings of the study is that after having their attitudes sought people tended to have higher support for nanotechnology. it is important that any policy formulation is based on more than top - line data, and the study showed that there are concerns relating to specific uses of nanotechnology, especially in foods, and that different applications evoke markedly different attitudes. ongoing public attitude studies should therefore continue to seek better quality data, through further engagement with the public, particularly through more two - way engagement, not just in enabling the public to have their say on issues relating to nanotechnology, but in enabling policy and technology developers to have a better sense of understanding of the public s needs and desires, and better match them with nanotechnology developments. | public debate on nanotechnology is a large topic within governments, research agencies, industry and non - government organisations. but depending who you talk to the perception of what the public thinks about nanotechnology can be very varied. to define coherent policy and to invest in research and development that aligns with public preferences, needs more than just perceptions of public perceptions. public attitude studies are vital in understanding what the public really think, but they need to go further than simplistic polling and should examine factors such as changes over time, drivers of attitude change, different attitudes to different applications and be supported by qualitative data. this paper summarises the findings of a three year tracking study of public attitudes towards nanotechnology, highlighting both concerns and aspirations for nanotechnology and discusses the impacts of that data on public engagement programs. |
robust data assessing the value of procalcitonin (pct) for monitoring treatment response in abdominal sepsis are rare, so the study of jung and colleagues published in the previous issue of critical care is most welcome. they concluded that a decrease of pct to 0.5 ng / ml lacked sensitivity to predict treatment response and a decrease of at least 80% from its peak failed to accurately predict treatment response. the value of the study is limited by the small number of patients included, the single - center approach and its observational character. nevertheless, it is to date among the best available evidence we have for these critically ill patients. international databases show that one in four cases of severe sepsis or septic shock is caused by intra - abdominal infection. abdominal sepsis is not just a single disease, but comprises a group of different entities. almost 90% of all intra - abdominal infections are so - called secondary peritonitis and require a primarily surgical approach (87% of patients had surgical intervention in the study of jung and colleagues). secondary peritonitis consists of community acquired and postoperative nosocomial forms, the latter one following a previous surgical intervention (anastomotic insufficiency following anterior rectum resection). tertiary nosocomial peritonitis is a persistent intra - abdominal infection without a surgically treatable focus, but this point is difficult to assess without an uncontributive reoperation proving that the patient indeed has a tertiary peritonitis. primary inadequate and inappropriate antibiotic regimens for both forms of nosocomial peritonitis are associated with substantially worse prognostic outcome for patients with intra - abdominal infections and result in substantial increases in health care costs. the difficulties associated with abdominal sepsis are complicated by uncertainty about surgical control of the source of the sepsis. do we have any reliable parameters that enable us to decide whether to perform a relaparotomy or not (in the jung and colleagues study about 20 out of 101 patients required a relaparotomy) ? an analysis of all investigated markers, including pct, failed to detect specific parameters that can be used under these difficult conditions. current guidelines recommend administration of broad - spectrum antimicrobials within 1 hour of the diagnosis of severe sepsis or septic shock. this recommendation is based on the evidence that delaying antimicrobial therapy in patients with sepsis- related hypotension is associated with increased mortality. pct has been evaluated over recent years as to whether it can be used to detect the presence of different types of infection, treatment failure or adverse outcome. the promising initial publications reporting the results of using serial serum pct concentrations to guide duration of antibiotic therapy in patients with community - acquired pneumonia have only partially been confirmed in critically ill patients. a recent randomized multicenter trial enrolling critically ill patients with mainly respiratory tract infection showed that duration of antibiotic therapy can be reduced by a mean of 2.7 days without impact on mortality. but unfortunately, pct had no influence on the duration of antibiotic therapy in patients with intra - abdominal infections. smaller randomized studies show that significant pct - guided reduction of antibiotic exposure can safely be accomplished in patients with severe sepsis and septic shock and in surgical patients with sepsis. more recently, it has been suggested that pct may be of value as a prognostic marker. daily measurements of pct in a general icu population in a recent large multicenter randomized study were associated with increased use of antibiotics and duration of mechanical ventilation. this commentary has been written by an abdominal surgeon and an intensivist, representing the two disciplines most frequently involved in the treatment of an important subgroup of critically ill patients. in this war against the high mortality from abdominal sepsis, surgeons and intensivists are brothers in arms. the study of jung and colleagues indicates that pct is still a valuable weapon in this war - but far from being the magic bullet. | the ideal management of infection includes not only the early identification and start of effective therapy but also the correct categorization of non - infected patients in order to avoid unnecessary use of antimicrobials. the availability of a specific and sensitive test for the presence of infection is of paramount importance to improve the prudent use of antimicrobial therapy. procalcitonin (pct) has been evaluated over recent years as to whether it can be used to detect the presence of different types of infection, allows reduced duration of antibiotic therapy, or predicts treatment failure or adverse outcome. in the previous issue of critical care, jung and colleagues report about the monitoring of treatment response in abdominal sepsis by repetitive determination of pct. |
increased pollution from rapid industrialization and increases in the smoking rate are gradually focusing public attention on respiratory disorders. breathing, the major function of the lung, is the process that alternately performs inspiration and expiration with gas exchange that is essential for humans life1. deteriorating lung function is a major cause of death among south koreans, and the number of patients with poor lung function is increasing2. managing lung function can improve dyspnea and enhance quality of life3, consequently public attention to respiratory physiotherapy is increasing. in respiratory physiotherapy, the evaluation of pulmonary function is performed to assess the lung s mechanical functions, volume, and capacity4. specifically, peak expiratory flow (pef), forced vital capacity (fvc), forced expiratory volume in 1 second (fev1), fev1/fvc, and forced expiratory flow between 25 and 75% of vital capacity (fef2575%) are related to the degree of disability and short - term and long - term prognoses in a variety of respiratory diseases and are frequently used as assessment tools5. in addition, maximal inspiratory pressure (mip) and maximal expiratory pressure (mep) are performed to evaluate pulmonary strength. these are known to be useful clinical indicators of the natural progress of chronic obstructive pulmonary disease (copd) patients6. various human organs, including the lung, exhibit circadian rhythms in which physiological functions are controlled according to a specific cycle. recently, postural control has been reported to be related to diurnal variation9, and diurnal variations exist in respiration10. the diurnal variation in respiration is known as the morning dip phenomenon, and evaluation of breathing at 4:00 pm gives the highest values, while the lowest values are measured in the morning11. a review of previous studies of the diurnal variations of pulmonary evaluation suggests conflicting viewpoints. hetzel12 reported changes in pulmonary function and pulmonary strength with time, whereas aguilar.13 reported that pulmonary function and pulmonary strength showed no statistically significant changes with time. the purpose of this study was to identify the changes in pulmonary function and pulmonary strength associated with time of day. the subjects were 20 healthy adults (11 men, 9 women) who had no cardiopulmonary - related diseases. the mean age, mean height, and mean weight of the subjects were 23.553.09 years, 169.909.61 cm and 64.4013.48 kg, respectively. the subjects were explained the purpose of this study and they voluntarily signed informed consent forms before participation in this study. this study obtained the approval of the bioethics committee of catholic university of pusan (cupirb-2014 - 010). the subjects pulmonary function and pulmonary strength were evaluated at three times, 9:00 am, 1:00 pm, and 5:00 pm, based on the hospital s working environment. the tests were performed at least 1 hour after the subjects had eaten a meal. pulmonary function was evaluated using microlab (micro medical ltd., uk). each subject was seated and looked straight ahead with the mouthpiece of the measurement device inserted in the mouth and a nose clip fixed on the nose. pulmonary strength was evaluated using microrpm (micro medical ltd., uk) to measure mip and mep. the mip and mep were measured while the subjects were seated and looked straight ahead with the mouthpiece of the measurement device inserted in the mouth. inhalation and exhalation were repeated three times, and the highest value was selected. if differences of more than 10% were found among the measured values, these values were excluded. the data collected during the process were encoded and analyzed using spss for windows ver. in addition, repeated measures analysis of variance (avona) was conducted to compare the differences in pulmonary function and pulmonary strength and contrast tests was used to compare between times of day. the results of pulmonary function at the different times of day are shown in table 1table 1.the changes of pulmonary function with time of day9:00 am1:00 pm5:00 pmfvc3.450.123.550.133.600.12fev1 3.150.103.350.103.450.09fef25754.400.224.500.194.450.18unit=. : statistically significant (p<0.05). fvc and fef2575% showed no statistically significant differences among the different times of day. the results of pulmonary strength at the different times of day are shown in table 2table 2.the changes of pulmonary strength with time of day9:00 am1:00 pm5:00 pmmip72.04.373.43.976.54.4mep74.35.774.44.676.94.6unit = cmh2o. different superscripts in a row indicate a significant difference.. mip and mep showed no statistically significant differences among the different times of day, but comparative testing of each variable found statistically significant differences between measurements taken at 9:00 am and 5:00 pm. human diurnal variations are controlled by the suprachiasmatic nucleus located in the anterior hypothalamus, which serves the role of the main circadian pacemaker that controls almost all human organs and behaviors15, 16. bagg and hughes11 reported that diurnal variations exist in the peak expiratory flow (pef) : the highest value was observed at 4:00 pm and the lowest value was observed in the morning, the morning dip phenomenon. their results displayed the morning dip phenomenon with significantly reduced values being observed in the morning. in the present study, the morning dip phenomenon could be observed. they did not tabulate the numerical values of their results, thereby limiting comparisons with our study. medarov.18 found that when fvc and fev1 were values measured in the afternoon they showed were the highest values. the difference in fvc between the highest and the lowest values was 11.9%, and the difference in fev1 between the highest and the lowest values was 15.7%. in the present study, fvc and fev1 measured in the morning were about 4.2% and 8.7% less than their respective values measured in the afternoon. the fef2575% measured in the morning was about 1.1% less than that measured in the afternoon, also displaying the morning dip phenomenon. teramoto.17 reported statistically significant diurnal variations in mip and mep, but, as mentioned above, they did not tabulate the numerical values of their results, thereby limiting comparisons with our study.. the mip and mep values in the morning were a little higher than those measured in the afternoon. while their study reported statistically significant differences, the small subject number limits its generalizability. aguilar.13 measured the diurnal variations of mip and mep of healthy adults for 12 hours. mip decreased about 4.6%, mep increased around 1.3% during the day, and mep exhibited the morning dip phenomenon. in the present study, mip and mep measured in the morning were respectively about 5.9% and 3.3% less than the mip and mep measured in the afternoon. thus, they demonstrated the morning dip phenomenon, but the differences were not statistically significant. the results of the present study demonstrate that pulmonary function and pulmonary strength show changes with time of day, confirming the morning dip phenomenon, though the differences were small. although the differences were small, the morning dip phenomenon appeared throughout the experiment, sometimes showing statistically significant difference. therefore, we consider the time of measurement is a matter of clinical concern. this study was performed with healthy asian adults in their 20s as subjects ; therefore, the results can not be generalized to the elderly or patients with lung disorders. some studies have reported that pulmonary evaluation measurements are influenced by gender, ethnicity, age, and height, and thus these factors should be taken into account in testing5, 20. follow - up studies should be conducted considering other factors, such as the age and disease of patients. | [purpose ] the purpose of this study was to identify changes in pulmonary function and pulmonary strength according to time of day. [subjects and methods ] the subjects were 20 healthy adults who had no cardiopulmonary - related diseases. pulmonary function and pulmonary strength tests were performed on the same subjects at 9:00 am, 1:00 pm, and 5:00 pm. the pulmonary function tests included forced vital capacity (fvc), forced expiratory volume in 1 second (fev1), and forced expiratory flow between 25 and 75% of vital capacity (fef2575%). pulmonary strength tests assessed maximal inspiratory pressure (mip) and maximal expiratory pressure (mep). [results ] fev1 showed statistically significant differences according to time of day. other pulmonary function and pulmonary strength tests revealed no statistical differences in diurnal variations. [conclusion ] our findings indicate that pulmonary function and pulmonary strength tests should be assessed considering the time of day and the morning dip phenomenon. |
the prevalence of intestinal parasitic infection is high in sub - saharan africa, where the majority of hiv / aids cases are from [2, 3 ]. diarrhea causing opportunistic parasites like cryptosporidium parvum and isospora belli is common among hiv positive persons with cd4 count less than 200 cells/l [4, 5 ]. antiretroviral treatment (art) increases the length and quality of life and productivity of patients by improving survival and decreasing the incidence of opportunistic infections in people with hiv through reduction of the viral load and increasing the level of cd4 cells. nevertheless, in ethiopia, few studies have tried to investigate the extent of intestinal parasitic infections in relation to art experiences and cd4 count [4, 5 ]. therefore, we investigated the prevalence of intestinal parasites among hiv positive persons who were naive and who were on art in hiwot fana specialized university hospital, eastern ethiopia. the study was conducted in hiwot fana specialized university hospital, harar, ethiopia. the hospital is found in harari national regional state which is one of the federal democratic republic of ethiopia regional states located 515 km from addis ababa. in harar a comparative cross - sectional study was conducted among naive and on art hiv positive persons in hiwot fana specialized university hospital from march to april, 2011. the study population was all the hiv positive individuals who were on art and pre art care in the art unit of the hospital during the study period. individuals who were on parasitic treatments during the data collection period were excluded. the prevalence of intestinal parasites among art - naive group hiv positive individuals was taken as 50% and to detect a difference of 15% between the two groups with the assumption of 95% confidence level (cl), power of 80% (0.84) and ratio 2 : 1 of those on art and art - naive group. double proportion formula was applied to calculate the sample size which was 274 on art and 137 art - naive group. small pieces of labeled clean plastic sheets and wooden applicator sticks were distributed and the participants were instructed to bring sizable stool specimen of their own. with the provision of specimen, then, it was examined by direct wet mount method using normal saline (0.85% nacl solution) at hiwot fana specialized university hospital laboratory in order to prevent the loss of motile stage of parasites. the remaining sample was preserved with 10% formalin and transported to the laboratory of medical laboratory sciences department haramaya university. in the laboratory, it was examined by formol ether concentration technique and modified zeihl - neelsen method (for detection of opportunistic parasites cryptosporidium species, cyclospora cayetanensis, and isospora belli). the most recent cd4 t - cells counts of the participants were obtained from their art fellow - up record in the hospital. data were entered into epideta version 3.1 and transported to spss version 16 software for analysis. the prevalence of intestinal parasites was determined in relation to different variables pearson 's chi square test was used to assess statistical significance difference between proportions. a statistical test result was reported as significant when its p value was less than 0.05. ethical clearance was obtained from haramaya university colleges of health and medical sciences institutional research and ethical review committee. all the participants were explained about the purpose and their right to participate or not to participate in this study. those who gave their written consent participated in this study. those participants who were found positive for intestinal parasitic infection were treated free of charge using the standard drugs by nurses in art unit. a total of 259 on art and 112 art - naive group hiv positive clients participated in the study, and the response rate was 86.7%. the mean age of the participants was 33.6 (sd 10.04) and many of the art - naive group (68.5%) and on art (70%) participants were female. the majority of the study subjects were in the age group of 2534, married, daily laborers, and urban dwellers (table 1). the overall prevalence of intestinal parasites among the study participants was 33.7% (125/371). it was significantly higher in the art - naive group (45.5% (51/112)) compared to those on art (28.6% (74/259)) (p = 0.002). some were infected with nonopportunistic (31.8%) and very few with opportunistic (3.5%) parasites. eleven parasite species were detected : entamoeba histolytica / entamoeba dispar (13.4% on art - naive group and 13.5% on art group (p > 0.05)) and giardia lamblia (7.1% on art - naive group and 8.5% on art group) were the common ones. opportunistic parasites like crypotospriudum (7.1%) and isospora belli (4.5%) were found only in art - naive group group. some of the art - naive group (26.8%) and few of the on art (17.4%) subjects were diarrheic (p 0.05), crypotospriudum species, i. belli, and s. stercolaris. the overall prevalence of intestinal parasites was significantly higher among diarrheic (53.3% (40/75)) as compared to nondiarrheic (28.7% (85/296)) study participants (p < 0.05) (table 2). about 31% (31/100), 17.3% (34/196), and 13.3% (10/75) of the study participants with cd4 count less than 200 cells/l, 200499 cells/l and greater than or equal to 500 cells/l, respectively, were diarrheic. the prevalence of diarrhea is significant in participants with cd4 count less than 200 cells/l (p = 0.005). in the bivariate analysis, age, sex, occupation, residence, education, and marital status of the participants did not show significant association with prevalence of intestinal parasites. participants with cd4 count less than 200 cells/l (cor : 1.93 ; ci : 1.03, 3.61), diarrheic (cor : 2.84 ; ci : 1.69, 4.77), and art - naive group (cor : 2.09 ; ci : 1.32, 3.31) had higher risk of intestinal parasites. the participants with cd4 count < 200 cells/l were 2.07 times more likely to be infected with intestinal parasites than those with cd4 count 500 cells/l. the diarrheic participants and the art - naive group ones were 2.30 and 2.60 times more likely to be infected with intestinal parasites (table 3). intestinal parasitic infections are the major causes of morbidity and mortality in hiv positive patients [47, 10 ]. in this study, about 33.7% of the participants were infected with intestinal parasites. the result is almost similar to the ones reported from afar, ethiopia, cameroon, and saudi arabia, but lower than those from jimma, hawassa, bahir dar, dire dawa and afar of ethiopia, india, kenya, and jakarta. the difference in the prevalence might be due to differences in geographical location, sensitivity of diagnostic techniques, study participants ' immunity status, environmental hygiene, socioeconomic status, access to safe water supply, or other. several species of protozoa and other intestinal parasites have been associated with acute and chronic diarrhea and even weight loss in hiv / aids patients [19, 20 ]. in this study, entamoeba histolytica / e. but it is higher than a report in ethiopia (4.2%), saudi arabia (5.2%), and jakarta (0.3%). it is lower than the one in kenya (58.3%) and ethiopia (23.8%). however, it is higher than report of 1.1% in ethiopia, 0.6% in senegal, and 1.9% in jakarta. it is also lower than report of 16.6% in kenya and 16% in ethiopia. the prevalence of crypotospriudum species was 2.2% and this is lower than the findings from other studies which are found in the range of 4.9% to 15.8% [5, 1215, 17 ]. while isospora belli was found at 1.3%. this is lower than other studies which are found in the range of 3.9% to 11.7% [5, 1315 ]. the lower prevalence of both parasites in this study might be due to that our study participants are in the art care who were taking art and/or treatment for opportunistic infection. the other reason might be due to difference in immunity, diarrheic status, environmental and personal hygiene of the study participants. other nonopportunistic intestinal helimiths were identified at the rate of ranging from 0.5% to 4.0%. this is similar to study carried out in ethiopia, but it is slightly lower than another study in ethiopia and saudi arabia. the effect of strongyloides stercoralis in hiv infected patients, which is disseminated strongloidiasis, was reported in another study. in this study, we tried to compare the prevalence of intestinal parasites with the diarrheic status, cd4 count, and art experience of hiv positive persons. the prevalence of intestinal parasites was significantly higher among diarrheic as compared to nondiarrheic groups. the most common diarrheal causing parasites were e. histolytica, gardia lamblia, crypotospriudum species, isospora belli, and strongyloides stercoralis. similarly, there are reports which showed that diarrhea can be caused by opportunistic and nonopportunistic parasites [5, 15, 22 ]. the association between opportunistic parasitic infection and hiv is widely reported [4, 15, 23 ]. however, in this study, most of the parasites identified were nonopportunistic. the relationship between nonopportunistic parasite and hiv was not well established. even though the defense against them might be damaged by hiv, the exposure to this parasites are likely to occur independent of hiv infection but heavier parasitic load might accumulate as well as experience of delayed clearance of parasite in individual with concurrent hiv induced immunosupperssion [23, 24 ]. the prevalence of intestinal parasites was highly significant among those study participants with cd4 count < 200 cells/l in this study. the correlation of cd4 count with opportunistic parasites could not be assessed because of small number of individuals infected with crypotospriudum species and isospora belli. however, the association of these two parasites with hiv positive persons, who have cd4 count < 200 cells/l, is reported in other studies [4, 5, 15, 23 ] the prevalence of intestinal parasites was significantly higher when art - naive group compared to those on art study participants. opportunistic parasites, which are crypotospriudum species and isospora belli, were found only in art - naive group group. art increases the immunity status hiv positive persons and decreases the incidence of opportunistic infections. the detection method for microsoporidia was not employed and sensitive diagnostic techniques for strongyloides stercoralis and enetrobius vermicularis were not used. therefore, the prevalence intestinal parasites in our study participant may have been underestimated. hiv negative control groups were not included in this study which was the other limitation. opportunistic and nonopportunistic parasites were identified with a different rate. the prevalence of intestinal parasites was higher among those hiv infected individuals with diarrhea, low cd4 count, and art - naive group groups. those results posit the need for considering early detection and treatment of intestinal parasites in hiv infected individuals in order to reduce their morbidity. this seeks great attention by those clinical service providers who are working in the art unit. adherence counseling of art, health information dissemination on environmental, and personal hygiene should also be given to hiv / aids patients. in addition further large scale study by using different diagnostic techniques, hiv negative control and assessing predisposing factors of intestinal parasites is recommended. | background. intestinal parasitic infection affects the health and quality of life of people living with hiv. this study was aimed to determine the prevalence of intestinal parasites among hiv positive individuals who are naive and who are on antiretroviral treatment (art) in hiwot fana specialized university hospital, eastern ethiopia. methods. a comparative cross - sectional study was conducted on 371 (112 art - naive group and 259 on art) hiv positive individuals. stool specimens were collected and examined by direct wet mount, formol ether concentration technique, and modified ziehl - neelsen methods. results. the overall prevalence of intestinal parasitic infections was 33.7% ; it was significantly higher among the study participants who were art - naive group (45.5%) (aor : 2.60(1.56,4.34)) and diarrheic (53.3%) (aor : 2.30(1.34,3.96)) and with cd4 count < 200 cells/l (46%) (aor : 2.07(1.06,4.04)). the most commonly identified parasites were entamoeba histolytica / e. dispar (13.5%), giardia lamblia (8.1%), strongyloides stercoralis (4.0%), and cryptosporidium species (2.2%). conclusion. hiv positive individuals with diarrhea and low cd4 count and art naive groups were more infected with intestinal parasites than their counterparts. early stool examination and treatment of intestinal parasites for hiv / aids patients is essential. |
recent advances in technology now permit the clinician to measure dynamic lip - tooth relationships and incorporate that information into the orthodontic problem list and treatment plan. digital photography along with videography is useful in both smile analysis and patient communication. there are two types of smile : the posed or social smile, and the emotional smile. if the smile is typical for a particular individual, a posed smile is natural, but the smile also can be forced to mimic an unposed smile. in the latter circumstance, the smile can not be sustained and will seem to be strained and unnatural. in the peck classification, stage ii smile is a forced or strained posed smile resulting in maximal upper lip elevation. when a person is asked to pose for a photograph, the smile that is desired is a voluntary, unstrained, static, yet natural smile. posed smiles gain importance in dentistry and orthodontics mainly because they are repeatable over time. an attractive, well - balanced smile is a paramount treatment objective of modern orthodontic therapy. vertical facial proportions in frontal and lateral views are best - evaluated in the context of the facial thirds, which the renaissance artists noted were equal in height in well - proportioned faces. in modern caucasians, the lower third can further be divided into thirds : the mouth should be at one - third of the way between the base of the nose and chin. throughout the orthodontic literature, one can find static profile photographs and lateral cephalograms have been the key diagnostic aids in analyzing patient 's profile and lip at rest. however, to best study a smile, and advance beyond static pictures, recent articles have established a new method of capturing a dynamic smile. desai. asked the subject to hold two rulers with cross configuration near their chin and were given instructions to say the verbal directive was give me a nice big smile, one that shows your teeth. visual directive was a poster with color photographs of six people smiling broadly and the subject was asked to smile like the people in the photo. van der geld. made the subjects to smile by showing practical jokes and spontaneous and posed smiles were obtained. a video camera was used and object to source distance was 4 feet, 4 feet 7 inches and 55 cm in studies by desai. analysis of the photo was done in adobe photoshop cs2 by desai. while selected video frames were measured with the help of digora program for dental radiography by van der geld. no literature review is available about the co - relation between posed smile width (sw) and lower facial height of the patient. the null hypothesis for the study is that posed sw and lower face height (lfh) are not related. the purpose of the study was to establish a ratio between the posed sw and lower facial height in south indian population and to add a new parameter that would be useful in orthodontic clinical evaluation and treatment planning. approval was obtained from institutional review board (dental college, kottayam, iec / m/06/2014/dck). bds students of government dental college, kottayam in the age range of 1825 years who were willing to participate in the study were screened after obtaining informed consent. the inclusion criteria were ethnic south indian origin, class i molar relationship, class i canine relationship, normal overjet and overbite, pleasing appearance, normal face height, orthognathic profile, and an average clinical frankfort mandibular angle (fma). all the subjects selected were having the mesoprosopic facial form (facial index of 84.087.9), well - proportioned and balanced faces. the exclusion criteria were subjects with the skeletal discrepancy, prior orthodontic treatment, history of extraction, high or low clinical fma. 79 students (44 females and 35 males) out of the 240 screened who met the selection criteria were taken as study subjects. each subject was asked to pose an enjoyment smile as realistically as possible, after being shown the proper way in a sample video. the video recording was done with a digital video camera (eos 600d, cmos sensor, digic-4, canon, tokyo, japan). during recording the subjects a scale mounted on an adjustable stand was positioned horizontally below the mandible in the same plane of smile to avoid magnification factor in recording [figure 1 ]. the video was then cut into 30 photos using a software (free studio). the widest commissure - to - commissure posed smile frame (posed sw) was selected as one of 10 or more frames showing an identical smile [figure 2 ]. the cut photo was put into adobe photoshop cs6 and perpendicular was dropped to the scale and readings were obtained. sn - me ' distance was measured by hanging a vertical string with a weight (subject in natural head position), and these points were marked accordingly on the string. the marked distance is measured as the true vertical distance between sn and me ' using digital vernier caliper. the subjects were videotaped on the same day as the measurement of lfh was taken. lfh and posed sw were calculated again on 20% of the sample population and reliability of the measurements was assessed. a scale mounted on an adjustable stand was positioned horizontally below the mandible posed smile photograph (obtained from video clip using free studio software) using spss for windows, (version 16.0. chicago). mean and standard deviations (sds) were calculated, and the ratio between lower facial height and smiling width was established. chicago). mean and standard deviations (sds) were calculated, and the ratio between lower facial height and smiling width was established. the results were expressed as mean and sd ; the mean lower facial height of females was 62.82 mm with an sd = 3.112. the mean lower facial height of males was 69.23 mm with a sd = 2.276. the difference between the above two means was significant with a t = 10.231 and p = 0.000 [table 1 ]. there was statistically significant difference between the mean posed sw of males and females with a t = 5.653 and p = 0.000 [table 2 ]. the co - relation between posed sw and lower facial height [table 3 ] was done, and a statistically significant (p < 0.01) result was obtained. the ratio of lower facial height and posed sw was calculated as 1.0016 in males with an sd = 0.04 and 1.0301 in females with an sd = 0.07 [table 4 ]. reliability of the measurements showed a cronbach 's alpha value (icc) of 0.972 and 0.869, respectively, for lfh and posed sw. mean and sd of lower facial height mean and sd of smiling width co - relation between lfh and smiling width shows a statistically significant (p<0.01) relation the scatter diagram also shows a positive co - relation (lfh : lower facial height ; sw : smile width) ratio between lfh and posed sw the artists of the renaissance period, primarily da vinci and durer, established the proportion that are used in drawing anatomically correct faces. they concluded that distance from hairline to base of nose, base of nose to bottom of nose, bottom of nose to chin should be the same. farkas ' showed that in modern caucasians of european descent, the lower third is slightly longer than the middle and upper third. the neoclassical canon of facial proportions divides the face vertically into fifths, with the width of each eye, the intercanthal distance, and the nasal width all measuring one - fifth. however, studies using direct anthropometry and photogrammetric analysis in whites and asian subjects found variations in these proportions, with the width of the eyes and nasal widths often being either less than or greater than the inter canthal distance. nasal tip projection can be measured using different parameters. the baum ratio is calculated by dividing the length of a line from the nasion to the nasal tip by the length of a perpendicular line from the nasal tip to a vertical line from the subnasale. the simons ratio also reflects nasal tip projection and is found by dividing the length from the subnasale to the nasal tip by the length from the subnasale to the superior labium. according to powell and humphreys, the ideal baum and simons ratios for whites are 2.8:1 and 1, respectively. the posed sw and lower facial height would be yet another ratio to this list of normal proportions for artistic facial evaluation. the videographic method of facial smile evaluation used in this study showed to be a reliable method of smile quantification because a more standardized smile could be obtained minimizing the inherent error of a single snapshot. van der geld. made the subjects to wear glasses with a clipped on reference standard to enable calibration. in a study by desai., subjects were asked to smile while holding rulers with cross configuration near their chin. once the video was cut and photo selected, the analysis was done by adobe photoshop cs2 and measurement was done by an indirect method by converting pixels into millimeter. in the above methods, the ruler and smile are in two different planes. the measurements obtained will differ from actual values. in order to eliminate this error, an adjustable stand was specifically made for the study, which could place the scale in the same vertical plane of smile. a direct method was followed while analyzing smile, that is, the cut photo was put into adobe photoshop cs6, and perpendicular was dropped to the scale and reading was obtained. the mean of the ratio between posed sw and lower facial height for south indian population was calculated as 1.00 in males and 1.03 in females. from this study, one can conclude that class i subjects with pleasing appearance, normal overjet and overbite should depict a 1:1 ratio between lfh (measured from subnasale to soft tissue menton) and posed sw (measured from outer commissure to outer commissure). (sd - 3.718) and 64.59 (sd - 3.706) for females in the present study. the lfh measured from subnasale to soft tissue menton in this study showed a value of 69.23 (sd - 2.276) for males and lfh of the various population is summarized in table 5. according to arnett., mean value for lower one - third facial height was 71 3.5 mm for females and 81.1 4.7 mm for males. the mean value of lower 1/3 of the face (iraq population) for males was found to be 68.63 4.21 mm and for females was found to be 63.03 3.72 mm. a turkish sample showed a mean lower facial height 76.5 5.5 mm for males and 68.7 4.5 mm for females. study in central indian population (madhya pradesh) found out that lower facial height for males was 61.08 0.5423 mm and for females was 55.40 3.92 mm. a study on andhra pradesh population by sinojiya. found that lower 1/3 of the face for females was 55.13 3.40 mm and for males was 58.88 2.20 mm. conducted a study to compare the standard soft tissue cephalometric analysis norms with norms derived for population of western uttar pradesh region of india. lower 1/3 of the face for females was found to be 66.500 4.7269 mm and for males was found to be 73.313 2.8040 mm. a study on south indian population by kalha. had results of lower facial height similar to the one obtained in the present study. lower 1/3 of the face for females was found to be 63.13 9.07 mm and for males was found to be 72.40 7.41 mm. lower 1/3 of the face in various population (in mm) the esthetic ratio between lfh and posed sw would be a new tool in the artistic facial evaluation. loss of vertical dimension in patients with chronic attrition as in bruxism or other parafunctional habit may benefit from this finding as the posed sw in such patients may be used as a guide in restoring their lost vertical dimension. in orthodontics, this parameter could be used in deciding the amount of bite opening in deep bite cases. from an orthognathic surgery perspective, the ratio might aid in determining lfh in vertical maxillary deficiency and the extent of maxillary inferior positioning, but further studies are required to validate this. in addition, this ratio can be added as a new factor in the evaluation of smile aesthetics. from this study, it is concluded that in class i subjects with normal overjet, overbite and fma, posed sw is equal to lfh. however, if lfh is intentionally increased or decreased by orthodontic therapy (molar extrusion / intrusion), how the smiling width change remains unclear and needs further study. chou. reported that with an increase in vertical dimension, there would not be any change in posed sw. further studies about posed sw are required in cases with loss of vertical dimension for evaluating whether it can be used in complete denture rehabilitations. the ratio was obtained in a particular population, and this is yet another limitation. lfh and posed sw relationships in class ii and class iii subjects with different growth patterns need to be assessed further. a study about the relationship of lfh and posed sw should be done a larger sample and on different population groups to finally use this ratio in clinical evaluation and treatment planning. this study focused on deriving a ratio between the posed sw and lower facial height in normal subjectsa 1:1 ratio could be established in normal individuals with a pleasing appearance and normal overjet and overbite. this parameter can be a new tool in the armamentarium of an orthodontist or a cosmetic dental surgeon in evaluating the dynamic facial esthetics for smile designingin full mouth rehabilitation cases due to bruxism, currently the vertical dimension measurement is done arbitrarily. the present result can be used as an additional parameter in determining the vertical dimension in such casesfurther studies in this area and to other age groups can confirm whether posed sw could be used as a parameter in determining vertical dimension at occlusion for edentulous patientsstudies with larger sample are warranted to determine the changes in smiling width in deep bite and open bite cases. this study focused on deriving a ratio between the posed sw and lower facial height in normal subjects a 1:1 ratio could be established in normal individuals with a pleasing appearance and normal overjet and overbite. this parameter can be a new tool in the armamentarium of an orthodontist or a cosmetic dental surgeon in evaluating the dynamic facial esthetics for smile designing in full mouth rehabilitation cases due to bruxism, currently the vertical dimension measurement is done arbitrarily. the present result can be used as an additional parameter in determining the vertical dimension in such cases further studies in this area and to other age groups can confirm whether posed sw could be used as a parameter in determining vertical dimension at occlusion for edentulous patients studies with larger sample are warranted to determine the changes in smiling width in deep bite and open bite cases. | objective : the present study is intended to add a new parameter that would be useful in orthodontic clinical evaluation, treatment planning, and determination of vertical dimension (at occlusion).materials and methods : standardized videographic recording of 79 subjects during posed smile was captured. each video was then cut into 30 photos using the free studio software. the widest commissure - to - commissure posed smile frame (posed smile width [sw ]) was selected as one of 10 or more frames showing an identical smile. lower third of the face is measured from subnasale to soft tissue menton using a digital vernier caliper. two values were then compared. ratio between lower facial height and posed sw was calculated.results:the co - relation between smiling width and lower facial height was found to be statistically significant (p < 0.01). the ratio of lower facial height and smiling width was calculated as 1.0016 with a standard deviation (sd) = 0.04 in males and 1.0301 with an sd = 0.07 in females. the difference between the mean lower facial height in males and females was statistically significant with a t = 10.231 and p = 0.000. the difference between the mean smiling width in males and females was also statistically significant with a t = 5.653 and p = 0.000.conclusion:in class i subjects with pleasing appearance, normal facial proportions, normal overjet and overbite, and average frankfort mandibular angle, the lower facial height (subnasale to soft tissue menton) is equal to posed sw. |
acinic cell carcinomas (acc) are usually low grade, uncommon neoplasms constituting 2.54% of parotid gland tumors. papillary cystic variant (pcv) of acc is a rare tumor, the diagnosis of which is based on histopathological examination. it is composed of tumor with papillary and cystic growth patterns, with varying proportions of one or more cell types. it has mostly been reported in younger patients (16 - 40 years) when compared to the classic type that characteristically presents in the fifth decade of life. although an uncommon tumor, it is important to recognize this variant as it has proved to be universally fatal within 10 years. we hereby report an unusual case of acc - pcv in a 20-year - old male which was considered a benign parotid mass both cytologically and radiologically. a 20-year - old male presented to the surgical clinic with a complaint of swelling on the left angle of mandible below the ear lobule since 4 months. it was 3.5 cm 2 cm in size, firm to hard with limited mobility and nontender. laboratory investigations revealed hemoglobin 16.2 gm%, total leucocyte count 6,800 cells/l, differential leukocyte count neutrophils 74%, lymphocytes 18%, eosinophils 5%, monocytes 3%, platelet count - 1.15 lac/l and blood sugar 120 mg / dl. contrast - enhanced computed tomography of neck showed a soft tissue nodular lesion isodense to the parotid (attenuation 40 hu) of size 2.3 cm 1.6 cm 1.3 cm with mildly enhancing peripheral walls in the left superficial parotid gland. few spiculated nodules were also seen in the right lung apart from patches of numerous centriacinar nodules. both lobes of the thyroid were normal in size, shape, attenuation and enhancement. grossly, the specimen measured approximately 6.5 cm 4.5 cm 3.5 cm with attached flap of skin measuring 3.5 cm 1.5 cm. cut surface showed a large cyst measuring 2.8 cm 1.5 cm filled with dark brown material [figure 1 ]. histopathological examination showed a large well - circumscribed cystic space lined by numerous papillary projections few of them showing thin vascular cores [figure 2 ]. many hemosiderin - laden macrophages and areas of hemorrhage are also seen [figure 3a ]. these papillae were mostly lined by hobnail cells with round, vesicular nuclei, central nucleoli and eosinophilic to vacuolated cytoplasm [figure 3b ]. gross specimen cut surface of which shows a large cyst measuring 2.8 cm 1.5 cm filled with dark brown material numerous papillae lying in cystic cavity (black arrow), fibro collagenous tissue and adjacent normal salivary gland tissue (white arrow) (a) papillary growth pattern along with hemosiderin within the papillae. acinic cell carcinoma is an uncommon salivary gland tumor, making up 1% of all salivary gland neoplasms. they most often arise in the parotid gland, but may occasionally involve the submandibular, minor salivary or seromucinous glands. it is the least aggressive of salivary gland cancers with low malignant potential, but several recurrences and metastasis have been reported. few high - grade variants of acc are known such as papillocystic carcinoma or carcinomas with undifferentiated cells in the medullary pattern. lesions of the tail of the parotid gland are difficult to assess clinically and provide a diagnostic dilemma on imaging. most benign parotid tumors present as slow - growing, painless masses often in the tail of the parotid gland. defines the tail of the parotid gland as inferior 2 cm of the superficial lobe of the gland. in a study by hamilton on 117 parotid tail masses, seventeen types of parotid tail masses were identified out of which benign lesions were the most common. also as it was a slow - growing painless mass present in the parotid tail, clinically it was thought to be a benign parotid mass which was also supported by cytological examination. accurate localization of these lesions on imaging is essential to assist the clinical diagnosis and to prevent inadequate / incomplete excision and complications, especially damage to the facial nerve. the diagnosis of pcv of acc usually poses a challenge because of the cytoarchitecture that is different from classic type. cystic fluid in the case of acc - pcv dilutes the overall cellularity leading to a mistaken diagnosis of benign lesion as was seen in the present case. histopathologically, acc shows a myriad of architectural patterns : solid, solid - lobular, acinar - microcystic, papillary cystic, tubuloductal, follicular and macrocystic, and dedifferentiated. the solid and microcystic are the most common subtypes and the pcv accounts for one - fourth of acc. varying proportions of one or more of five cell types are seen including hobnail, acinar, intercalated, vacuolated, nonspecific glandular and clear cells. this is due to bulging of the apical portions of lumen lining cells into the lumen presumably after release of secretions. the clinical picture is not specific, and diagnosis is based on the histopathologic examination. the mean age of occurrence is in the fifth decade, but the pcv is reported to occur in younger patients compared to the classic type. the most significant differential diagnosis of acc - pcv is papillary carcinoma of the thyroid. thyroid ultrasonography, hormonal assays, and immunohistochemistry for thyroglobulin are helpful in differentiating these two lesions. timely diagnosis and treatment of acc - pcv is essential as it has been found to be universally fatal within 10 years. although most parotid masses are benign, removal is required for histopathologic confirmation because of the clinical and radiologic overlap. to conclude, the present case emphasizes the importance of histopathological examination in parotid masses as the imaging features as well as cytology lack the necessary specificity to differentiate benign from malignant masses, especially in a case of papillary cystic acc. it also highlights the need to consider malignant lesion in the differential diagnosis of parotid tail tumor. | acinic cell carcinoma (acc) is an uncommon low - grade tumor of the salivary glands that constitutes 2.54% of parotid gland tumors. papillary cystic variant (pcv) of acc is even rarer and can be diagnosed on histopathological examination only. it is important to diagnose this variant as it carries a poor prognosis when compared with other variants of acc and is known to be universally fatal in 10 years. the present case describes acc - pcv in a 20-year - old male, which presented as a slow growing parotid tail tumor and was misdiagnosed as a benign lesion both cytologically and radiologically. this case emphasizes the importance of histopathological examination in parotid masses as well as the need to consider malignant lesion in the differential diagnosis of a parotid tail tumor. |
as the number of applicants to medical schools always exceeds the number of available places, there has to be a means of selecting candidates for those places. applicants are usually well qualified and highly motivated. the task of selection and rejection is always challenging, and fraught with uncertainty. appropriate selection has been the subject of unending debate among professionals in the field of medical education. it entails the selection of good students who eventually would become good doctors. in spite of a lack of a general consensus in the selection process, a variety of diverse protocols has been suggested. places in medical schools continue to be highly sought after though only 52% of applicants have a clear ambition to do medicine.1 these applicants should be encouraged to meet with staff and students of the medical schools they have applied to, and be given a realistic indication of what the medical profession entails devoid of the romantic ideas of the life of a doctor.1 some applicants are ill informed on the implications of being in a medical school. they are sometimes under pressure from their parents to apply to a medical college, particularly if they obtained high matriculation scores that should not be wasted by choosing a different course.2 these students or their parents view medicine as a reward for obtaining a high score at matriculation rather than as a demanding career. many students who fall into that category are unlikely to do well ; they fail or even withdraw from the medical course. student alienation and disaffection, rather than academic incompetence, is the usual major cause of failure to complete the course of the study.1 the risk of students withdrawing or failing to complete their medical studies, and the high cost of wastage resulting therefrom, can be minimized through proper selection procedures. academic achievements and intellectual ability are both considered important cognitive skills in the selection of medical students. the academic matriculation score remains a major predictor of who will and who will not be selected for medical education.3 applicants with a strong science background especially in chemistry, physics and biology are believed by some to have a better chance of success in medical schools.45 others argue that the academic score based on science courses should not be the sole parameter for student evaluation, and that it should be complemented by education in humanities or experience in social sciences as well as a proficiency in the english language where english is the medium of instruction for medicine.6 this combination would enhance significantly student perception of the medical educational process.7 the highest predictor of performance in the first year subjects at dammam medical college was a combination of secondary school scores and admission test scores in pre - medical subjects.8 students with a background knowledge of both the humanities and science were found to be twice as likely to complete their medical degree as those who had studied science alone.9 gunn recommended a broad pre - medical education in such subjects as history, philosophy and literature to enhance students ability to think for themselves on important issues.10 the selection of medical students based on their academic achievements alone prevails in many medical schools.11 it is argued, however, that academic achievement should be used only as a filter rather than as the major selection criterion.12 intellectual abilities or non - cognitive skills are equally important. there is a general agreement that a medical student who would respond well to national health needs should be mentally capable, self - disciplined and emotionally committed to the process of medical education. non - cognitive skills include intellectual flexibility, inquisitiveness, critical reasoning, logical thinking, tolerance, the ability to cope with uncertainty and problems.1 these skills also have a positive impact on medical education. the student should be motivated, mature, emotionally stable, self- confident, have low anxiety levels, possess good judgment and perception, show a high degree of decisiveness and assertiveness and be moderately extroverted.1316 interestingly, these qualities correlated significantly with good clinical performance, but did not correlate with prior academic achievement as measured by grade and cognitive tests.1517 applicants for enrollment in medical schools to undertake a course as stressful as medicine should possess good physical and mental health. disabilities ranging from dyslexia to being carriers of infectious diseases have raised serious concerns in the selection of medical students.18 hiv - positive students and carriers of hepatitis b are now excluded by the selection committees in many medical schools in the united states and the united kingdom.19 on the other hand, good athletic or cultural records are positive predictors for student selection. preference for students with these skills will inevitably increase the number of medical students who can participate in social, athletic or cultural extracurricular activities, and are always encouraged in a medical school environment.1 psycho - social issues in medicine should therefore, be taken into account in the selection of medical students.20 selection should be based on the ability of the applicants to cope with their professional demands, and manage their patients with compassion, a trait much appreciated by patients.2122 personality and attitudes, as well as tolerance of ambiguity are important predictors of success with doctor - patient relationship.23 tolerance of ambiguity relates to the ability to make appropriate and justifiable decisions on incomplete data, and be comfortable with those decisions even if they later turn out to be wrong.1 these qualities together with the humanities lead to greater sensitivity, insight and humane understanding among medical students. students who have relations who are medical doctors were more inclined to choose the same profession than those who had no family members in medicine.24 demographic data such as age, gender, race, religion, socio - economic status and schooling of applicants to medical schools should not influence the selection process. a study among first year medical students showed that overseas students had initial academic difficulties, but managed to overcome them by the end of the first year.25 the lack of certain qualities or attitudes at the time of application to a medical school is frequently overcome as medical studies progress. this applies to medical students from socially disadvantaged backgrounds who are not very impressive at the time of application. much benefit can be derived from a wider social range of doctors.3 gender and religion sometimes have a negative influence on interview ratings in some studies.92627 candidates who apply to medical schools are usually in their late teens, an age that is hardly ideal for making sound decisions for a life - long commitment to a profession like medicine. in the united states, personal quality assessments are commonly practised at many medical schools for the selection of medical students.1228 personal qualities are assessed by asking applicants to complete a confidential questionnaire, or by interviews. interviews rank second only to academic evaluation.29 when conducted in an objective fashion, their ratings are positive predictors of successful clinical performance.30 the interviewers should first be trained for the task3132 of asking questions that are pre - designed professionally, and making an assessment made on model answer sheets.1 the interview would lose significant credibility if these criteria were left to the interviewer 's subjective views. it is also preferable to assign two interviewers to each candidate to elicit two independent ratings for a more objective assessment.33 single interviewers yield less consistent ratings, and should not be used for definitive evaluations.34 to the best of the author 's knowledge, there are no published studies on the admission criteria in other saudi universities for comparative evaluation. however, the current selection to all medical colleges in the kingdom of saudi arabia is largely based on a combination of the matriculation score and a national written admission exam. the admission policy of new medical students at that time was based solely on cognitive skills related to the matriculation scores as outlined by whitehouse in 1977.3 this policy did not address other important admission criteria,1 and resulted in about 50% students dropout from the medical college. king faisal university medical college now believes that high scores in the matriculation exam should be coupled with other evaluation criteria for the proper selection of medical students.1228 the matriculation score in the new policy constitutes about 30% only of the total evaluation score, and stresses the scientific subjects. this change in the admission policy has resulted in a dramatic reduction in student dropout to less than 5% in the last few years. applicants initially submit their certificates on completion of the final year secondary schools and the marks obtained in physics, biology, chemistry and mathematics. even though a minimum score of 90% in these subjects is a basic prerequisite, only a tenth of this total number (about 180) is eventually accepted each year. selection of these applicants is based upon a written admission examination that is composed of two parts. part one comprises multiple choice questions in four subjects already taught in secondary school : physics, biology, chemistry and mathematics, as well as in islamic sciences and the english language (english is the official teaching language in the college of medicine). part two measures the iq, personal capabilities and general knowledge of the students, with a 30 mark score. these parameters are considered mandatory by many centers.13122021 evaluation is conducted by a specialized organization called the the total score of this exam (65 marks) is added to the final year secondary school score (35 marks), making a total score of 100. students with the highest scores are scheduled for an interview that is preceded by the completion of a detailed questionnaire form. this questionnaire, evaluated by a psychiatrist comprises five headings : (i) the principal motivation for becoming a medical student ; (ii) preliminary knowledge on the nature and duration of medical education and the teaching process in the medical college as compared to the secondary school ; (iii) aspirations and positive attributes in the applicant 's personality ; (iv) negative and weak points in the applicant 's personality ; and (v) any volunteer activities engaged in by the applicant and lessons learnt from them. the interview is carried out by two or more faculty members, and lasts 45 - 60 minutes with each applicant. conduct of the interview by a group rather than an individual allows for maximum objectivity in the selection process.3334 the interview focuses on the following non - cognitive criteria : (i) personal awareness of the applicant of the major health problems in the saudi community ; (ii) expression, listening and communications skills of the applicant ; and (iii) personal characteristics of the applicant as general interests, motivation, discipline, respect for others, self - respect, self - esteem, and ability to work in a team. in accordance with many medical schools in the united states and the united kingdom,1819 all nominated students have to pass a medical check - up before being accepted in the college of medicine. | the appropriate selection of medical students is a challenging task. it requires that important assessment criteria principally based upon cognitive skills that include the matriculation and admission test scores of the applicants be fulfilled. non - cognitive skills are also important, but used to a lower degree include intellectual flexibility, inquisitiveness, critical reasoning, logical thinking, tolerance, ability to cope with uncertainty and problem solving. other criteria that are also considered important for selection include personal qualities and attitudes of the applicants that reflect directly on doctor - patient relationship. in contrast, such demographic factors as age, gender, race, religion, socio - economic status and schooling should not influence the selection process. the admission criteria adopted at king faisal university medical college focus basically on cognitive criteria. other criteria whether non - cognitive or personal quality assessment are also taken into account through interviews and completed questionaires. |
atopic dermatitis (ad) is a chronic and recurrent disease which concerns 1020% of population. the onset of ad is usually before 2 years old, and in approximately 60% of the patients skin lesions of different intensity remain for the whole life. ad is characterized by typical morphology and distribution of skin lesions, severe pruritus, and familial atopic history. clinical phenotype of the disease depends on multiple interactions between genetic and immunological disturbances, epidermal barrier impairment, and environmental factors [3, 4 ]. histopathologically skin lesions present mainly with a dermal infiltrate of mainly cla+ memory t cells and langerhans cells. leukocyte trafficking into the skin in ad patients is probably mainly regulated by adhesion molecules and chemoattractive proteins, so - called chemokines [6, 7 ]. chemokines are small secreted proteins involved in migration and activation of lymphocytes t. recently published papers indicate their role in pathogenesis of multiple inflammatory skin diseases including atopic dermatitis. in active skin lesions in the course of ad infiltrates of th-2 lymphocytes releasing il-4 and/or il-13 lymphocytes th-2 migration is selectively induced by such chemokines as ccl17 and ccl22, which are highly overexpressed on the keratinocytes in ad patients epidermis. these phenomena lead in a consequence to development of local inflammation [9, 10 ]. in the studies analyzing serum concentration of these chemokines, increased levels of ccl-17 and ccl-22 were found in ad patients, and their concentrations strongly correlated with disease activity [11, 12 ]. shimada. found increased levels of th-2 (ccl-17 and ccl-22) and th-1 (cxcl-9) chemokines in ad patients. they also found a positive correlation between th-2 chemokines serum level and total ige concentration, moreover th-2 chemokines correlated with th-1 ones. in another study performed in infantile ad patients (mean age 4.5 months) also increased levels of ccl-17, ccl-20, ccl-27 were observed which strongly correlated with disease activity, however the most prominent correlation was observed for ccl-27. interleukin (il)-18 is involved in pathogenesis of type-2 helper cells - mediated diseases including atopic dermatitis. according to literature, its serum concentration is significantly elevated in ad patients and correlates with clinical severity of the disease [15, 16 ]. most literature data point out an important role of chemokine network imbalance in development of atopic dermatitis, however there are scarce data on the complex analysis of serum levels of th-1- and th-2-derived chemokines in ad patients. thus, the aim of the paper was to assess the serum level of cxcl-9, cxcl-10, cxcl-11, cxcl-12, ccl-17, ccl-20, ccl-21, ccl-22, ccl-27, il-18 in two ad patient groups, below and over 10 years old. additionally we analyzed serum levels of the chosen chemokines in two groups of healthy volunteers, aged - matched, to note any age - dependent variations in healthy population. forty patients (mean age 11.4 years old ; 23 f, 15 m) with ad and 50 healthy controls, age and sex matched were enrolled into the study. the enrolled patients were divided into two age categories : under 10 years old (group 1 ; n = 23) and over 10 years old (group 2 ; n = 17). according to this criterion, the control group was divided as well (control 1 under 10 years old n = 30 ; and control 2 over 10 years old n = 20). we used criterion of 10 years old as a cut - off point because this age is believed to initiate adolescence life period. / her parents gave written informed consent before entering the study, and all the experiments were approved by the local ethics committee. the investigations were carried out in accordance with declaration of helsinki. before entering the study the subjects underwent thorough physical examination, and scoring atopic dermatitis (scorad) the patients enrolled to the study had moderate ad (mean scorad index 23 range 1639). serum samples were analyzed for cxcl-9, cxcl-10, cxcl-11, cxcl-12, ccl-17, ccl-20, ccl-21, ccl-22, ccl-27, il-18 concentration with elisa assay (r&d system, mineapolis, minn, usa) according to manufacturer 's instructions. data were analyzed using the mann - whitney u test, and correlations coefficients were determined by using the spearman rank correlation test. median concentrations of all the analyzed proteins : cxcl-9, cxcl-10, cxcl-11, cxcl-12, ccl-17, ccl-20, ccl-21, ccl-22, ccl-27, il-18 are presented in table 2. the median serum cxcl-9, cxcl-10, ccl-17, and il-18 level was statistically significantly lower in ad patients from group 1 when compared to the control 1 (56.7 pg / ml versus 87.3 pg / ml ; p =.003 ; 84.8 pg / ml versus 98.0 pg / ml ; p =.04 ; 405.2 pg / ml versus 620.1 pg / ml, p =.04 ; 64.8 pg / ml versus 94.7 pg / ml, p =.0001 ; resp.). in group 1 the median serum concentration of cxcl-12 and ccl-27 was significantly higher than that in the control 1 group (2444.9 pg / ml versus 2135.8 pg / ml, p =.004 ; 463.5 pg / ml versus 406.6 pg / ml, p =.03 ; resp.). for other chemokines median serum values did not differ statistically when compared to the control 1 (p >.05 for all comparisons). the median serum cxcl-12, ccl-17, and ccl-22 levels were significantly higher in ad patients from group 2 than in the age - matched control 2 (2553.5 pg / ml versus 2361.1 pg / ml, p =.01 ; 357.6 pg / ml versus 178.3 pg / ml, p =.04 ; 1152.5 pg / ml versus 606.1 pg / ml, p =.001 ; resp.). for other chemokines median serum values did not differ statistically when compared to the control 2 (p >.05 for all comparisons). comparing serum median levels of analyzed chemokines between two ad groups (group 1 versus group 2) the only significant difference was found for ccl-20 which median value was higher in children below 10 years old than in older population (group 2) (7.8 pg / ml versus 7.4 pg / ml ; p =.03). comparing cxcl-9, cxcl-11, ccl-17, ccl-20, ccl-22, and il-18 serum median concentration between controls 1 and 2 we found significantly higher values in younger population (control 1) than in control 2 (87.3 pg / ml versus 17.9 pg / ml, p =.00004 ; 66.9 pg / ml versus 43.3 pg / ml, p =.049 ; 620.1 pg / ml versus 178.3 pg / ml, p =.001 ; 8.3 pg / ml versus 6.9 pg / ml, p =.008 ; 1410.2 pg / ml versus 606.1 pg / ml, p =.00003 ; 94.7 pg / ml versus 97.2 pg / ml, p =.049 ; resp.). for other chemokines median serum values did not differ statistically when compared two control groups (p >.05 for all comparisons). positive correlation between median serum concentration of cxcl-11 and cxcl-9 (r = 0.44, p =.03), cxcl-9 and cxcl-10 (r = 0.42, p =.04), and cxcl-10 and il-18 (r = 0.8, p =.000001) was found in ad patients below 10 years old (group 1). in group 2 (ad patients over 10 years old) we found positive correlations of the median serum levels of the analyzed proteins for the following parameters : cxcl-11 and cxcl-9 (r = 0.85, p =.00001), cxcl-11 and cxcl-10 (r = 0.8, p =.00006), cxcl-11 and ccl-20 (r = 0.96, p <.001), cxcl-9 and cxcl-10 (r = 0.59, p =.01), cxcl-9 and ccl-20 (r = 0.84, p =.00002), cxcl-10 and ccl-20 (r = 0.79, p =.0002), and ccl-17 and il-18 (r = 0.5, p =.03). moreover in this group we observed a positive correlation between serum median concentration of ccl-22 and patients ' age (r = 0.52, p =.003). in control 1 we found positive correlations between serum levels of the following chemokines : cxcl-11 and ccl-21 (r = 0.4, p =.03), cxcl-11 and ccl-22 (r = 0.37, p =.04), cxcl-11 and ccl-17 (r = 0.36, p =.048), cxcl-9 and cxcl-10 (r = 0.47, p =.008), ccl-17 and ccl-22 (r = 0.68, p =.00003). contrary, negative correlation between serum level of ccl-21 and cxcl-9 (r = 0.36, p =.04) was noted. analysing correlation between serum chemokine levels and patients ' age we found a negative link for ccl-21 and cccl-22 (r = 0.5, p =.004 ; r = 0.36, p =.048 ; resp.). in the control 2 positive correlations were found only between cxcl-10 and ccl-21, and cxcl-17 and ccl-20 (r = 0.84, p =.002 ; r = 0.9, p =.0003 ; resp.). taking the whole ad group (group 1 and 2) into statistical analysis we found no correlation between serum levels of analyzed parameters and patients ' age, while doing the same analysis for whole control group (control 1 + control 2) we found negative correlations between age of the subjects and the following proteins : ccl-17, ccl-22 and il-18 (r = 0.38, p =.01 ; r = 0.67, p =.000002 ; r = 0.3, p =.045 ; resp.). in group 1 a positive correlation between mean total ige serum concentration and ccl-20 was found (r = 0.52, p =.009), while in group 2 it was found for ccl-17 (r = 0.68, p =.002), ccl-27 (r = 0.049, p =.046) and il-18 (r = 0.58, p =.01). analysing all the subjects from ad group (group 1 and 2) ige serum concentration correlated positively only with ccl-17 (r = 0.49, p =.01). in ad patients below 10 years old (group 1) a positive correlation between eosinophilia and ccl-20 (r = 0.53, p =.009) and il-18 (r = 0.45, p =.03) was noted while in group 2 eosinophilia correlated positively with ccl-27 (r = 0.52, p =.03). analyzing all the subjects from ad group (groups 1 and 2) eosinophilia correlated positively only with ccl-22 (r = 0.36, p =.02). cxcl-9, cxcl-10, and cxcl-11 recruit lymphocytes mainly to th-1 type inflammatory sites while chemokines such as ccl-11, ccl-17, ccl-22 lead to th-2 dominated pattern of cell recruitment. ccl-27 selectively attracts cla + memory t cells via ccr-10 receptor expressed on these cells [20, 21 ]. recent data indicate increased serum level of ccl-27 in ad patients, correlating with disease activity, what suggests its role in inflammatory process [22, 23 ]. hon. assessed ccl-27 serum level in children aged 111 years with mean scorad [29.7 ] and found its higher serum concentration, what is in line with our results, however contrary to the authors we found no correlation between ccl-27 and ccl-17 and ccl-22 concentrations. also level of ccl-17 in group 1, although statistically different than that in control 1, was lower in ad patients than in healthy ones. such discrepancy between our results and published ones in other papers, indicates that it is still unclear either increased or decreased ccl-17 level is a characteristic for ad patients, however its distinct level when compared to the control groups testifies its role in ad pathogenesis. in our study interestingly, in group 2 (patients over 10 years old) we found elevated serum levels of ccl-17, ccl-22, and cxcl-12 what also proves their role in ad pathogenesis. other authors also showed significantly higher concentrations of ccl-17, ccl-22, and eotaxin in ad patients than in healthy control. they found positive correlations between serum level of ccl-17 and ccl-22 and total ige concentration and as well these chemokines correlated positively with scorad index. in our study ccl-17, ccl-27 and il-18 serum levels correlated positively with total ige in group 2, and eosinophilia correlated positively with ccl-27.. revealed correlation between ige and ccl-17 and eosinophiles count but not with ccl-27 what is only partially consistent with our results. in skin biopsies taken from ad patients ccl-20 expression this protein is a strong chemoattractant for immature dendritic cells and memory t cells via interactions with ccr6. ccl20 may be induced on keratinocytes under proinflammatory cytokines such : il-1 or tnf-. in healthy epidermis ccl20 is constitutively expressed in epidermal basal layer, however its expression is significantly lower than in inflammatory skin [26, 27 ]. although we found no differences between ccl-20 serum concentration in ad patients and controls, its higher concentration was observed in group 1 than in group 2. moreover, in younger population (group 1) ige and eosinophilia correlated with ccl-20. to our knowledge, there are no data in literature on the subjects, however observed association and data mentioned above provide its role in ad pathogenesis in younger population and its level normalization in line with the age. to our knowledge, there are no data on ccl-21 serum levels in ad patients. we examined this chemokine as it is strongly chemoattractive to lymphocytes t, enhances expression of lfa-1 on these cells, and mediates cell - to - cell adhesion. besides, ccl-21 and ccr7 receptor influence naive t cell migration to lymph nodes where antigen is presented. in healthy skin immunostaining for ccl21 is negative, however it is expressed on dermal endothelial cells in atopic dermatitis.. showed that ccl21 expression on blood vessels positively correlated with the presence of cd45ra+ t cells in the inflammatory infiltrate. although ccl21 expression was found in inflammatory t cell - mediated diseases, its exact role in their pathogenesis is not elucidated. our study in which we found no differences in ccl-21 serum concentration between ad patients and controls does not prove the role of the chemokine in ad pathogenesis. to assess chemokines serum levels depending on age in healthy population, we attempted to check differences between controls 1 and 2. our analysis revealed a distinct pattern in healthy population than that in ad groups. in younger healthy population we found increased levels of cxcl-9, cxcl-11, ccl-17, ccl-20, ccl-22, and il-18 while comparing groups 1 and 2 the only significance concerned ccl-20. in the study published by furusyo. no differences in serum level of ccl-17 between ad patients (children 05 years old) and age - matched healthy control were revealed. moreover in healthy children they observed that serum ccl-17 concentration decreased with age while serum ccl-17 in ad patients did not differ in relation to age. these data are partially consistent with ours, as we also observed a decrease in ccl-17 with age in healthy population and no such age - dependence in ad group. to our knowledge there are no more data analyzing chemokines in this aspect. lack of these naturally occurring changes in chemokine serum levels in ad patients provides their role in the disease pathogenesis. this hypothesis may be partially proven by our observation on the lack of correlation between age and chosen chemokines serum levels in ad patients and the presence of multiple negative correlations between ccl-17, ccl-22, il-18, and age in the whole control group. concluding, we may assume that in younger children with ad a decreased serum level of th-1-derived chemokines is one of the factors involved in the disease development. the imbalance between th-1 and th-2 is probably involved in ad pathogenesis as well, what in our paper is especially emphasized by differences in chemokine concentration between two ad groups and two age - matched controls. our study, similar to others, revealed significant changes between chemokine levels in ad patients and controls, however not always consistent with other authors what may result from two main reasons. the first one is a new aspect of ad pathogenesis, mostly focused now on the impairment of epidermal barrier and innate immune defense as the primary causative factors involved in ad ; only these disturbances lead secondary to induction of adaptive immune response, inflammation development involving chemokines disturbances. the second reason may be the lack of objective and standardized method for ad clinical evaluation, thus the patients enrolled to the studies in different centers, although with the same scorad index, may have a little different clinical picture. based on literature and our results we conclude that chemokine imbalance is involved in ad pathogenesis, however discrepancies obtained in many studies and relatively small number of the patients included in our study do not allow to draw equivocal conclusions. in our opinion further studies correlating chemokine serum levels, their expression in the skin, and ad clinical picture are required and probably will give new light on the disease pathogenesis. | atopic dermatitis (ad) is an inflammatory skin disease in which pathogenesis chemokines are partially involved. the aim of the paper was to assess the serum level of cxcl-9, cxcl-10, cxcl-11, cxcl-12, ccl-17, ccl-20, ccl-21, ccl-22, ccl-27, and il-18 chosen in ad patients by elisa assay. forty patients (mean age 11.4 years old) with ad and 50 healthy controls were enrolled into the study. the patients and controls were divided into two age categories : under 10 years old (group 1 and control 1) and over 10 years old (group 2 and control 2). significantly lower serum concentration of cxcl-9, cxcl-10, ccl-17, and il-18 and higher concentration of cxcl-12 and ccl-27 were found in group 1 when compared to control 1. in group 2 serum concentration of cxcl-12, ccl-17, ccl-22 was higher than in control 2. the obtained results indicate the imbalance in chemokine serum levels in ad what suggests their role in the disease pathogenesis. |
the knowledge of medicinal plants in india has been accumulated in course of many centuries based on several ancient medicinal systems, including ayurveda, unani and siddha. according to the survey report of world health organization, india, one of the richest floristic regions of the world has diverse socio - economic, ethnic, linguistic and cultural areas. therefore, the indigenous knowledge of medicinal plants and their use in treating several ailments might reasonably be expected in this country. have reported that nearly about 70% of tribal and rural inhabitants of india are to a large extent depended on medicinal plants for their primary healthcare management due to either insufficient or inaccessible or less availability of modern healthcare system. the information regarding the medicinal properties of plants came down traditionally generation after generation through traditional healers. apart from the tribal groups, many other forest dwellers and rural people also possess unique knowledge regarding plant utilization. malda district of west bengal, india [figure 1 ] is situated between the latitude and longitude of 244020n to 253208n and 882810e to 874550e respectively with a total geographical area of 3455.66 sq km. the district is characterised by its great archaeological relics such as mourya empire, gupta dynasty and pala dynasty. most of the remote villages are covered by jungles, which consist chiefly of thorny scrub bush and large trees showing wide distribution of flora. the soil of the western region of the district is particularly suited to the growth of mulberry and mango, for which malda has become famous. various ethnic communities, including santala, rajbanshi, namasudre, polia, oraon, mundas, malpaharias etc. they are quite popular to treat several types of local ailments of human and veterinary purposes. they also earn their livelihoods by selling milk, egg, flesh, etc., which plays a significant role in the rural economy of this district. map of study area (malda district) preliminary floristic survey and a few numbers of folk usages of local plants had been studied for malda district by sur., pal and das and chowdhury and das, whereas saha however, no detailed ethnomedicinal practices by local tribal communities had been done so far for this province. hence, this is the first hand information on the ethnomedicinal usage by the ethnic people of malda district as per author s best knowledge. now - a - days the traditional knowledge is in the way of erosion due to environmental degradation, deforestation, agricultural expansion and population pressure. traditional knowledge of medicinal plants and their use by indigenous cultures are not only useful for conservation of cultural traditions and biodiversity but also for community healthcare and drug development at present and in the future. the objective of this study was to interact with local traditional healers and to document their knowledge on utilization of medicinal plants, their usage and the types of diseases treated, etc. the practice of medicinal plants is widespread among the tribal people of malda district, and it is deeply rooted in their socioeconomic culture. considering the great cultural and ethnolinguistic diversity of the tribal people of the province, several field interviews were designed to cover as broad an area of the region as possible, in order to maximize the diversity of knowledge and the plant species employed in traditional remedy. different interviewing procedures, including direct interview, group discussion, open - ended conversations, semi - structured questionnaire etc. were followed to get the information from the local traditional healers, known as kavirajs, baidyas or ojhas and aged knowledgeable persons regarding the use of different medicinal plants curing several ailments. the purpose of this survey was explained to them in details, and prior informed consent was taken as per ethical guidelines of the international society of ethnobiology. the villages were visited in different seasons to get the plant in its flowering condition. plants were pointed out by the informants and their local names, used plant parts, formulation and dosages were also recorded. the plants were properly photographed, and herbarium was prepared for each specimen and deposited at raiganj university college, raiganj, india. the collected specimens were identified with the help of central national herbarium, kolkata, india. the survey method followed in this study was that of the guided field - walk method as described by jain and the collection of voucher specimen, preservation, herbaria technique was followed as per jain and rao. during the survey, we interacted with more than 100 informants and retained the information only from 74 informants. among these, 55 were male (74.33%), and 19 were female (25.66%). more emphasis was given to the aged knowledgeable healers due to their vast experience in treating the local diseases and disorders. mandal (53 years), nargis bibi (48 years), farshed ali (58 years), fatema begum (68 years), basudeb rajbanshi (55 years) md. subed ali (44 years) etc. were the healers in the study area that we found. to analysis the data more clearly, obtaining from the informants, we set up our own database using microsoft access version 2007 and the parameters were name of the taxon, family name, voucher number, vernacular names, parts used, diseases treated, mode of administration or medicinal uses. we also analyzed the percentage between the used parts of plant species, growth forms of the species by putting them in the graph. the practice of medicinal plants is widespread among the tribal people of malda district, and it is deeply rooted in their socioeconomic culture. considering the great cultural and ethnolinguistic diversity of the tribal people of the province, several field interviews were designed to cover as broad an area of the region as possible, in order to maximize the diversity of knowledge and the plant species employed in traditional remedy. different interviewing procedures, including direct interview, group discussion, open - ended conversations, semi - structured questionnaire etc. were followed to get the information from the local traditional healers, known as kavirajs, baidyas or ojhas and aged knowledgeable persons regarding the use of different medicinal plants curing several ailments. the purpose of this survey was explained to them in details, and prior informed consent was taken as per ethical guidelines of the international society of ethnobiology. the villages were visited in different seasons to get the plant in its flowering condition. plants were pointed out by the informants and their local names, used plant parts, formulation and dosages were also recorded. the plants were properly photographed, and herbarium was prepared for each specimen and deposited at raiganj university college, raiganj, india. the collected specimens were identified with the help of central national herbarium, kolkata, india. the survey method followed in this study was that of the guided field - walk method as described by jain and the collection of voucher specimen, preservation, herbaria technique was followed as per jain and rao. during the survey, we interacted with more than 100 informants and retained the information only from 74 informants. among these, 55 were male (74.33%), and 19 were female (25.66%). more emphasis was given to the aged knowledgeable healers due to their vast experience in treating the local diseases and disorders. mandal (53 years), nargis bibi (48 years), farshed ali (58 years), fatema begum (68 years), basudeb rajbanshi (55 years) md. to analysis the data more clearly, obtaining from the informants, we set up our own database using microsoft access version 2007 and the parameters were name of the taxon, family name, voucher number, vernacular names, parts used, diseases treated, mode of administration or medicinal uses. we also analyzed the percentage between the used parts of plant species, growth forms of the species by putting them in the graph. the present study revealed that a total of 53 medicinal plants belonging to 37 families were frequently used in the treatment of 44 types of local ailments with 88 phytotherapeutic uses in the territory. the number of species most frequently used in the treatment of several disorders by each family was mentioned as euphorbiaceae-6 species, fabaceae-5 species, whereas acanthaceae, amaranthaceae, vitaceae, malvaceae, solanaceae, mimosaceae, and zingiberaceae contributed 2 species to each family. the scientific names of recorded species, their families, vernacular names, voucher number, used parts, mode of administration and local ethnic uses were illustrated in table 1. our study also exhibited that herbs were the most dominant growth forms with 17 species (32%), followed by 13 shrubs (24%), 12 trees (23%), 9 climbers (17%) and only 2 parasitic species (4%) treating different ailments as shown in figure 2. andrographis paniculata, amaranthus spinosus, alstonia scholaris, cuscuta reflexa, jatropha gossypiifolia, caesalpinia crista, tamarindus indica, sida rhombifolia etc. were the most important plant used in the treatment of several diseases. growth forms of utilized species various preparations of roots were used most number of occasions with 18 times (25%), followed by leaves with 15 times (21%), seeds with 12 times (17%), barks with 8 times (13%), whole plants with 6 times (8%), fruits with 4 times (6%), latex and gum with 3 times (4%) etc. as shown in figure 3 in the treatment of several human disorders. a total of 88 types of formulations was being administrated to heal 44 types of ailments including azoospermia, diabetes, bone crack or ankle sprain, several types of pain, menstrual disorders, rheumatism, dysentery, etc. it had been observed that 20 types of diseases were healed by leaves, whereas 26 types of ailments cured by roots [table 1 ]. a single plant part of same plant species was involved in treating different ailments and vice - versa. pie chart of used plant parts the majority of remedies were prepared from fresh plant material in the form of a decoction, infusion or a paste. the most frequently used mode of remedy administration is oral ingestion, followed by external use. most of the diseases and pains were usually treated either with a single plant or a mixture of plant parts. in some cases, and other ingredients such as black peeper, ginger, curcuma, milk etc. were also used to make ethnic formulations along with the parts of plant species. a total of 44 types of diseases were reported to be cured in the present study. azoospermia with 8 times was mostly healed disease in the study area, followed by different types of pains with 6 times, ankle sprain and diabetes with five occasions each whereas dysentery, inflammation, menstrual disorder, rheumatism, skin disorders, leucorrhea with 4 times each. further, it can be concluded from table 1 that the most of the preparations were oral except a few of external use. various methods of preparation like crushing, grinding, direct use and homogenizing in water or with other plant extracts were used to prepare the traditional remedy. mustered oil or ghee (a remedy from milk) was being utilized as an ointment at the time of external use such as itching, eczema, inflammation, pus, etc. the present study revealed that a total of 53 medicinal plants belonging to 37 families were frequently used in the treatment of 44 types of local ailments with 88 phytotherapeutic uses in the territory. the number of species most frequently used in the treatment of several disorders by each family was mentioned as euphorbiaceae-6 species, fabaceae-5 species, whereas acanthaceae, amaranthaceae, vitaceae, malvaceae, solanaceae, mimosaceae, and zingiberaceae contributed 2 species to each family. the scientific names of recorded species, their families, vernacular names, voucher number, used parts, mode of administration and local ethnic uses were illustrated in table 1. our study also exhibited that herbs were the most dominant growth forms with 17 species (32%), followed by 13 shrubs (24%), 12 trees (23%), 9 climbers (17%) and only 2 parasitic species (4%) treating different ailments as shown in figure 2. andrographis paniculata, amaranthus spinosus, alstonia scholaris, cuscuta reflexa, jatropha gossypiifolia, caesalpinia crista, tamarindus indica, sida rhombifolia etc. were the most important plant used in the treatment of several diseases. various preparations of roots were used most number of occasions with 18 times (25%), followed by leaves with 15 times (21%), seeds with 12 times (17%), barks with 8 times (13%), whole plants with 6 times (8%), fruits with 4 times (6%), latex and gum with 3 times (4%) etc. as shown in figure 3 in the treatment of several human disorders. a total of 88 types of formulations was being administrated to heal 44 types of ailments including azoospermia, diabetes, bone crack or ankle sprain, several types of pain, menstrual disorders, rheumatism, dysentery, etc. it had been observed that 20 types of diseases were healed by leaves, whereas 26 types of ailments cured by roots [table 1 ]. a single plant part of same plant species was involved in treating different ailments and vice - versa. pie chart of used plant parts the majority of remedies were prepared from fresh plant material in the form of a decoction, infusion or a paste. the most frequently used mode of remedy administration is oral ingestion, followed by external use. most of the diseases and pains were usually treated either with a single plant or a mixture of plant parts. in some cases, and other ingredients such as black peeper, ginger, curcuma, milk etc. were also used to make ethnic formulations along with the parts of plant species. a total of 44 types of diseases were reported to be cured in the present study. azoospermia with 8 times was mostly healed disease in the study area, followed by different types of pains with 6 times, ankle sprain and diabetes with five occasions each whereas dysentery, inflammation, menstrual disorder, rheumatism, skin disorders, leucorrhea with 4 times each. further, it can be concluded from table 1 that the most of the preparations were oral except a few of external use. various methods of preparation like crushing, grinding, direct use and homogenizing in water or with other plant extracts were used to prepare the traditional remedy. mustered oil or ghee (a remedy from milk) was being utilized as an ointment at the time of external use such as itching, eczema, inflammation, pus, etc. the prevalent diseases identified in the study area were azoospermia, ankle sprain, pain, diabetes, menstrual disorders, rheumatism, dysentery, skin disorders, etc. to expel ankle sprain or bone crack of local people, different plant parts like whole plant of cissus quadrangularis, roots of tragia involucrata, bark of litsea glutinosa, bark of acacia catechu, rhizome of alocasia macrorrhiza, fruits of terminalia chebula were administrated whereas eight plant species namely roots of bombax ceiba, seeds of c. reflexa, ocimum kilimandscharicum and abrus precatorius, roots of curculigo orchioides etc. diabetes was cured by means of leaf of a. paniculata, seeds of trigonella foenum - graecum, seeds of syzygium cumini, fruit of alpinia zerumbet and whole parts of oxalis corniculata. to treat menstrual disorders several plants had been utilized by the local traditional healers as explained in table 1. there were few species used more than one occasion to prepare medicinal preparations curing different ailments, viz. c. quadrangularis known as harjora was used in bone crack and ankle sprain ; a. spinosus was used to treat menstrual disorders, rheumatism, cuts and wounds ; t. foenum - graecum was used against kidney stone, diabetes and dandruff problems. as the tribal people remain busy throughout the year with their practice of livelihood from the agricultural sector, they rarely visit the hospitals in towns. it has also been observed that some of the villages are in such remote areas where transportation facilities are inaccessible or sometimes become detached due to some natural calamities. the ethnomedicinal practices are popular in the study area as it is more accessible, easy to prepare, low costs, and eco - friendly. besides, the practice of medicinal plants treating the patients is an alternative source of income for the healers. the present study exhibited that how different interviewing procedures helped to gather the information regarding the name of the diseases treated, plant resources and their usage, including their mode of administration. a total of 44 types of local ailments was treated with 88 phytotherapeutic uses in this district. the making procedure of herbal preparation is yet a secret and passed on generation after generation verbally. proper analysis of herbal formulations and phytoconstituents of used plants can open new door for the researchers. however, ethnobotanical data is the basis of further validation of practices and plant uses in the context of a professional approach to develop new herbal drug. | aim : the present study was aimed at exploring the indigenous knowledge of native tribes on the utilization of wild plant species for local healthcare management in malda district of west bengal.materials and methods : successive field surveys were carried out from july 2012 to august 2013 in search of traditional healers or practitioners who ceaselessly use their worthy knowledge to treat several ailments for human purposes. the information was collected by means of open - ended conversations, semi - structured questionnaire, group discussion, etc. information obtained from the informants was also cross verified to check the authenticity.results:the present study revealed that a total of 53 medicinal plants belonging to the 37 families are frequently used to treat 44 types of ailments with 88 herbal preparations. of 53 plants, herbs possess the highest growth forms (32%) that were used in making traditional preparation, followed by shrubs (24%), trees (23%), climbers (17%), and parasites (4%). roots comprised the major plant parts used (25%), followed by leaves (21%), seeds (17%), bark (13%), whole plant (8%) and fruits (6%) to prepare the medicinal formulations. the chief ailments treated in this province were azoospermia, diabetes, menstrual disorder, dysentery, rheumatism, etc.conclusion:it can be concluded that the documentation of the ethnobotanical knowledge in management of local healthcare is the first step, which will open new door for the researchers in the field of modern drug development. |
allosteric regulation is a key mechanism whereby proteins respond to environmental stimuli that modulate their activity. classic models of allostery (e.g., the mwc and knf models) suggest that a binding event at an allosteric site induces substantial conformational changes in the primary catalytic site. however, allostery has since been observed in the absence of large - scale conformational changes, suggesting that subtle alterations in protein dynamics can induce a population shift in the conformational ensemble without substantially changing the mean conformation of the protein. recent advances in both correlated - residue clustering and dynamical network analysis have helped computationally quantify allosteric states. dynamical network models of allostery often focus on the single most direct path of residues leading from the allosteric to the primary active site. however, few researchers have considered the state changes of slightly longer (suboptimal) allosteric pathways. the statistical distribution of these additional pathways may be useful for locating accessible residues that, if disrupted via pharmacological or mutational means, could modulate the allosteric regulation of important drug targets. in this paper, we introduce weighted implementation of suboptimal paths (wisp), a tool that compliments current dynamical network models of allostery by rapidly calculating the primary communicating path between two residues as well as the slightly longer suboptimal paths. we illustrate the utility of the wisp method using the biological system hish - hisf, a well - characterized glutamine amidotransferase enzyme. to facilitate the broader adoption of this method, we have also created a wisp plugin for the popular visual molecular dynamics (vmd) package. wisp has been specifically tested on several operating systems, using several versions of python, numpy, scipy, and networkx (table 1). wisp has been tested on a number of operating systems, using various versions of numpy, scipy, and networkx. we note that installation of necessary packages under windows was difficult ; however, the command - line version of the program was successfully executed after installing the appropriate dependencies using the activepython software package. as input, wisp accepts an aligned molecular dynamics trajectory in the common multiframe pdb format. trajectory postprocessing is necessary prior to wisp analysis, as most trajectories are not initially aligned or pdb formatted. the freely available visual molecular dynamics (vmd) software package can be used to perform the necessary alignment and conversion. wisp, similar to other dynamical network analysis tools, is based on the dynamic interdependence among protein constituents (e.g., amino acids). a protein system is first simplified by representing each constituent as a single node. for example, depending on user - specified wisp parameters, an amino acid can be represented by a node positioned at the residue center of mass, the side - chain center of mass, the backbone center of mass, or the carbon. as a default the interdependence among nodes is represented as a connecting edge with an associated numeric value that reflects its strength. there are numerous methods for describing the interdependence among nodes in a protein network. typically, this interdependence is represented by a matrix c with values corresponding to the weights of each edge. by default, wisp generates an n matrix c by calculating the correlated motion among node node pairs as shown in eqs 1 and 2:12where n is the number of nodes, i and j are indices corresponding to individual nodes, ri(t) is the location of node i at time t, and cij is the matrix element at position (i, j). the absolute value of cij is larger when the motions of two nodes are highly correlated or anticorrelated. in order to compute signaling pathways, it is useful to construct a matrix where the opposite is true, i.e., where small values indicate highly correlated or anticorrelated motions. consequently, the correlation matrix is functionalized according to eq 3, as outlined in previous works.3 as a point of clarification, each wij can be thought of as a distance in functionalized correlation space. throughout the remainder of this paper, concepts like length and distance will refer to spans in this space, unless specifically described as we further note that, while wisp s default functionalized correlation matrix is generally useful, any user - specified matrix that defines signaling strength as inversely proportional to edge length can be used. in order to improve the speed of subsequent path - finding steps, the complexity of the functionalized correlation matrix w must be reduced. to this end first, a contact - map matrix mcontact is used to separate entries in w that represent pairs of physically distant residues from those that represent adjacent residues. by default, mcontact is constructed using pcutoff, a user - specified cartesian cutoff distance that represents physical proximity. the average location of each atom over the course of the aligned molecular dynamics trajectory is first calculated, followed by a pairwise cartesian distance comparison. two nodes are considered to be in physical contact if the average locations of any of their associated residue atoms come within pcutoff of one another. mcontact entries are set to zero for all node node pairs that are not in physical contact. a simplified, functionalized correlation matrix wsimp is then constructed by multiplying w and mcontact element - wise. the entries of wsimp that equal zero represent node node interactions that are subsequently ignored. second, to further reduce the complexity of the functionalized correlation matrix w, a pruning algorithm identifies nodes that only participate in pathways having lengths in network space that are greater than another cutoff (dcutoff). as the ultimate goal is to identify suboptimal paths with lengths less than dcutoff, these nodes can be effectively discarded as well. to identify these nodes, an fnp is the optimal pathway between two user specified nodes na and nb that is forced to pass through a given third node ni. for any two fixed nodes na and nb, each third node ni is associated with a single fnp. the set of all fnps can therefore be generated by iterating over all the nodes, ni, of the system. to calculate an fnp, dijkstra s algorithm, included in networkx, is first used to identify the optimal paths between na ni and nb ni, respectively. the fnp has a length equal to the sum of these two constituent paths. any path between na and nb that passes through ni must have a length equal to or greater than that of the associated fnp. consequently, if the length of the fnp is greater than dcutoff, all entries in wsimp associated with ni are set to zero, so that ni is effectively ignored. having generated wsimp, we are now ready to search for both the single optimal and multiple suboptimal paths between na and nb. the optimal path is fairly easy to identify using dijkstra s algorithm, mentioned above. in contrast, identifying all suboptimal paths is difficult because the number of possible pathways between na and nb grows rapidly as the total number of nodes increases. to identify suboptimal paths, a recursive simultaneous searches start from na and nb (figure 1, in blue and red, respectively) and recursively traverse the nodes of the dynamical network. the recursive algorithm ignores the connections / correlations between nodes that are physically distant (figure 1, gray lines). additionally, nodes eliminated using the fnp technique described above are likewise ignored (figure 1, gray circles), resulting in substantial speedups. as soon as any of the lengthening paths grows longer than dcutoff, that branch of the recursion is killed (figure 1, red x). simultaneous searches start from na and nb (blue and red, respectively) and recursively traverse the nodes of the dynamical network. connections / correlations between nodes that are physically distant are ignored (gray lines). nodes eliminated using the fnp technique are also ignored (gray circles). as soon as any of the lengthening paths grows too long, that branch of the recursion is killed (red x). at each recursive step, all branches originating from na and nb are compared for common nodes (asterisk). if a common node exists, the two paths are joined. if the length of this composite path is sufficiently short, a suboptimal path has been identified. at each recursive step, all branches originating from na and nb are compared for common nodes (figure 1, the node marked with an asterisk). if a common node exists, the two paths are joined at this node. if the length of this composite path is less than dcutoff, a suboptimal path has been identified. as wisp has been developed to take advantage of multiple processors, running the program on a multicore system can lead to further speedups beyond the software optimizations described above. the program output is a directory containing multiple files, including the specific w and mcontact matrices used. the primary output file is a tcl script that, when loaded into vmd, draws three - dimensional splines representative of the optimal and suboptimal paths. user defined parameters control the relationship between spline thickness, color, opacity, and path length. useful information is also given as comments in the tcl file, including path lengths and participating protein residues. in addition to the command - line program, we have also developed a visual molecular dynamics (vmd) plugin and tcl - based gui for easy preparation and visualization of wisp results. the main window of the wisp gui (figure 2) allows the user to specify the molecular trajectory as well as the allosteric - signal source and sink residues. several additional window interfaces allow the user to modify more advanced program options if needed. all options available through the wisp command - line interface are available to users of the gui. the gui is used to visualize the allosteric pathways between leu50:hisf and glu180:hish. in the main window (top left), the user selects the relevant molecule and which residues to use as the source and sink. the user may also select to load the visualization into vmd upon job completion. the setting option windows (left and bottom right) allow the user to specify additional wisp arguments. once satisfied with the run specifications, the user may click the run wisp the plugin loads the visualization of the allosteric pathways into the main vmd window, where the appearance can be further modified according to the user s preferences. the molecular dynamics simulations of hish - hisf used in the current study have been described previously. in brief, a model of the hish hisf apo dimer was prepared from the 1gpw crystal structure (thermotoga maritima). to generate the corresponding holo structure, the 1ox5 crystal structure (saccharomyces cerevisiae), which contains a cocrystallized prfar allosteric effector molecule, was aligned to the apo model, effectively positioning prfar within the 1gpw : hisf allosteric site. the aligned 1ox5 prfar was then merged with the 1gpw - based apo model to yield the corresponding holo structure. following solvation with tip3p water molecules and 1 ns of harmonic constrained equilibration, 20 ns of production dynamics with a 2 fs time step were run for both the apo and holo systems using namd, the charmm27 force field, and the same prfar parametrization used previously. consequently, one natural strategy for rational drug design is to impede or agonize protein function via allosteric modulation. classic views of allostery suggest that the binding of an effector molecule at an allosteric site induces large conformational shifts that alter the activity of the primary site. however, as allostery is not necessarily limited to large shifts, this reasoning does not explain some examples of regulation at a distance. recently showed that significant backbone deformations are not required for an allosteric effect ; rather, in the absence of large conformational changes, subtle shifts in local dynamics driven by entropic effects govern certain types of allostery. quasi - harmonic analysis (e.g., like that used by software packages such as carma to calculate entropy) is commonly used to build dynamical network models that quantify signaling pathways among protein constituents. optimal and suboptimal pathways are calculated that connect protein constituents believed to be important for allostery (i.e., sources and sinks). an optimal pathway is the shortest distance traversed between source and sink along weighted edges (e.g., as determined by correlated motions), and suboptimal pathways are those closest in length to, but not including, the optimal path. existing tools can compute optimal and suboptimal pathways between residues ; however, these programs lack the speed required to compute more than 50 suboptimal pathways within a reasonable amount of time (several hours or days). as statistics related to suboptimal pathways may provide important insights that can not be gleaned from the single optimal pathway, faster algorithmic advances must be made. wisp is designed to facilitate the calculation of hundreds of suboptimal pathways in minutes, thereby permitting fast and robust statistical analysis of biological systems modeled as dynamical networks. for example, using a modern workstation with 24 cores, we recently used a 20 000-frame trajectory to identify 750 pathways. wisp loaded and analyzed the trajectory, generated the functionalized correlation matrix, and identified the 750 pathways in 21 min and 52 s. when the calculation was repeated using a copy of the functionalized correlation matrix saved from the first run, the 750 pathways were identified in only 5 min and 44 s. to demonstrate the utility of the wisp algorithm, we used it to study hish - hisf, a multidomain globular protein known to exhibit allostery. the activity of hish - hisf, which regulates the fifth step of the histidine biosynthetic pathway in plants, fungi, and microbes, is substantially altered by the allosteric effector n1-[(5-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (prfar). guided by previous work, we investigated the suboptimal pathways between residues leu50:hisf and glu180:hish using 20 ns molecular dynamics simulations of both apo and holo hish - hisf. a total of 700 pathways (figure 3) between leu50:hisf and glu180:hish were calculated using wisp s default correlation (eqs 13) and contact - map matrices, described in the materials and methods. had only the two optimal pathways (apo vs holo) been considered, we would have concluded that communication between the allosteric and primary site is fundamentally different in the presence and absence of the prfar effector molecule (figures 3 and 4). the optimal pathway between leu50:hisf and glu180:hish in the apo state was leu50:hisf phe49:hisf phe77:hisf the 700 shortest paths between leu50:hisf and glu180:hish, shown as red splines, derived from (a) the apo trajectory and (b) the holo trajectory. wisp allows the user to choose between a number of graphical settings to better visualize signaling among nodes. a histogram of the 700 path lengths associated with the apo and holo trajectories is shown. the path distribution is largely shifted to the left for the holo (allosteric) state. this shift likely results from a more coherent signal in the holo simulation, indicating a possible decrease in the entropy along the pathways due to prfar binding. however, when we considered multiple suboptimal paths, it became apparent that allosteric signaling may be far more intricate. the optimal path in the apo simulation is the shortest suboptimal path in the holo simulation (top 0.3%), and the optimal path in the holo simulation is the 13th shortest suboptimal path in the apo simulation (top 2.0%). in light of this multipathway analysis, the idea that prfar binding fundamentally alters a solitary line of communication between the allosteric and primary site becomes less tenable. rather, the binding of the effector molecule likely has small effects on multiple pathways, both optimal and suboptimal, that when taken together yield a substantial allosteric effect. the lengths of the two optimal pathways of the two systems did not differ substantially (apo, 2.97 ; holo, 2.84). consequently, had only these two pathways been considered, some might have mistakenly concluded that the allosteric consequences of prfar binding are minor. in contrast, when hundreds of suboptimal paths were also considered, a large prfar - dependent shift in communication between the allosteric and primary site became apparent. to demonstrate this shift, we generated a histogram of all path lengths for both the holo and apo simulations (figure 4). the distribution derived from the holo trajectory is substantially skewed toward shorter path lengths, suggesting that the motions of the residues connecting the allosteric and primary sites are more tightly correlated when prfar is bound. an overall dynamical tightening and loss of entropy along the pathways may therefore explain the allosteric signal. to identify protein residues critical for allosteric transmission, we counted the number of times each residue appeared in any of the 700 paths associated with the apo and holo trajectories, respectively (i.e., the degeneracy of each node, figure 5). notably, a number of residues had large effector - molecule - dependent shifts in degeneracy, i.e., hisf : leu47 (shifts down), val69 (shifts up), ala70 (shifts up), ile73 (shifts up), asp74 (shifts up), pro76 (shifts down), and ala97 (shifts down) and hish : lys181 (slight shift down) (table 2). importantly, these residues, which may be crucial for the regulation of protein activity, did not all appear in the optimal apo and holo paths and so would not have been identified had the suboptimal paths been ignored. previous studies in evolutionary conservation have shown that hisf : leu47, val69, ala70, and ile73 are partially or strongly conserved and hisf : pro76 and ala97 and hish : lys181 are strictly conserved across the entire glutamine amidotransferase family of enzymes. hisf : asp74 is not conserved, but this amino acid is still predicted to play a role in allostery. compounds that target (i.e., selectively bind) these critical residues may serve as useful precursors to future allosteric - modulating small molecules. the total number of times a given residue participates in any of the 700 paths (i.e., node degeneracy) is shown for (a) hisf and (b) hish. green indicates the holo state, blue indicates the apo state, and cyan indicates an overlap. note that leu50:hisf and glu180:hish are present in all 700 paths. we note that our decision to specifically analyze the 700 shortest paths between leu50:hisf and glu180:hish was arbitrary. in order to better assess the minimum number of paths required to reliably predict node degeneracy, we analyzed the holo trajectory by varying the number of paths considered and calculating the degeneracy of selected residues / nodes implicated in the allosteric mechanism (figure 6). we note that the degeneracy of these nodes had largely converged by 350 paths. a similar result was obtained when the apo simulation was analyzed (data not shown). given that the relative importance of suboptimal paths in determining the competency of an allosteric signal is likely highly system dependent, we do not necessarily recommend this exact number of paths for all analyses. however, we are hopeful that this general benchmark will help guide future researchers in their efforts. normalized node degeneracy as a function of the number of suboptimal paths calculated (holo simulation). the degeneracy of selected residues / nodes as a function of paths searched was calculated and normalized by dividing by the total number of paths. similar results were obtained when the apo simulation was analyzed (data not shown). we present wisp, a program that rapidly calculates both optimal and suboptimal communication pathways between distinct protein residues. the program is available as a vmd plugin or a standalone command - line script. wisp outputs path members and lengths that can be subsequently used in the analysis of path distributions, node degeneracy, and other metrics of interest to scientists studying the molecular mechanisms of allostery the utility of our program was presented by performing a dynamical analysis of the hish - hisf protein. in our test case, allosteric modulation was likely the result of subtle changes in multiple suboptimal pathways rather than large changes in a single optimal path. additionally, we showed that prfar binding causes a large shift toward shorter path lengths (i.e., more correlated motions) in 700 communication pathways between residues hisf : leu50 and hish : glu180. this shift explains the strong allosteric effects of the prfar modulator (figure 4). remarkably, the significant shift in collective correlated dynamics occurred even at relatively short (tens of nanoseconds) time scales, suggesting that the allosteric signal is rapidly transmitted. the multiple suboptimal pathways are dominated by a few select residues, as indicated by the shift in node degeneracy between the apo and holo states (figure 5 and table 2). wisp has been successfully tested on a number of platforms (table 1). we are hopeful that the program will be a useful tool for the computational - biology community. a numerical representation of the same data from figure 5. the comparison between the apo and holo states suggests that certain residues are more sensitive to the allosteric effector prfar than others (shaded columns). | allostery can occur by way of subtle cooperation among protein residues (e.g., amino acids) even in the absence of large conformational shifts. dynamical network analysis has been used to model this cooperation, helping to computationally explain how binding to an allosteric site can impact the behavior of a primary site many ngstroms away. traditionally, computational efforts have focused on the most optimal path of correlated motions leading from the allosteric to the primary active site. we present a program called weighted implementation of suboptimal paths (wisp) capable of rapidly identifying additional suboptimal pathways that may also play important roles in the transmission of allosteric signals. aside from providing signal redundancy, suboptimal paths traverse residues that, if disrupted through pharmacological or mutational means, could modulate the allosteric regulation of important drug targets. to demonstrate the utility of our program, we present a case study describing the allostery of hish - hisf, an amidotransferase from t. maritima thermotiga. wisp and its vmd - based graphical user interface (gui) can be downloaded from http://nbcr.ucsd.edu/wisp. |
certain marine algae produce potent toxins that impact human health through the consumption of contaminated shellfish and finfish and through water or aerosol exposure. over the past three decades, the frequency and global distribution of toxic algal incidents appear to have increased, and human intoxications from novel algal sources have occurred. this increase is of particular concern, since it parallels recent evidence of large - scale ecologic disturbances that coincide with trends in global warming. the extent to which human activities have contributed to their increase therefore comes into question. this review summarizes the origins and health effects of marine algal toxins, as well as changes in their current global distribution, and examines possible causes for the recent increase in their occurrence.imagesfigure 2figure 3 |
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insights into the toxicity caused by the intracellular accumulation of mutant serpins largely comes from work on 1-antitrypsin deficiency, the best characterized of the serpinopathies. 1-antitrypsin is synthesized in the liver and is constitutively released into the circulation where it protects tissues against damage from the neutrophil enzyme elastase. the z mutant of 1-antitrypsin is found in 4% of the north european population and causes some of the newly synthesized protein to misfold and form ordered polymers (fig. 1) that are retained within the endoplasmic reticulum (er) of hepatocytes (3, 4). the resulting protein overload predisposes individuals homozygous for the z mutation to neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. other mutants of 1-antitrypsin (51phe, ser53phe) also cause the protein to form ordered polymers and create a similar degree of protein overload. these mutations probably also predispose to liver disease, but their rarity makes it difficult to undertake detailed epidemiological studies. the s (glu264val) and i (arg39cys) mutants of 1-antitrypsin form polymers much less readily than the z mutant and thus can be cleared by the normal disposal pathways within the cell. these mutants are not associated with liver disease unless they are inherited along with the more severe z allele. thus, there is a threshold effect whereby the mutation must cause sufficient accumulation of polymers to cause disease. mutants of the brain - specific serpin neuroserpin also form ordered polymers that accumulate within the er of neurons. these mutations cause an autosomal - dominant dementia known as familial encephalopathy with neuroserpin inclusion bodies (fenib) (5). similar to 1-antitrypsin mutants, the neuroserpin mutants display a striking genotype phenotype correlation : more severe mutations lead to faster polymer formation, more inclusions, and disease onset at a younger age. how intracellular accumulation of mutant 1-antitrypsin and neuroserpin polymers causes the cell death and inflammation that characterize cirrhosis and fenib is not completely clear. a majority of the mutated protein fails to fold and is degraded by the proteasome (6), and 1015% of the protein folds normally and is secreted into the plasma (in the case of z 1-antitrypsin). however, a small proportion of the mutant protein folds into ordered polymers, some of which are degraded by autophagy (7) and the remainder of which accumulates within the er. accumulation of the mutant protein in the er activates the transcription factor nuclear factor b (nf-b) and caspases (810), thereby inducing inflammation and apoptosis. the mechanism that prevents the exit of polymers from the er and the consequent signaling that activates nf-b and caspases has yet to be elucidated. not all pathogenic serpin mutations result in the build up of toxic polymers. in some cases this is exemplified by naturally occurring mutations in the plasma proteins c1-inhibitor, antithrombin, and 1-antichymotrypsin, which control the complement, coagulation, and inflammatory cascades, respectively. these mutations destabilize the protein 's architecture, allowing the formation of unstable intermediates and inactive polymers within the er of hepatocytes. however, as these proteins are less abundant (synthesized at 10% the rate of 1-antitrypsin) and the mutations are usually heterozygous, the aberrant protein can be effectively cleared by degradative pathways and does not form toxic inclusions. however, intrahepatic clearance of these mutant proteins leads to reduced secretion and a lack of functional protein in the circulation. c1-inhibitor deficiency results in uncontrolled activity of the complement cascade and angio - edema ; antithrombin deficiency causes thrombosis ; and 1-antichymotrypsin deficiency renders tissues vulnerable to proteolytic attack, which can lead to inflammation and chronic obstructive pulmonary disease. a mutation in the serpin heparin cofactor ii, which normally inhibits coagulation, is associated with plasma deficiency of the protein, but so far has not been shown to cause disease (11). this mutation is of particular interest as it is analogous to the z allele that causes polymerization of 1-antitrypsin. the same mutation in the drosophila serpin necrotic causes temperature - dependent polymerization and inactivation of the protein (12). we therefore predict that episodes of fever will precipitate the inactivation of unstable serpins and thus exacerbate the tissue damage of the serpinopathies. although mutation - induced polymerization often results in the retention of the protein within hepatocytes, some mutant protein can traffic through the secretory pathway and reach the circulation or local tissues. this protein often retains function as a proteinase inhibitor but still carries the mutation and thus the propensity to form polymers once secreted. indeed, polymers of 1-antitrypsin, antithrombin, and c1-inhibitor have been identified in the plasma of individuals with mutations in these proteins (1315). moreover, polymers of neuroserpin predominate in the culture media of cells transfected with mutants of neuroserpin (16). it is clearly important to consider whether extracellular polymers are inactive bystanders or whether they can themselves exacerbate the tissue damage of the serpinopathies. recent work has shown that polymers of 1-antitrypsin are chemotactic for neutrophils in vitro and cause a neutrophil influx when instilled into the lungs of mice (1719). in contrast, the monomeric protein has little effect on the migration of neutrophils in vitro or in vivo. the influx of inflammatory cells in mice treated with polymers is not mediated by chemokines, but appears to be a direct effect of 1-antitrypsin polymers on neutrophils. neutrophil chemotaxis caused by systemic polymers may explain the well - recognized association between vascular disease associated with the presence of antibodies against neutrophil cytoplasmic components and the z allele of 1-antitrypsin (20). in this case, polymers may cause the aberrant leukocyte migration that underlies the arteritis and capillaritis that causes organ damage. for example, the presence of neuroserpin polymers at the neuronal synapse may cause local inflammation, thus contributing to neuronal dysfunction and disruption of synaptic plasticity in fenib. inflammation caused by the tissue deposition of c1-inhibitor polymers may exacerbate the vascular permeability that characterizes angio - edema. it is clear that all three mechanisms of serpin - mediated tissue damage may contribute to the pathogenesis of disease, although their contribution is likely to vary between serpins and serpinopathies. the interaction of the different pathways is perhaps best characterized for the type of emphysema that is associated with 1-antitrypsin deficiency. individuals who are homozygous for the z mutation typically develop panlobular emphysema in their 40s and 50s (or 30s in those who smoke). for many years, this type of emphysema was largely attributed to the lack of effective antiproteinase activity and control of inflammation within the lung (mechanism 2). however, the recognition that other pathways contribute to tissue damage in the serpinopathies requires a reconsideration of the pathogenesis of disease. in emphysema, noxious stimuli such as cigarette smoke are thought to trigger the initial inflammation within the lung. a chemotactic gradient of interleukin (il)-8 and leukotriene - b4 recruits large numbers of macrophages and neutrophils from capillaries into the small airways and alveoli (fig. 2). to reach the alveoli, the cells must migrate through an interstitial space that contains elastin, proteoglycans, and collagen, and then through the junctions between epithelial cells (21, 22). in patients with emphysema, neutrophils are initially concentrated in the centrilobular regions of the lung parenchyma where they release serine and cathepsin proteinases, which degrade elastin and other structural proteins and thus contribute to disease (23). degraded elastin fragments themselves act as chemoattractants and emphysema is further exacerbated by free radicals contained in cigarette smoke (10 per puff) and superoxide released by activated neutrophils, which damage proteins, lipids, and dna (26) and cause cell death. (a) the alveolar wall is composed of structural type i (green) and type ii (blue) pneumocytes (reference 22). these are separated from the capillary endothelium (yellow) by interstitial matrix that is maintained by fibroblasts (red) and also contains macrophages. (b) neutrophils (light yellow) migrate through the interstitium (arrow) into the lung (black oval) in response to inflammatory mediators, such as ltb4 (blue), il-8 (gold), and elastin fragments (black). interstitial polymers (inset, repeating units of blue, red, and yellow) bind to neutrophils and cause them to degranulate, thus amplifying and accelerating tissue destruction in emphysema. as the major inhibitor of neutrophil elastase, 1-antitrypsin is critical for defense against such proteolytic lung damage. 1-antitrypsin enters the lung by passive diffusion from the circulation and is also secreted locally by macrophages and bronchial and alveolar epithelial cells. in individuals homozygous for the z mutation, however, all of the 1-antitrypsin in the lungs has the propensity to form polymers, regardless of its source. indeed, polymers of 1-antitrypsin are found in lavage (18, 27) and tissue sections (19) from the lungs of these individuals. deposits of polymers are particularly prominent around capillaries (consistent with a circulating source of polymers) and epithelial cells (consistent with local synthesis). the greatest resistance to diffusion is the interstitium, where z 1-antitrypsin is concentrated and where it forms polymers. the chemotactic property of 1-antitrypsin polymers can then trap neutrophils within the interstitium as they migrate from the vascular space to the alveolar compartment in response to chemokines (fig. 2). once neutrophils are retained within the interstitium, the polymers cause the cells to adhere, degranulate, and release proteolytic enzymes (17), thereby maximizing damage to the extracellular matrix and spreading the focus of inflammation throughout the lobules of the lung. the destructive effects of neutrophil - derived proteolytic enzymes are amplified in individuals with 1-antitrypsin deficiency, as the small amount of monomeric z 1-antitrypsin that is present is less efficient at inhibiting neutrophil elastase. the activity of 1-antitrypsin is likely to be further reduced by oxidation of the key 358met residue by superoxide radicals. in addition to exacerbating proteolysis, the lack of active 1-antitrypsin leads to uncontrolled activation of intracellular caspase-3 and hence alveolar cell apoptosis and emphysema (28). thus, there is evidence that both uncontrolled proteolytic activity due to loss of 1-antitrypsin function (mechanism 2) and tissue damage due to extracellular deposition of z 1-antitrypsin polymers (mechanism 3) contribute to the pathogenesis of emphysema. although less well investigated, cell death and inflammation caused by intracellular accumulation of z 1-antitrypsin (mechanism 1) is also likely to contribute to tissue damage in the alveolus and bronchial epithelium. as in the liver, the intracellular production of z 1-antitrypsin may activate nf-b signaling cascades in lung cells (8, 9), which could increase the production of inflammatory mediators and further amplify neutrophil recruitment and tissue damage. the chronic activation of nf-b is also likely to accelerate apoptosis within all alveolar cells, which would help explain the widespread destruction of the alveoli and the panlobular distribution of this type of emphysema. although the relative importance of each of the three types of tissue damage remains to be clarified, this integrated model of emphysema associated with 1-antitrypsin deficiency suggests some novel therapeutic strategies. small molecule inhibitors have been developed that block the polymerization of z 1-antitrypsin in vitro and clear protein aggregates in cell models of disease (29). however, the current generation of small molecules also inactivates 1-antitrypsin as a proteinase inhibitor. thus, although these drugs may help reduce the risk of liver disease associated with 1-antitrypsin deficiency, they would be predicted to exacerbate emphysema. it is therefore important to develop refined versions of these molecules that block polymerization of the protein without affecting its inhibitory activity. such drugs could be administered directly into the lung to prevent polymerization and so ameliorate the inflammatory response. small molecules that block the binding of polymers to neutrophils would be an alternative approach. this strategy would block the ability of polymers to activate neutrophils and thus restore the normal migration pathway of neutrophils from the circulation to the alveoli. strategies that target the downstream effects, rather than the polymer itself, provide a third possibility. given the central role of free radicals in emphysema, antioxidant agents are required to reduce oxidative stress within the lung. although the current generation of antioxidants is not sufficiently potent, they may be effective if coadministered with agents that block polymer formation and/or the interaction of polymers with neutrophils. finally, inhibition of the inflammatory response by blocking intracellular (nf-b dependent) and extracellular pathways that are activated by polymers might also be a successful approach to ameliorating the lung disease associated with 1-antitrypsin deficiency. | members of the serpin (serine proteinase inhibitor) superfamily play a central role in the control of inflammatory, coagulation, and fibrinolytic cascades. point mutations that cause abnormal conformational transitions in these proteins can trigger disease. recent work has defined three pathways by which these conformers cause tissue damage. here, we describe how these three mechanisms can be integrated into a new model of the pathogenesis of emphysema caused by mutations in the serpin 1-antitrypsin. |
studies focusing on protein interfaces have revealed that binding energies are not uniformly distributed along the protein interfaces. instead, there are certain critical residues called hot spots. these residues comprise only a small fraction of interfaces yet account for the majority of the binding energy (13). these residues are observed to be critical for function and stability of the protein association (1). thorn and bogan (4) deposited hot spots from alanine scanning mutagenesis experiments, in a database called asedb. bid is an effort to organize protein interaction data compiled from the literature and presents amino acids at the protein protein binding interfaces (5). yet computational methods can introduce alternative approaches to experimental techniques to detect and catalog hot spots (6). several groups have developed energy - based methods to predict hot spots (79). molecular dynamics studies can also be used to investigate the energetic contributions of interface residues (1012). while both energy and md - based methods are very efficient, they are at the same time costly and not applicable in large - scale hot spot prediction. residues in protein interfaces (13) and functional sites (14) were observed to be mutating at a slower pace compared to the rest of the protein surface. a very recent study based on sequence environment and evolutionary profile of residues predicts computational hot spots (15). correlation between hot spot residues and structurally conserved residues were found to be remarkable (1619). these hot spots are also found to be buried and tightly packed with other residues (18) resulting in densely packed clusters of networked hot spots, called hot regions. here, we present hotsprint, a database documenting computational hot spots in the protein interfaces combining conservation and solvent accessibility of residues in the protein interfaces. hotsprint contains protein interfaces extracted from the structures in protein data bank (pdb) and is the first database, to our knowledge, which exploits sequence conservation to detect hot spots on a large scale. total 49 512 interfaces are extracted from 34 817 pdb entries as of february 2006. conserved naccess is used to obtain the solvent accessibility of residues (21). in summary, hotsprint marks residues that are highly conserved and tightly packed in protein interfaces as hot spots. the interfaces, used for the identification of the computational hot spots in the hotsprint, are taken from the updated version of interface dataset generated by keskin. interfaces were generated by the atomic distance criteria : if the distance between any atoms of two residues, one from each chain, is less than the summation of their van der waals radii plus a tolerance 0.5, these residues are named as interface residues. if the distance between non - interacting and interacting residues in the same chain is smaller than 6, the non - interacting residue is named a nearby residues are important for the information about the architecture of the interface and provided in our database. all 15 268 multi - chain pdb structures are used to extract two chain interfaces and then interfaces having less than 10 residues are eliminated. the resulting dataset contains 49 512 two - chained interfaces that are denoted by six - letter nomenclature where the first four letters denote the pdb i d, and the last two letters are the chain identifier. hotsprint database can be accessed through a web interface where users can search for computational hot spots in protein interfaces. rate4site makes use of topology and branch lengths of the phylogenetic trees constructed from multiple sequence alignments (msa) of proteins and estimates conservation rates of amino acids based on the empirical bayesian rule. msas of proteins constituting interfaces are taken from hssp (homology - derived secondary structure of proteins) (23) database as of 14 january 2006. all msas obtained from hssp are converted to fasta format to be used in rate4site step. in addition, some residues are more frequently observed to be hot spots, so each of the 20 amino acids has a different propensity to be a hot spot. further, hot spots prefer to reside in protein cavities (24), therefore surface area accessibility of interface residues are incorporated into our hot spot scoring formula. the computational hot spot score of ith residue in a chain is defined as pscorei = scorei x pk, where scorei is the conservation score from rate4site (25), pk is the propensity of residue type k (i.e, k = ala, val, etc.) to be conserved in the interface (details are given in the supplementary data). for an amino acid in a protein interface to be considered as a computational hot spot, we propose that following formulation should be satisfied : pscorei > t and asa > tasa and asacomplex t and asa > tasa and asacomplex < tasax where t, tasa and tasax are user - defined thresholds, the default values are set to 6.2, and 49 and 12, respectively. asa is the asa change of the residue upon complexation, asa = asamonomer asacomplex, asa of the residue in the monomer and complex form, respectively. in asa calculations, naccess (21) is used and buried asas of interface are calculated for each interface. thus, this formulation combines amino acid conservation scores obtained from rate4site [scaled with amino acid conservation propensities (e.g. aromatic residues are observed to be hot spots independent of their sequence position) ] and asa of the residue. figure 1.the flowchart of the procedure to predict hot spots and deposit them in the hotsprint. the flowchart of the procedure to predict hot spots and deposit them in the hotsprint. we have evaluated prediction performance of our formulation by comparing the results with the experimental hot spot data extracted from asedb (4). we assessed success of the formulations using the statistical analysis using accuracy and f - measure. our formulation yields 76.83%, 60.1%, 86.56%, 63.06% and 65.69% for accuracy (percentage of correctly predicted hot spot and non - hot spot residues over all interface residues), sensitivity (ratio of correctly predicted hot spots to all hot spots residues on the interface), specificity (ratio of correctly predicted non - hot spots to all non - hot spot interface residues), positive predictive value (number of correctly predicted hot spots divided by number of interface residues predicted as hot spot) and f - measure [2 sensitivity ppv/(sensitivity + ppv) where ppv is the positive predictive value ], respectively. ofran and rost recently developed a sequence environment and evolutionary profile - based method to predict computational hot spots (15). when we adopt the same convention, their positive predictive value (referred as positive accuracy in their text) of 60%, outperforms ours (46%). however, our sensitivity (57%, coverage in their text) is remarkably higher than theirs (15%). hotsprint provides an easy query screen with three distinct query boxes : (i) hot spot search in protein interfaces for a given pdb i d, (ii) advanced search box and (iii) conservation and asa querying of the complete protein (including non - interface residues). the computational hot spots in the interfaces can be identified based on one of the three options mentioned in supplementary data. one may either choose (i) the default hot spot criterion as defined in the methods section (pscore + asa, conservation score rescaled with conservation propensity + contribution of asa), (ii) only conservation criterion (score) or (iii) conservation score rescaled with conservation propensity (pscore) in the query page. the first query box allows the user to fetch associated interfaces of a given protein using its pdb identifier. the default thresholds in these expressions can also be modified by the user. if there exists only a single interface associated with the input pdb identifier (e.g. for pdb i d : 1axd), then information for that interface (1axdab) is displayed. however, there may be more than one interface extracted from that protein. in this case, interface identifiers of interfaces associated with that pdb are displayed (e.g. for the pdb i d 1yp2, four interfaces are available 1yp2ab, 1yp2ad, 1yp2bc and 1yp2cd). when one selects one of the interface identifiers listed, information for that interface is presented. figure 2 demonstrates the result page yielded after querying the interface 1yp2ab among the associated interfaces of 1yp2. overall properties (number of computational hot spots, number of conserved residues, average conservation score, buried asa and a link to interface information in the original dataset), individual residues and graphical representation of the interface are all displayed in this page. using the link to the original dataset, users can get detailed information about interfaces : whether it is a biological or crystal interface, and interface amino acid composition. the graphical representation part contains snapshots of the interface and its hot spots from four different perspectives and a jmol plugin is loaded in a new window when these images are clicked. overall properties (number of computational hot spots, number of conserved residues, average conservation score, buried asa and a link to interface information in the original dataset), individual residues and graphical representation of the interface are all displayed in this page. using the link to the original dataset, users can get detailed information about interfaces : whether it is a biological or crystal interface, and interface amino acid composition. the graphical representation part contains snapshots of the interface and its hot spots from four different perspectives and a jmol plugin is loaded in a new window when these images are clicked. the page presenting interface information overall properties of the interface such as number of computational hot spots on the interface, number of conserved residues on the interface, average conservation score of interface residues and buried asa of the interface are presented. the next section lists residues of the interface along with their position, name, conservation score, asa in monomer, asa in complex, type (contacting interface residue, neighboring interface residue or none). a residue is highlighted with a red background if it is a computational hot spot. static snapshots of the interface from four different perspectives are shown using rasmol (26) at the bottom of the page (figure 3). it is possible to include only contacting residues in the presented results using the check box at the bottom of the query box. figure 3.one of the four snapshots displayed in hotsprint generated by rasmol for interface 1yp2ab. an interface is composed of two sides (chain a and chain b of potato tuber adp - glucose phyrophosphorylase with pdb i d 1yp2) from two interacting proteins. interface residues are shown as balls whereas the rest of the protein is shown as the trace. the purple and red residues represent interface residues of the a and b chains of the interface, respectively. the yellow and green residues are predicted hot spots on the chains a and b, respectively. one of the four snapshots displayed in hotsprint generated by rasmol for interface 1yp2ab. an interface is composed of two sides (chain a and chain b of potato tuber adp - glucose phyrophosphorylase with pdb i d 1yp2) from two interacting proteins. interface residues are shown as balls whereas the rest of the protein is shown as the trace. the purple and red residues represent interface residues of the a and b chains of the interface, respectively. the yellow and green residues are predicted hot spots on the chains a and b, respectively. the second query box allows advanced search with different options. one can find structures satisfying given criteria among all the structures stored in the database. interfaces with certain number of computational hot spots, number of conserved residues and average conservation score can be fetched. furthermore, one may also be interested in finding interfaces with specified conserved propensities or buried accessible surface areas (asa) in a given range. for example, if interfaces with more than seven hot spots and which have 1000 asa 2000 are queried, a table listing the interface ids with respective properties is provided. at the bottom resides the final query box that can be used to access residue information (position, name, conservation score, monomer asa) of the whole protein including both the interface and non - interface residues. the results for the given structure identifier will be output by the server. as a case study, we compare the experimental hot spots of the numb ptb domain with hotsprint predictions. figure 4 displays the ribbon diagram of the numb ptb domain that is in complex with numb - associated kinase (nak)-c (pdb i d : 1ddm) (27). numb ptb domain is known to interact with a diverse set of peptides through a large hydrophobic cavity on its surface (28). the left figure presents the predicted hot spots by using pscore only, whereas the right panel illustrates the results when the pscore + asa is used. red and yellow residues are the identified as hot spots by alanine scanning substitutions on the protein complex. considering only propensity scaled conservation scores of the residues (left figure) in the interface of 1ddmab, 8 of the 10 experimentally identified hot spots (red residues) are predicted computationally. including asa further filters some of the hot spot predictions (5 of the 10 hot spots right figures present the results for the prediction of hot spots using pscore and pscore + asa, respectively. right figures present the results for the prediction of hot spots using pscore and pscore + asa, respectively. in this article, a database of computational hot spots in protein interfaces (hotsprint) is introduced. 49 512 protein interfaces are extracted from the 34 817 structures in protein data bank (pdb) as of february 2006. we defined a hot spot as an interface residue that is conserved and buried in the complex form. it is the first database, to our knowledge, which exploits sequence conservation to detect hot spots on a large scale. we believe study and characterization of hot spots will help to unravel insights of protein associations and will constitute an important step in understanding recognition and binding processes. the dataset can be downloaded as a single sql file from the website. a non - redundant subset of the database (40% homology with respect to blast) is also provided for retrieval. | we present a new database of computational hot spots in protein interfaces : hotsprint. hot spots are residues comprising only a small fraction of interfaces yet accounting for the majority of the binding energy. hotsprint contains data for 35 776 protein interfaces among 49 512 protein interfaces extracted from the multi - chain structures in protein data bank (pdb) as of february 2006. the conserved residues in interfaces with certain buried accessible solvent area (asa) and complex asa thresholds are flagged as computational hot spots. the predicted hot spots are observed to correlate with the experimental hot spots with an accuracy of 76%. several machine - learning methods (svm, decision trees and decision lists) are also applied to predict hot spots, results reveal that our empirical approach performs better than the others. a web interface for the hotsprint database allows users to browse and query the hot spots in protein interfaces. hotsprint is available at http://prism.ccbb.ku.edu.tr/hotsprint ; and it provides information for interface residues that are functionally and structurally important as well as the evolutionary history and solvent accessibility of residues in interfaces. |
in the last decade, the increased use of microarrays for global expression analyses made large - scale transcript analyses possible. using publicly available microarray datasets, several studies showed that genes with similar function tended to be transcriptionally coordinated (stuart., 2003 ; ihmels., 2004). based on this observation, co - expression approaches have been used to assign functions for genes involved in cell wall formation, isoprenoid and glucosinolate biosynthesis, and different metabolic pathways in arabidopsis (wille., 2004 ; brown., 2005 ; persson., 2005 ; wei., 2006 ; hirai., 2007). interestingly, certain co - expression relationships appear to also be conserved across different species across the kingdoms of life (stuart. for example, orthologous genes involved in protein synthesis, cell cycle, and protein degradation formed comparable co - expressed clusters in different species (stuart., 2003). in the above studies, the microarray analyses still had to be quality - controlled and evaluated by the investigators themselves to obtain co - expressed relationships. however, web - based co - expression tools now enable users to easily mine publicly available microarray dataset to investigate their gene(s) of interest. in this review we present several co - expression tools and describe recent developments and updates for them. we also give an overview of how these tools have been used to identify novel cell wall - related genes, and exemplify the use of comparative co - expression analyses across species to infer lignin - related genes. several co - expression tools have been developed for plant biology, including act (manfield., 2006), asidb (rawat., 2008), atted - ii (obayashi., 2009), cressexpress (srinivasasainagendra., 2008), csb.db (steinhauser., 2004), and genecat (mutwil., 2008), which have been comprehensively described and compared elsewhere (usadel., 2009). in addition, several recent platforms have emerged, such as aranet, cornet, genemania, planet, and ricearraynet. aranet aims at annotation of arabidopsis genes by integrating available large - scale experimental data, e.g., co - expression and protein protein interaction data, as well as gene associations inferred from other species and literature queried data (lee., 2010). similarly, genemania is predicting gene function based on available genomics and proteomics data sets for arabidopsis, yeast, and several animal model species (warde - farley., 2010). for user - defined selection of microarray experiments and cut - offs for assessing co - regulated genes in arabidopsis. protein interaction, functional annotation, and localization data (de bodt., planet provides a platform for gene co - expression network analysis for seven plant species and includes information about significant enrichment for functional annotation using mapman ontology terms (mutwil., another interesting co - expression tool is ricearraynet, which calculates positive as well as negative correlation of gene expression profiles in rice (lee., 2009). this tool has later been extended to brassica and arabidopsis, referred to as plantarraynet. in principle, the platforms above calculate co - expression relationships between two genes of interest by comparing their respective expression profiles. the pearson s correlation coefficient (pcc) is a commonly used measure to estimate transcriptional co - ordination. using this measure as a basis, co - expression relationships between many genes can be determined, and can be visualized as networks in which nodes represent genes and the connection between nodes indicates the transcriptional co - ordination of the genes. such co - expression networks can be divided into rank - based and value - based networks (aoki., 2007). previously, the most widely used approach was value - based networks, i.e., edges were established between genes that were co - expressed above a certain pcc - value threshold (lee., 2004 ; oldham., 2006). one major drawback of this method arises from the fact that some biological processes are tightly transcriptionally co - regulated, while other processes are not. therefore, when a stringent global pcc - value cut - off is applied many genes involved in weakly transcriptionally coordinated processes that may be biologically relevant become disconnected. in contrast, a lower pcc - threshold will in many instances result in excessively large gene clusters, containing thousands of genes (mao., 2009). to avoid such problems, some tools have introduced the rank - based method, which is based on the ranks of two given genes in their mutual co - expression lists (atted - ii : obayashi and kinoshita, 2009 ; planet : mutwil., 2010,2011). although both the value- and rank - based networks are derived from pcc, rank - based networks appear to lead to a network topology that closer resembles biological networks (ruan., 2010). however, it is important to note that both approaches have their advantages and drawbacks (usadel., 2009). most of the co - expression tools have focused on the model plant arabidopsis, and have incorporated the main bulk of publicly available microarray datasets, thus representing condition - independent co - expression relationships (usadel., 2009). however, some biologically relevant transcriptional relationships may be revealed only under specific experimental conditions, or in certain tissues. to find such relationships, tools like for user - defined selection of microarray experiments to calculate co - expression relationships. another example of condition - dependent databases is the updated version of atted - ii, which enables the user to analyze co - expression relationships of genes under five predefined conditions : tissue and development, abiotic stress, biotic stress, hormone treatment, and different light regimes (obayashi., 2011). furthermore, seednet is a relatively new tissue - specific database, which returns co - expression relationships of genes during seed development (bassel., 2011). one possible caveat with co - expression analyses is the rate of false positives, i.e., co - expressed genes that might be co - expressed by chance rather than being functionally related. a useful approach to minimize the rate of such false positives may be to investigate whether orthologous genes are also co - expressed in related species. as mentioned above, co - expression relationships are often conserved across species (stuart., 2003). hence, two co - expressed genes from one species often have orthologs in another species that in turn are also co - expressed. in theory it should therefore be possible to enrich for co - occurring co - expression relationships across species, and hence minimize false positives. therefore, across species co - expression analyses might improve the reliability of co - expression - based functional annotation. consequently, several tools, such as starnet, cop, and atted - ii, allow pairwise comparison between species (jupiter.. moreover, comparison of several species at the same time was introduced by the networkcomparer - tool of planet (mutwil., 2011). this tool bins genes into gene families according to their pfam annotation (finn., 2010), and then finds recurring pfams in co - expression networks across species (mutwil., 2011). cellulose is produced by the cellulose synthase (cesa) complex (csc), which is comprised of cesa1, 3, and 6-related proteins in the primary wall and cesa4, 7, and 8 in the secondary wall (gardiner. interestingly, these primary and the secondary cesas display similar expression patterns, respectively (brown., 2005 ; persson., 2005). in addition, these studies were able to show that many genes involved in xylan and lignin synthesis were co - expressed with the secondary wall cesas (brown., 2005 ; persson., 2005). hence, co - expression analyses may be useful to identify new genes involved in secondary cell wall - related synthesis. figure 1 shows a truncated node - vicinity network (nvn) of genes co - expressed with the primary wall - related arabidopsis cesa1-gene, which was obtained from planet (mutwil., 2011). several primary wall cesa genes (cesa2, cesa3, cesa5, and cesa6) may be found in close vicinity of cesa1. in addition, many other genes important for cellulose synthesis, such as cobra (cob), chitinase - like (ctl)1, cellulose synthase interacting (csi)1/pom - pom2, and korrigan (kor), are present in this network (nicol., 1998 ; 2002 ; roudier., 2005 ; gu., 2010 ; this result confirms previous findings which were based on a smaller number of microarrays (persson., 2005), and is similar to results obtained from other co - expression tools. for example, the many of the genes in this network are also present in the atcesa1-top 300 list of co - expressed genes in atted - ii (77 out of 190 genes ; see also the genes marked in bold in figure 1). subset of the co - expression gene vicinity network of atcesa1 (turquoise node). nodes indicate individual genes, and edges indicate whether two genes are co - expressed above a certain mutual rank threshold. node colors indicate whether mutations in the gene cause gametophytic lethality (yellow), any biological phenotype (green), or if no mutant phenotype information currently is available (gray) according to tair. green, orange, and red edges indicate a mutual rank relationship < 10 (green), 1019 (orange), and 2029 (red), respectively, for each connected gene pair. genes marked in bold are also present in the top 300 co - expression list of atcesa1 obtained from atted - ii (http://atted.jp/). the respective gene family according to pfam annotation is given below the gene names / agi codes (finn. in addition to cellulose synthesis, co - expression approaches have been used to identify genes involved in the synthesis of hemicelluloses. (2007) found that the arabidopsis cslc4 gene, which presumably is involved in glucan backbone synthesis of xyloglucan, was co - expressed with the xylosyltransferase atxt1, which has previously been shown to attach xylose residues to a glucan backbone (faik., 2002). moreover, expression profiling was used to identify irx15 and an irx15-like gene, with corresponding single mutants showing a mild irregular xylem phenotype (jensen., 2010 ; both genes belong to a gene family with a domain of unknown function (duf) 579 and only the corresponding double mutant showed decreased levels of xylan and altered cell wall morphology in stems (jensen., 2010 ; although their exact function is unknown, these results suggest an important role of this duf579-gene family in xylan biosynthesis (jensen., 2010 ; brown., 2011). interestingly, also many genes that are important for lignin synthesis are co - expressed. (2005) showed transcriptional co - ordination of many putative and bona fide genes involved in monolignol biosynthesis, transport, and polymerization. (2005) that found laccase genes, which are probably involved in polymerization of lignin, in the list of genes that are co - expressed with secondary wall - related cesas. (2007) was one of the first studies to compare transcript relationships of cell wall - related genes across monocots and dicots. based on est data, the authors found that members of the gt43-, gt47-, and gt61-family are enriched in monocots and therefore might be involved in biosynthesis of the grass - specific glucuronoarabinoxylan (mitchell., 2007). indeed, the same group could recently show that several gt61-members are essential for arabinosylation of xylan (anders., 2012). another recent study compared the co - expression relationships of xylan - related genes from arabidopsis and rice (oikawa., 2010). using the arabidopsis genes irx9, irx10, and irx14, which have previously been associated with xylan backbone synthesis (brown., 2005 ; pea., 2007), the tentative rice orthologs were identified based on sequence homology and expression patterns (oikawa., 2010). these genes were then utilized as baits in the atted - ii tool and revealed many re - occurring homologs in the respective co - expression lists (oikawa., 2010). a similar co - expression approach has been undertaken for cellulose - related genes by comparing the co - expression networks of primary and secondary cesas from seven species using planet (ruprecht. interestingly, many gene families are consistently co - expressed with cesa genes across species. the function of most of these gene families remains unknown ; however, their conserved transcriptional co - ordination suggests that they fulfill important functions during cellulose synthesis (ruprecht., 2011). based on the results, an arabidopsis mutant corresponding to pinoresinol reductase (prr) 1 was identified that displayed distorted xylem vessels (ruprecht., 2011), probably due to decreased lignin or lignan production. while comparative transcriptional analyses across species have been undertaken for cellulose and hemicelluloses - related genes, conserved co - expression relationships among lignin - related genes have been paid less attention. similar to previously published results (ehlting., 2005), we found that the co - expression network of arabidopsis phenylalanine ammonia lyase (pal) 1, which marks the initial step for monolignol synthesis, comprises many genes involved in lignin formation figure 2b). using atpal1 as bait in the networkcomparer - tool of planet, we identified similar co - expression networks for atpal1-orthologs in barley, medicago, poplar, rice, soybean, and wheat. the resulting consensus network indicates that most of the gene families needed for monolignol synthesis are conserved across species (figure 2). surrounding the pal-gene family, we found gene families corresponding to the subsequent steps in the lignin pathway such as c4h (which belongs to gene family p450 according to pfam annotation, finn., 2010), 4cl (amp_binding), hct (transferase), c3h (p450), ccoaomt (methyltransf_3), ccr (epimerase), and cad (adh_zinc_n). in addition, genes functioning upstream of pal in the phenylalanine synthesis pathway were previously identified as transcriptionally linked with the phenylpropanoid - related genes in arabidopsis (tohge and fernie, 2010), which appeared to be also conserved in other species (i.e., gene families dahp_synth_2, udpgt, corresponding to glycosyltransferases, was present in almost all pal1-derived co - expression networks across the seven species (figure 2). members of this family have been shown to glucosylate various monolignols (lim., 2001) and we therefore hypothesize that the respective co - expressed genes of this family might have a similar function in the different species. recently, miao and liu (2010) showed that abc - transporters are likely to mediate transport of monolignols, and their glucoconjugates, across plasma and vacuolar membrane, respectively. the atpal1-network contained three different genes from the abc - transporter family (abc2_membrane) which might mediate this function in arabidopsis. while single knock - out mutants for one of them (atabcg33) did not show any obvious phenotypes (kaneda., 2011), it is plausible that the other two members may functionally compensate for the loss of atabcg33. interestingly, the consensus network also comprises several highly conserved gene families, whose function in lignin biosynthesis is still elusive, for example, the gene families hydrolase, abhydrolase_1, and abhydrolase_3. one of the co - expressed genes from the hydrolase-family in arabidopsis is responsive - to - antagonist (ran) 1, which encodes for a copper transporter involved in ethylene signaling (hirayama., 1999). copper is a co - factor of laccases, which are important for polymerization of monolignols to lignin in the cell wall (boerjan., 2003). we therefore speculate that ran1 has an additional function in providing copper co - factors for enzymatic lignin formation. abhydrolase_1-family (at3g03990) are methylesterases, suggesting that these genes might function antagonistically to caffeic acid o - methyltransferases (comts). peroxidase-family, which one might associate to monolignol polymerization in the cell wall. however, the respective co - expressed gene in arabidopsis is ascorbate peroxidase (apx)1, a cytosolic enzyme that has an important role in scavenging hydrogen peroxide (davletova., 2005). apx1 might therefore function in preventing premature radical coupling of monolignols already in the cytosol. thus, exemplified by the lignin - related analysis, we propose that comparative co - expression analyses might be useful in the future to reveal novel players for different aspects of cell wall biosynthesis, but also in high - lighting conserved and divergent elements in different biological processes. a) consensus co - expression network of lignin formation across seven plant species obtained from the networkcomparer - tool from planet (http://aranet.mpimp-golm.mpg.de/aranet/networkcomparer). co - expression network of following genes / probeset ids were used : arabidopsis, at2g37040 ; rice, loc_os02g41650 ; medicago, medtr1g076720 ; barley, contig1805_s_at ; poplar, ptpaffx.1672.3.a1_a_at ; wheat, taaffx.45277.1.s1_x_at. (b) monolignol biosynthesis pathway (adopted from boerjan., 2003). arabidopsis genes present in the co - expression network of atpal1 were mapped on the pathway. question marks indicate that the function of the respective genes has not been shown yet. co - expression approaches have been especially valuable for identifying new genes involved in secondary cell wall synthesis (brown., 2005,2011 ; persson., 2005 ; jensen., 2010 ; ruprecht., a comparison of the candidate genes from several different studies showed that similar results were obtained regardless of the underlying microarray datasets (i.e., condition - independent or stem - specific samples) and the bait genes (xylan or cellulose synthesis - related) used (oikawa., 2010). this is probably due to the fact that secondary cell wall formation is a highly coordinated process that is mainly restricted to certain tissue and cell types. however, many genes that are transcriptionally associated with secondary cell wall formation have already been investigated, and mutant analyses targeting only one gene are likely to only yield mild or no phenotypes (jensen., 2010 ; brown., 2011 ; ruprecht., 2011). one likely reason for this is genetic redundancy, and hence, mutant combinations or knock - down approaches that target several homologous genes might be needed in the future to generate informative phenotypes. in addition, detailed comparative transcriptional studies across and within species might be needed to obtain more reliable candidate genes related to cell wall synthesis. 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. | global transcript analyses based on publicly available microarray dataset have revealed that genes with similar function tend to be transcriptionally coordinated. indeed, many genes involved in the formation of cellulose, hemicelluloses, and lignin have been identified using co - expression approaches in arabidopsis. to facilitate these transcript analyses, several web - based tools have been developed that allow researchers to investigate co - expression relationships of their gene(s) of interest. in addition, several tools now also provide the possibility of comparative transcriptional analyses across species, which potentially increases the predictive power. in this short review, we describe recent developments and updates of plant - related co - expression tools, and summarize studies that have successfully used expression profiling in cell wall research. finally, we illustrate the value of comparative co - expression relationships across species using genes involved in lignin biosynthesis. |
ena / vasp proteins regulate cell migration by promoting actin polymerization at the plasma membrane via antagonizing actin filament capping and acting as processive actin polymerases (barzik., 2005 ; bear., 2002 ; breitsprecher., 2008, 2011 ; hansen and mullins, 2010 ; pasic., 2008). each family member consists of an n - terminal evh1 domain, a central proline - rich region, and a c - terminal evh2 domain (bear and gertler, 2009). the evh2 domain, which contains monomeric and f - actin binding sites, is responsible for promoting actin polymerization (barzik., 2005 ; breitsprecher. in contrast, the evh1 domain mediates intracellular targeting of ena / vasp proteins by interacting with a sequence (d / e)fppppx(d / e)(d / e), which is referred to as the fpppp motif because these residues are essential for binding (bear., 2000 ; niebuhr. ena / vasp proteins are recruited to focal adhesions by zyxin, which contains four fpppp motifs (drees., 2000 ; the ability of ena / vasp proteins to control cell migration, however, depends on their recruitment to the leading edge (bear., 2000, 2002), by fpppp motif containing mrl proteins (mig10, riam, and lamellipodin ; col., 2005 ; krause., 2004 ; lafuente., 2004 ; quinn., 2006). of all the proteins interacting with the evh1 domain of ena / vasp proteins, tes, a focal adhesion protein, stands out as the only one that lacks an fpppp motif (coutts., 2003 ; garvalov., 2003). tes negatively regulates the localization of mena at focal adhesions and also inhibits mena - dependent cell migration (boda., 2007). tes interacts with mena via its c - terminal lim3 domain and is unique in being the only protein that binds a single ena / vasp family member (boda. 2003). given the interaction of tes with mena, we sought to identify additional atypical evh1 binding partners that also lack we found that the evh1 domain interacts directly with abi, a component of the wave regulatory complex (wrc), which plays an essential role in driving cell migration by activating the arp2/3 complex in response to rac signaling (bisi., 2013). our observations demonstrate that the evh1:abi interaction enhances cell migration and the ability of rac - activated wrc to promote arp2/3-mediated actin polymerization as well as the function of wrc in vivo in drosophila. to identify ena / vasp binding proteins lacking fpppp motifs we performed pull - down assays with gst - tagged evh1 domain of mena on lysates from mv cells, which lack endogenous mena and vasp (figure 1a). mass spectrometry analysis of the resulting bands identified zyxin and four subunits of the wrc : abi1, nap1, pir121, and wave2 (figure 1a). western blot analysis confirmed that the mena evh1 domain interacts with the wrc (figure 1b). furthermore, this interaction depends on its fpppp binding activity, because preincubation of the resin with the fpppp region of zyxin (evh1-fpppp) or the lim3 domain of tes (evh1-lim3) inhibited binding (figure 1b). using bacterially expressed proteins, we found that the evh1 domains of mena, evl, and vasp are all capable of interacting directly with abi1 (figures 1c and 1d). the evh1 domain of mena can also bind abi2 and abi3 (figure 1e). human and mouse abi1 lack fpppp motifs, but do contain extensive proline rich regions that may contain alternative evh1 binding sites. consistent with this, pull - down assays reveal that a wave complex containing abi1(1 - 159) lacking its c - terminal proline rich region and sh3 domain can bind rac1 but not the vasp evh1 domain (figure 1f). evh1 binding motif was first identified in acta by screening a series of overlapping peptides using a far western approach (niebuhr.,, we found that the mena evh1 domain interacts with a series of peptides covering residues 352394 in the proline rich region of human abi1 (figure 2a). the evh1 domains of evl and vasp also bound the same region although the peptide binding patterns were not completely identical (figures s1a and s1b available online). probing three overlapping peptide arrays containing systematic amino acid substitutions at each position we found that mutation of proline residues 366368 (peptide 1) and 383385 (peptide 3) as well as phenylalanine 375 (peptide 2) significantly reduced binding (figure 2b). substitution of prolines 366368 and 383385 with glycine as well as phenylalanine 375 to alanine (termed abievh1) abrogated all peptide interactions with the mena evh1 domain (figure 2c). pull - down assays on lysates from cells expressing gfp - abievh1 confirmed that these mutations disrupt the interaction of abi1 with mena but not the rest of the wave complex (represented by the pir121 subunit ; figure 2d). to investigate the impact of the loss of the interaction between abi1 and ena / vasp proteins on cell migration, we stably expressed gfp - tagged abi or abievh1 in ht1080 cells (figure s1c). both gfp - tagged proteins are recruited to the leading edge of migrating cells (figure 2e). expression of abievh1 but not abi, however, retarded the migration of ht1080 cells into a scratch (figures 2f and s1d). conversely, in the absence of endogenous abi1, gfp - tagged abievh1 was less effective at promoting cell migration than the wild - type protein (figures 2f and s1d). given the impact of abievh1 on cell migration, we wondered whether the interaction of evh1 with abi1 modulates the activity of vasp and/or the wrc. to explore this possibility, we performed in vitro actin polymerization assays using recombinant vasp and wrc (figures 3 and s2). in the absence of its canonical activator, rac1, the wrc can not stimulate arp2/3-mediated actin assembly (figure 3a, red solid curve). in contrast, vasp promotes actin assembly (figure 3a, black dotted curve). further addition of the wrc produced no further changes in actin assembly (figure 3a, red dotted curve), indicating that vasp does not activate the inhibited state of the wrc, or vice versa. in our assay conditions (100 mm kcl), the wave1 vca peptide only slightly increases actin assembly by the arp2/3 complex (figure 3b, green solid curve). saturating concentrations of rac1 increase wrc - mediated actin assembly to this same level (figure 3b, red solid curve). addition of vasp to the rac1-activated wrc significantly increases the extent of actin polymerization (figure 3b red dotted curve) in a dose - dependent manner (figure 3c). this increase is not the result of simple summation of the activity of vasp and the arp2/3 complex, as when the activated wrc is substituted with the vca peptide, the increase in actin polymerization is only additive (figure 3b, green curves). to examine whether direct interaction between vasp and the wrc is responsible for the enhancement in actin assembly, we tested a variant of the wrc (wrc - abi1[1159 ]), which can not bind the evh1 domain as it lacks the proline rich region of abi1 (figure 1f). wrc - abi1(1159) can be activated by rac1, but failed to recapitulate the increased activity with vasp seen for the full - length wrc (figure 3b, compare blue and red dotted curves). we next sought to understand how the vasp - wrc interaction promotes enhanced actin assembly. in principle, this could arise from increased activity of either vasp or the wrc. when the arp2/3 complex is removed from assays containing both vasp and activated wrc, the total activity dropped to the level of vasp alone (figure 3b, orange curve). therefore, the increased activity depends on the arp2/3 complex, suggesting that vasp increases the activity of wrc, but not vice versa. because the isolated evh1 domain of vasp can not increase the activity of the wrc (figure 3d), we sought to discover which other regions of vasp are required for this effect. in addition to the evh1 domain, vasp has a c - terminal evh2 domain, which includes a g - actin binding motif (gab), an f - actin binding motif (fab), and a coiled coil maintaining vasp as a constitutive tetramer (bear and gertler, 2009). deletion of the coiled coil, which generates a monomeric form of vasp (vasp [1335 ]), fails to recapitulate the increased activity seen with the full - length vasp (figure 3d). furthermore, the fab motif is also necessary, since its mutation, which completely disables vasp in actin assembly (breitsprecher., 2008, 2011 ; hansen and mullins, 2010 ; pasic., 2008), no longer allows vasp to enhance actin assembly by the wrc even at a concentration of 5 m (figure 3e). in contrast, mutation of the gab motif alone, which significantly decreases the activity of vasp in actin polymerization, still allows vasp to stimulate wrc activity (but only at high concentrations of 15 m range ; figure 3f). taken together, our data suggest that disrupting the actin polymerization activity of vasp impairs its ability to enhance wrc activity. to facilitate further analysis of the physiological role of the interaction between ena / vasp proteins and the wrc, we switched to drosophila because it only has a single isoform for each protein. drosophila abi (dabi) and the evh1 domain of ena have 38% and 72% sequence identity to their respective human counterparts (figure 4a). drosophila abi also lacks the evh1 binding motif we identified in human abi1 (figure 4a). nevertheless, the evh1 domain of ena still retains the wave complex from drosophila s2 cell lysates (figure 4b). using a far western approach, we found that the evh1 domain of ena bound two sets of dabi peptides containing an fpppp motifs (ball., 2000 ; niebuhr., 1997 ; peterson and volkman, 2009). far western analysis of peptide arrays containing systematic substitutions at each position confirmed that both lpppp motifs are important for evh1 binding (figure 4e). to establish if these motifs are important in the context of dabi, we performed pull - down assays on wild - type and mutants in which the two lpppp motifs were mutated to agggg, either alone or in combination (figure 5a). substitution of residues 311315 (mut1) substantially weakened but did not completely abolish the interaction of dabi with the ena evh1 domain. in contrast, mutation of residues 374378 (mut2) resulted in a loss of binding (figure 5a). motifs did not, however, disrupt the ability of dabi (dabiena) to colocalize with wave at the plasma membrane of drosophila s2 cells (figure 5b). the dabiena mutant was also as effective as the wild - type protein at rescuing the spiky morphology of drosophila s2 cells induced by rnai - mediated loss of endogenous dabi (figure 5c ; kunda., 2003 ; rogers., 2003). to examine whether the interaction between ena and dabi helps to stabilize the wrc at the plasma membrane, we performed fluorescence recovery after photobleaching (frap) experiments on drosophila s2 cells treated with rnai targeting the 3 utr of endogenous dabi and expressing gfp - tagged abi or abiena. we found that the loss of ena binding results in a statistically significant 1.38-fold increase in the exchange rate of dabi at the plasma membrane (figure 5d). importantly, pull - down assays on s2 lysates demonstrate that the loss of the interaction with ena did not disrupt the ability of dabi to incorporate into the wave complex (figure 5e). to investigate the physiological significance of the interaction between ena and abi in regulating wrc functions in vivo, we took advantage of abi mutant flies (stephan., 2011). we first examined the consequences of the loss of the abi : ena interaction in drosophila macrophages. structured - illumination microscopy analysis of spreading wild - type macrophages reveals a highly polarized actin cytoskeleton with a broad lamellipodial cell front (figures 6a and 6b). macrophage - specific knockdown of wave in larval macrophages using the hemolectin - gal4 driver completely disrupts lamellipodia formation (figure 6a ; sander., 2013). the abi mutant macrophages also had a similar spiky morphology (figures 6a and 6b). these defects in cell morphology were substantially rescued by ubiquitous reexpression of wild - type abi but not by the mutant lacking both ena binding motifs (abiena) from the same genomic locus (figures 6a and 6b). to further analyze differences in lamellipodia protrusions and cell shape of rescued macrophages we found that cells expressing abi exhibit more stable and periodic membrane protrusions, whereas the abiena population are significantly less circular (more spiky) and have a reduced rate of membrane protrusion, (figure 6c ; movie s1). furthermore, in contrast to wild - type macrophages, ena is no longer at the leading edge of lamellipodial protrusions but becomes relocalized to the tips of filopodia - like protrusions and along stress fiber - like actin bundles when it can not interact with abi (figure 6d). in vivo, abi and a functional wrc are required in the drosophila larval visual system for the correct axonal targeting of photoreceptor neurons (r - cells) to their respective optic ganglions in the fly brain (stephan., 2011). remarkably, we found that the loss of the ability of abi to interact with ena resulted in a similar defect in r - cell targeting as the complete loss of abi (figures 7a and 7b). we also examined the impact of an abi mutant lacking its ena binding sites and the c - terminal sh3 domain (abienash3) because an abi transgene lacking its c - terminal sh3 domain is able to substantially rescue abi mutant phenotypes (stephan., 2011). in contrast to abiena, the expression of abienash3 surprisingly rescued the abi - dependent r - cell targeting defects (figures 7a and 7b). overexpression of the abi variants in a wild - type background, however, did not affect r - cell targeting excluding any dominant effects (figure s3). next, we analyzed the cellular requirement for the abi : ena interaction during oogenesis because, in contrast to the visual system, wrc and ena control cell - autonomous actin - based structures that are essential for normal egg development (gates. loss of wave and arp2/3 in the germline results in small and abnormally shaped eggs (hudson and cooley, 2002 ; zallen., 2002). a similar dumpless mutant phenotype is also observed in flies lacking abi in the germline (zobel and bogdan, 2013). rescue experiments using a puast - abi transgene that is only expressed in somatic follicle cells and not the germline rescues the lethality of abi mutants (zobel and bogdan, 2013). however, the integrity of cortical actin in nurse cells within the egg chamber is disrupted and the rescued female flies are completely sterile (figures 7c and 7d). to overcome this problem, we generated puasp transgenes for efficient germline expression (rrth, 1998) and performed rescue experiments with the different abi variants. reexpression of wild - type abi rescues the egg morphology defects and female sterility of abi mutant flies (figures 7c and 7d). in contrast, abi mutant flies expressing abiena are completely sterile, containing smaller and abnormally round eggs (figures 7c7e). this round - egg phenotype resembles those seen in rac1, rac2 mutant follicle cell clones defective for egg chamber elongation rather than flies lacking abi in the germline (conder., we observed a similar round - egg phenotype when we suppress wrc function in follicle cells using an sra-1 rnai transgene (figure 7f). late - stage abiena mutant egg chambers also show additional defects in nurse cell cortical actin integrity, resulting in detached cytoplasmic actin bundles and ring canals (figure 7c). these defects closely resemble phenotypes found in abi mutants lacking germline abi (puast - abi rescue ; zobel and bogdan, 2013) and wave germline mosaics (zallen., 2002). because overexpression of abiena in a wild - type background did not affect fertility, dominant effects can again be excluded (figure 7d). finally, we examined the impact of an abi mutant lacking its ena binding sites and the c - terminal sh3 domain (abienash3). consistent with our observations in the larval visual system, abienash3 rescued the egg morphology defects and female sterility of abi mutants (figures 7c7f). in summary, our data demonstrate that the abi : ena interaction plays an important role in vivo in regulating diverse actin - based structures and morphogenetic processes that require a functional wrc. the wrc binds and activates the arp2/3 complex to drive actin polymerization at the plasma membrane in response to rac signaling during cell migration (bisi., 2013). in contrast, ena / vasp proteins stimulate cell migration by antagonizing actin filament capping and acting as processive actin polymerases (barzik., 2005 ; bear and gertler, 2009 ; bear., 2002 ; breitsprecher., 2008, 2011 ; hansen and mullins, 2010 ; pasic., we have now demonstrated that ena / vasp proteins can be linked to the function of wrc by virtue of a direct interaction between their evh1 domains and abi, an integral component of the wrc. our results have confirmed and extended previous yeast two - hybrid data and pull - downs from cell lysates demonstrating that the evh1 domains of mena and vasp can interact with human and mouse abi1 (dittrich., 2010 ; hirao., 2006 ; maruoka., 2012 ; tani., the structure of several evh1:fpppp complexes reveals that the fpppp motif adopts a type ii polyproline helix that is coordinated by three aromatic residues present in all ena / vasp family members (peterson and volkman, 2009). in contrast, the evh1 domain interacts with an extended proline - rich binding site in human abi1. consistent with their ability to bind, abi2 has an almost identical sequence whereas abi3 has two lpppp motifs in this region. in many respects, the extended nature of the abi1 interaction resembles that of the n - wasp wh1 binding site in wip, which also involves three regions of contact (peterson., 2007). in classical evh1 interactions, the acidic residues flanking the fpppp motif play an important role in determining the affinity, orientation and specificity of evh1 binding (ball., 2000 ; peterson and volkman, 2009). in contrast, the evh1 binding site in human abi1 contains two pairs of aspartic acid residues flanking the central phenylalanine in the middle of the motif as well as a downstream acidic patch (dyedee ; figure 4a). the molecular basis of the evh1 human abi1 interaction, including the extended peptide orientation and role of acidic residues, must await structural determination of the complex. nevertheless, our data clearly demonstrate that the evh1 domain can bind additional proline rich ligands beyond fpppp motifs. interestingly, the meander region of wave1 contains an lpppp motif that is capable of interacting with mena (okada., 2012). the ability of mena to bind abi in the wrc presumably explains why it still associates with wave lacking its proline rich region (okada., 2012). consistent with the presence of lpppp motifs pull - downs with recombinant proteins demonstrate that the evh1 domain of mena can interact with wave 1 and 2, but not wave 3 (figure s2b). our observations, however, suggest that the interaction with abi is more important for mena interactions with the wrc than wave (figure 4d). moreover, our in vitro assays clearly demonstrate that the ability of rac to activate wrc - mediated actin polymerization via the arp2/3 complex is significantly enhanced by vasp binding to abi. in contrast to the full - length protein, monomeric vasp or its isolated evh1 domain is unable to activate the wrc to stimulate arp2/3-mediated actin polymerization even at high concentrations. this difference may reflect the ability of the vasp tetramer to induce oligomerization of the wrc, an effect that would enhance wrc potency toward the arp2/3 complex (padrick., 2008 ; padrick and rosen, 2010). it is possible that the simultaneous engagement of a vasp tetramer with abi and the lpppp motif in wave increases the activity of the wrc. however, oligomerization alone can not account for our data because mutating the actin binding elements of vasp, which should have no effect on tetramerization, abrogates activity. furthermore, the vasp effect does not appear to be simple allosteric activation of the wrc (i.e., release of the vca), because this should produce activity equal to that of the vca alone. while not definitive, our collective data are most consistent with a model in which vasp binds the rac - activated wrc with high affinity based on tetramerization - mediated avidity and also interacts with actin filaments, thus increasing the association of the wrc with filaments. because both the released wave vca and actin filaments activate the arp2/3 complex (machesky., 1999), assembling these two elements should enhance their cooperative actions and increase actin assembly. in contrast to the situation in humans, the interaction between the evh1 domain of ena and abi in drosophila is mediated by two lpppp the loss of these two lpppp motifs increases the dynamics of the wrc at the plasma membrane but did not affect lamellipodium formation in s2 cells in culture. in contrast, the consequences of disrupting the interaction of ena with abi in vivo are more dramatic, as primary macrophages expressing abiena have reduced lamellipodial membrane protrusions and defects in cell morphology. unlike the situation in s2 cells, which have been treated with dsrna and transiently transfected with gfp - tagged expression constructs, the abi transgenes (abi and abiena) are expressed from the same genomic locus. these in vivo rescue experiments therefore allow for a more precise analysis of the requirement of the interaction between ena and abi rather than in the hypomorphic situation in s2 cells. the ability of abiena to rescue lamellipodium formation in s2 cells might reflect an incomplete abi knockdown or a difference in its expression level compared to endogenous abi in untreated cells. consistent with this, in macrophages, we found that strong expression of abi in earlier larval stages using the da - gal4 driver results in a more robust rescue of lamellipodia protrusion and cell morphology defects as compared to macrophage - specific expression (hml-gal4) at late larval stages. given that our in vitro actin polymerization assays indicate that vasp (ena) is not an essential activator but rather acts cooperatively with rac1 to promote wrc activation, it is likely that in vivo the requirement for this interaction depends on the level of abi. this explanation may also partially account for the more dramatic phenotypes observed in the multicellular context. remarkably, we found that the loss of the ability of abi to interact with ena resulted in a similar defect in r - cell targeting as the absence of the complete protein. this suggests that ena has a nonautonomous role in the larval brain, as we have previously shown for wrc function in targeting of early retinal axons (stephan., 2011). mosaic mutant analysis further supports a nonautonomous function for ena in retinal axon targeting (data not shown). thus, we propose that the interaction between ena and the wrc is required to regulate actin dynamics in the target area neurons. however, since the precise projection pattern of early retinal axons depends on complex interactions between different populations of glia cells and neurons in the target field, it remains unclear how ena and the wrc function together in this developmental context. in contrast, drosophila oogenesis provides an excellent model to study the cell autonomous function of the interaction between ena and the wrc. previous phenotypic analyses of mutant egg chambers suggest ena and wrc have both distinct and overlapping functions during oogenesis (gates., 2009 ; zallen., 2002) both are required for the integrity of the cortical actin in nurse cells and mutant egg chambers become multinucleated as the plasma membrane breaks down due to a loss of cortical actin integrity. in contrast, to wave mutant egg chambers, disruption of ena function does not affect ring canal morphology but rather leads to a reduced and delayed formation of cytoplasmic actin filament bundles (gates., 2009 ; zallen., 2002). similar to wave germline clones, the loss of abi in the germline results in a dumpless mutant phenotype and female flies are sterile (zobel and bogdan, 2013). we have now found that these defects in egg morphology and female fertility can not be rescued by reexpression of a full - length abi deficient in ena binding. abiena mutant egg chambers have defects in the integrity of the nurse cell cortical actin resulting in detached cytoplasmic actin bundles and ring canals. the rupture of nurse cell membranes is even more obvious at later stages when the fast transport of nurse cell contents starts, as recently observed for ena, wave, and abi mutants (gates., 2009 ; zallen., 2002 ; zobel and bogdan, 2013). in addition to nurse cell dumping defects, we also observed a striking egg chamber elongation defect. mutant eggs lacking the interaction between abi and ena fail to elongate and remain spherical as similarly found in rac or pak mutants (conder., 2007). the round egg phenotype observed in flies expressing abiena suggests that there might be a defect in the basal actin cytoskeleton of the follicle cells that drives egg chamber elongation (bilder and haigo, 2012 ; gates, 2012). consistently, reexpression of abiena in somatic follicle cells (abi, da > uast - abiena) also results in a round - egg phenotype. these data suggest a requirement of wrc function in follicle cells during egg elongation. supporting this notion, we found that a follicle cell - specific knockdown of sra-1 function results in a strong round - egg phenotype. our rescue experiments additionally imply a more complex interaction network among ena, abi, and sh3 interacting proteins. whereas a minimal abi fragment lacking the ena - binding or proline - rich region and the c - terminal sh3 domain is able to rescue substantially abi mutant traits in drosophila and dictyostelium (davidson., 2013 ; stephan., 2011) thus, we propose a scenario in which the influence of ena on wrc activity depends on additional proteins interacting with the abi - sh3 domain. the most prominent candidate is the nonreceptor tyrosine kinase abelson (abl) that binds abi and ena proteins (dai and pendergast, 1995 ; gertler., 1989). based on the antagonistic genetic interaction between ena and abl, it has been hypothesized that a precise balance between abl and ena activity is required for fly viability. however, it is still unclear how abl affects the function of ena, because mutation of all known abl phosphorylation sites only has a modest effect on ena function in vivo (comer., 1998 ; similarly, abl and abi have opposing roles in drosophila (lin., 2009). thus, we propose a model in which ena synergizes with rac to activate the wrc, but also inhibits abl function. abl in turn inhibits wrc function as previously shown (lin., 2009) thus, the disruption of ena binding to dabi would simultaneously decrease wrc stimulation by ena and increase its inhibition by abl. such a scenario would explain why loss of ena binding to abi (wrc) phenocopies the abi mutants. this also suggests that the interaction among wrc, abl, and ena function is of more general relevance for actin - based processes in multicellular contexts. furthermore, recent data also suggest that lamellipodin, which cooperates with the wrc to promote cell migration in vivo, is also likely to be part of this complex regulatory network, because it can bind both the evh1 domain of vasp and the sh3 domain of abi (krause. our in vitro data clearly demonstrate that ena / vasp proteins can directly affect the activity of the wave complex, whereas our observations in drosophila have revealed that, in vivo, the function and activity of ena / vasp proteins and the wave complex are intimately linked. bacterial expression vectors for gst - tagged evh1 domains of vasp, evl, and mena as well as the lim3 domain of tes and fpppp region of zyxin have been described (boda., 2007). the evh1 domain of drosophila ena (residues 1115) as well as human abi1 - 3, wave1 - 3, vasp, nap1, and pir121 were amplified by pcr and cloned into the not1-ecor1 sites of pmw172-gst-3c or pmw172-his-3c to generate a gst or his - tagged e. coli expression vectors. the vasp 1335 and the gab (l226a / i230a / l235a / r236e / k237e) and fab (r273e / r274e / r275e / k276e) mutants (breitsprecher., 2011 proteins were typically expressed in bl21(de3) rosetta cells and purified as previously described (boda. all his - tagged vasp proteins were expressed in bl21 (de3) t1 cells at 18c and purified by ni - nta agarose beads, a source sp15 column, and finally a superdex200 or superfex75 column. other proteins, including arp2/3 complex, actin, vca, rac1 q61l, and tev protease were purified as previously described (ismail., 2009). pull - down assays with recombinant proteins on mv and s2 cell extracts were performed as previously described (boda., 2007, 2011). stained protein bands (imperial protein stain, thermo scientific) were reduced, alkylated, and digested with trypsin, as previously described (collazos., 2011). the digests were analyzed with liquid chromatography - tandem mass spectroscopy (lc - ms / ms) on an agilent 6510 mass spectrometer (agilent). lc - ms / ms data were searched against a protein database (ncbinr 20080210) using the mascot search engine (matrix science). the human wrc was purified as previously described (chen., 2014). gst pull - down was performed by mixing 380 pmol of bait proteins (gst or gst - tagged evh1 or rac1) with equimolar prey proteins (wrc or wrc - abi1[1159 ]) and 20 l of glutathione sepharose beads (ge healthcare) in 1 ml of binding buffer (20 mm hepes, 100 mm nacl, 5% [w / v ] glycerol, 2 mm mgcl2, 1 mm egta, and 5 mm -mercaptoethanol, ph 7) at 4c for 30 min. bound proteins were eluted with 30 mm reduced glutathione and examined with sds - page. actin polymerization assays were performed at 22c using a pti fluorometer (photon technology international) as previously described with slight modifications (ismail., 2009). reactions contained 4 m rabbit muscle actin with 5% labeled with pyrene, 10 nm bovine arp2/3 complex, 100 nm human wrc, and/or other proteins of interest in 10 mm imidazole, 100 mm kcl, 1 mm mgcl2, 1 mm egta, 20% (w / v) glycerol, and 1 mm dithiothreitol, ph 7.0. note that under these conditions, the wrc fully activated by rac1 (or equivalently, 100 nm vca) only exhibits modest activity toward the arp2/3 complex. monoclonal antibodies gfp 3e1 (cancer research uk), actin ac-74 (sigma), and zyxin 164d4 (sysy) as well as rabbit antibodies against abi1 (sigma a5106) and vasp (cell signaling 3132s) were used. antibodies against human abi1, nap1, and pir121 (steffen., 2004) were kindly provided by dr theresia stradal (university of muenster, germany). immunofluorescence and immunoblot analyses were performed as described (boda., 2007). projection patterns of all photoreceptor axons of the indicated genotypes were depicted using mouse anti-24b10 (-chaoptin, dshb ; stephan., 2011). human abi1 or abi1evh1 were cloned into the not1/ecori sites of a modified plvx - puro - gfp vector and used to generate lentiviruses. ht1080 cell lines stably expressing gfp or the gfp - tagged proteins were selected with puromycin and facs sorting. for scratch assays, the cell lines were transfected with qiagen allstars negative control or sirna hs abi1 9 which targets the 3utr of abi1. a day later, 5 10 cells were seeded into each well of a 96-well imagelock microplates (six replicate wells / condition ; essen bioscience) and after 24 hr, a scratch was made in each well using the essen instruments woundmaker 96. the cells were washed three times with pbs and serum - free dulbecco 's modified eagle 's medium was added. the plates were equilibrated at 37c for 30 min before scanning in the essen instruments incucyte flr. images were captured every 30 min for 20 hr and percentage relative wound density was calculated using incucyte software. a representative graph and images from one experiment are shown. numbers are mean of six wells sem and similar results were obtained in five other experiments. all abi mutants were generated using the quikchange site - directed mutagenesis kit (stratagene). abi and evh1 binding mutants were cloned into cb6-n - gfp and pac - c - gfp (ac5 promoter) modified from pac5.1/v5-his (invitrogen) for expression in mammalian and drosophila cells, respectively. 293 t and s2 cells were transfected with abi expression vectors using calcium phosphate or effectene (qiagen). coimmunoprecipitations and pull - down assays using gst - evh1-ena were performed as described previously (boda., 2007, 2011). far western analysis of overlapping 20-mer human and drosophila abi peptide arrays were probed with gst or gst - tagged evh1 domains of ena, evl, mena, and vasp as described previously (postigo. the 3utr and orf of dabi were amplified from a drosophila cdna library by pcr using primers containing a t7 rna polymerase binding site at their 5 ends. purified pcr products served as templates for single - stranded rna (ssrna) synthesis using ambion megascript high yield transcription t7 kit. the ssrnas were annealed and the resulting double - stranded rna (dsrna) was purified using the ambion megacleartm kit. s2 cells were treated with dsrna as described previously (kiger., 2003). after 3 days, dabi expression was assessed with qrt - pcr and immunoblot. to express dabi - gfp clones, for frap assays, drosophila s2 cells were seeded on to concanavalin a - coated mattek dishes for 30 min and imaged using a zeiss lsm 710 confocal with a 63/1.4na objective. a region of 300 100 pixels was recorded using a scan speed of 1.27 s / pixel with a pinhole of 90 m. a selected region within the imaging area of 90 25 pixels covering the protruding lamellipodia was imaged 5 times before bleaching with 30 iterations of the 488 nm laser at full power. immediately after bleaching, 260 images were acquired. analysis of fluorescence recovery and curve fitting was performed as previously described (weisswange., 2009). all drosophila strains and crosses the following strains were used : frt82b abi20 (stephan., 2011), elavc155gal4, da - gal4, hml-gal4, and egfp (bloomington stock center). uast / p - abi, uast / p - abiena and uast / p - abienash3 transgenes were generated by c31-integrase - mediated integration into the landing site m{3xp3-rfp.attp}zh-68e as previously described (stephan., 2011). full - length abi, abiena, and abienash3 fragments were amplified by pcr and cloned into gateway entry vectors (pentr dtopo, invitrogen). the inserts were sequenced and subcloned into puast - attb - rfa, puasp - attb - rfa puast - attb - rfa - egfp (drosophila genomics resource center) by in vitro lambda recombination (invitrogen). to force early expression and to simultaneously depict cell dynamics, we combined the ubiquitous da - gal4 driver with the macrophage - specific expression of cytoplasmic egfp (hml-gal4, uas - egfp). macrophages were isolated by dissecting larvae in a drop of m3 medium (invitrogen). glass slides were pretreated with 0.5 mg / ml concanvalina (sigma) for 30 min. cells were fixed for 15 min in 4% paraformaldehyde, and shortly rinsed in pbs and 0.1% triton (pbst). after fixation, cells were incubated for 2 hr with primary antibody, rinsed twice with pbs, and incubated for 1 hr with secondary antibody and with phalloidin - alexa488 (1:100) and dapi (1:1000, invitrogen). wild - type ovaries (w) and ovaries from rescued abi20 mutant females (abi20, da - gal4/abi20, uasp - abi ; abi20, da - gal4/abi20, uasp - abiena and abi20, da - gal4/abi20, uasp - abienash3) were dissected in cold pbs, fixed in 4% paraformaldehyde in pbs for 20 min, and stained with alexa - fluor-488 phalloidin and dapi (invitrogen). to test fertility, sim images were taken with an elyra s.1 microscope (zeiss) with the software zen 2010 d (zeiss). for image acquisition,, cells were plated on chambered cover glass (lab - tek) without concanvalina. movies were taken with a spinning disc cell observer sd zeiss microscope and cropped so that only a single cell was present within the field of view. following kalman - filtering to suppress noise, cells were segmented in each frame using a region - growing algorithm implemented as a plug - in for imagej and velocity maps constructed as described previously (dbereiner., 2006). the circularity of cells (c = 4[area]/[perimeter ]) was estimated in each movie frame and an average taken for all frames. the dynamic dimension is a measure we define as the slope of the line of best fit to the histogram of a velocity map, plotted logarithmically, for velocities greater than or equal to zero (r2 > 0.9). the software necessary to perform these calculations was coded in java and implemented as a plug - in for imagej. x.j.c. identified and mapped all ena / vasp evh1 interactions in human and drosophila abi. x.j.c also generated all abi mutants and vasp 1 - 335 and performed all experiments in s2 cells, including the frap analysis. b.c. purified the wrc and vasp proteins and performed gst - evh1/wrc pull downs, as well as all in vitro actin polymerization assays, which were overseen by m.k.r. generated ht1080 cell lines, analyzed their migration, generated vasp gab and fab mutants, and performed abi1 - 3 and wave1 - 3 in vitro evh1 pull - down assays. | summaryena / vasp proteins and the wave regulatory complex (wrc) regulate cell motility by virtue of their ability to independently promote actin polymerization. we demonstrate that ena / vasp and the wrc control actin polymerization in a cooperative manner through the interaction of the ena / vasp evh1 domain with an extended proline rich motif in abi. this interaction increases cell migration and enables vasp to cooperatively enhance wrc stimulation of arp2/3 complex - mediated actin assembly in vitro in the presence of rac. loss of this interaction in drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of ena to the tips of filopodia - like extensions. rescue experiments of abi mutants also reveals a physiological requirement for the abi : ena interaction in photoreceptor axon targeting and oogenesis. our data demonstrate that the activities of ena / vasp and the wrc are intimately linked to ensure optimal control of actin polymerization during cell migration and development. |
francisella tularensis causing zoonotic infectious disease " tularemia " is gram - negative facultative non - motile intracellular coccobacillus bacteria considered as a potential bioterrorism agent because subspecies tularensis classified as type a is a high - risk pathogen which can cause severe symptoms when infected with only 10 colony - forming units or less [1 - 3 ]. f. tularensis is largely classified as four subspecies : subspecies tularensis, subspecies holarctica, subspecies mediasiatica, and subspecies novicida (table 1). among the subspecies, subspecies tularensis is called as type a causing high fatality if not properly treated with antibiotics, and another subspecies holarctica is called as type b showing comparatively much less severe symptom. subspecies novicida is used for study of microbiological characteristics and vaccine research without biosafety level 3 facilities because it is known as a nonpathogenic in human. the results from current bioinformatic study revealed that there are remarkable differences in genomic sequence between subspecies, and some taxonomists insist on the distinguished new species as francisella novicida instead of the francisella tularensis subsp. both strains of tularensis and holarctica among four subspecies are responsible for human pathogenesis of tularemia, and subspecies tularensis causes severe pathogenic symptoms such as fever, edema and influenza - like illness comparing mild symptoms raised by subspecies holarctica. in some cases, subspecies tularensis finally can lead the infected patients to death if they are not properly treated with antibiotics though the symptoms and severity are different according to the infection routes. although major route of infection is dermal penetration by tick - biting from rabbits as a vector, f. tularensis can infect human through the respiratory ducts or oral routes. usually man - to - man transmission is not considered to be happened because any cases infected by the bacteria transmission through the air flow were not reported yet, however it is not totally impossible to infect human within close distance by the saliva droplets from infected patient 's cough to another human 's nostrils or mouth. ticks from rabbits are the most common vectors for tularemia infection in united states, but the deer flies from small rodents are also can be a vector to deliver the bacteria in western area of north america and mosquitoes are vectors and reservoir in northern europe including russia and sweden (fig. 1). f. tularensis generally enters the inside of phagosome by the phagocytosis of macrophage (fig. the survived bacteria usually replicate themselves enough to infect other adjacent cells, and then the quickly growing population of bacteria raises the pathogenic symptoms after eruption from infected cells. mutation of the genes in francisella pathogenic islands (fpi) and key virulence factors usually inhibits the ability of bacteria escaping from phagosome, and the mutants are considered as possible live vaccine strains. tularemia was firstly discovered as an infectious disease from wild animals such as rabbits by edward francis at tulare county of united states in 1912. since the first diagnosis of human tularemia was happened and the bacteria was isolated by g. w. mccoy at the plaque laboratory of the us public health services in 1912, some levels of endemic cases were continuously reported in north america, and eurasia, especially in the regions with cold winter. the oldest history about human outbreak by tularemia in canaan region was recorded during the 17th century bc, also history tells the long - lasting endemic tularemia in eastern mediterranean area and middle east asia in the 14th century bc. in japan, a rabbit - transmitting infectious disease named " yatobito"was described in its medical history written in the 18th century. as a modern history, the outbreak with one fatality at martha 's vineyard in 2000 reminded the us center for disease control and prevention that the aerosolized f. tularenisis can be a fatal bio - threat and another outbreak occurred in kosovo of europe in 2000. well - known tularemia endemic place is gori region in eurasian country georgia and there was the last outbreak in 2006. f. tularensis can be used as a bioterrorism agent because of its high infectivity and mortality without treatment. furthermore, it is easy to make aerosolized f. tularensis and the decontamination is easy comparing bacillus anthracis or other agents. there are some reports that former soviet union developed f. tularensis as a bio - weapon, and other countries such as united states and japan focused on f. tularensis and made provision against risk of bio - threat by the bacteria. kenneth alibek, a bio - weapon scientist of soviet union, has alleged the tularemia among german soldiers in stalingrad by release of the bacteria from soviet forces. in 1954, united states also performed the research for practical use of f. tularensis as a biological weapon. although vaccination is the best prevention for bioterrorism, there is no officially approved vaccine to date against tularemia due to the various issues including safety concern about live bacteria candidates. tularemia vaccine development was started by several western countries from 1940 's when the f. tularensis was firstly considered as a potential biological weapon. similar to the common history of any vaccine development, development procedure for tularemia vaccine was also tried in both types of whole bacteria vaccine and subunit vaccine including split or recombinant forms. among the types of whole vaccine, killed vaccine may not be considered possible format to be developed because the most trials including the heat - inactivated or the formalin - treated did not show enough efficacies to protect against f. tularensis infection. actually the first trial for tularemia vaccine development was split vaccine. in 1940 's, foshay group tried to develop killed vaccine, which was conserved in phenol after acid extraction of the bacteria. this candidate was considered as a very safe form and it was tested for human volunteers in 1933 and 1941. however the researchers did not get clear results about protective efficacy because of the limited clinical information about the vaccinated group. foshay 's this trial was the first vaccine trial against tularemia even though it did not show enough efficacies in the animal models either. after 1940 's, live attenuated strains were developed to be used as a vaccine by former ussr, and this type of live attenuated vaccine strain (lvs) is considered as the most efficient vaccine candidate which is limitedly used for tularemia researchers as an unofficial vaccine. however, the lvs is live bacteria still having potential risk to replicate while the immunogenicity is also reduced by the attenuation of pathogenicity. most scientists studying tularemia vaccine still keep trying to develop better mutant strains with higher immunogenicity and lower pathogenicity. currently, advanced countries for tularemia vaccine research, such as united states and sweden, are trying to develop recombinant vaccine which is considered as a type bearing high protection efficacy with guaranteed safety. the first meaningful attempt for tularemia vaccine development was accomplished by former ussr before 1950 's. scientist of ussr have repeatedly cultured f. tularensis and selected colony which have less virulence with various conditions, and f. tularensis lvs was isolated. in 1960 's and 1970 's, f. tularensis lvs was used as vaccine for tularemia in united states governmental research institutes and the results showed good efficacy. during this time, many experiments using mice model suggested f. tularensis lvs has good efficacy for prevention of tularemia. first, f. tularensis lvs can not completely prevent pneumonic infection of type a bacteria. second, the exact mechanism of attenuation in f. tularensis lvs is not fixed yet. these characters can raise concern about safety, the most important consideration for vaccine, and f. tularensis lvs is not approved by us food and drug administration nowadays. recently, with great advance of genetic engineering technique, exact gene mapping or targeted mutagenesis are possible in f. tularensis biology. it allowed mutated f. tularensis with deletion of specific virulrence factor, and many tests using those bacteria as live vaccine have reported (table 2). swedish group and national institutes of health (nih) group developed various mutant strains with deleted or changed genes for virulence factors on the fpi for live vaccine with higher safety which are not confirmed simultaneously conserving the high protection efficacy yet ; mainly omp gene for capsule formation was deleted, or mglab and pmra that are responsible for transcriptional regulation during bacterial replication were mutated. canadian research group and another united states research group tried to develop new types of vaccine with mixing some of these attenuated strains. as a new candidate for live vaccine development, f. novicida was also tesed after mutated for antigen - mimicking. however, the trials with the mutants of f. novicida did not produce enough protection efficacies for the challenge using type a strain. until now, lvs strains attenuated from f. tularensis subsp. otherwise some tularemia scientists think the subspecies tularensis (experimentally confirmed type a strains such as schu s4) rather than other subspecies should be the best candidate for live vaccine if safety can be guaranteed. it was reported that the immune responses during lvs infection was much different from the responses to the infection of type a strains, especially about the responses against the respiratory infection by bacteria. the difference may imply that the development of tularemia vaccine would be focused on the type a strains for vaccine materials instead of genetically different type b strains. at the early time of subunit vaccine development, research was focused on the protective proteins or components of the bacteria surface, and lipopolysaccharides (lps) is one of the major targets for subunit vaccine because f. tularensis is gram - negative bacteria containing lps on the outer membranes. foshay 's split vaccine trial also was continued for a while even though the results were not very clear. while in vitro research on the cell level indicated increase of proteins activating t - cell - mediated immune responses were secreted by lps injection into the animals or human, the split vaccine components boosting these immune responses did not generate enough protection efficacy on the whole organism level comparing to lvs injection (table 3). other antigenic proteins in addition to the lps did not show remarked efficacy against the type a strains while they induced somewhat protective capacity against lower pathogenic type b strains. fopa is major component of outer - membrane protein in f. tularensis, however the results from the efficacy test using the protein still did not get expected efficacy. antigens such as groel, katg, and tul4 did not suggest any successful results of vaccination efficacies either, and the continuous failure of subunit vaccine trials from foshay 's split vaccine raises the assumption ; it should be required that host system could recognize the multiple antigens to induce combined immune responses including cellular immunity. based on this idea, combined vaccination of lvs and lps is currently tested for the generation of higher efficacy. types of recombinant vaccine using subunits as immunogens can guarantee the safety comparing currently developed lvs, also they can maximize the immunogenicity mixing multiple antigens from different variants. f. tularensis is intracellular bacteria, and its pathogenesis raises both antibody - mediated and cell - mediated immunity. not only antibody generation but also induction of t - cell response would be required to obtain sufficient vaccine efficacies. to induce the combined immune response, research considering cellular immunity the first meaningful attempt for tularemia vaccine development was accomplished by former ussr before 1950 's. scientist of ussr have repeatedly cultured f. tularensis and selected colony which have less virulence with various conditions, and f. tularensis lvs was isolated. in 1960 's and 1970 's, f. tularensis lvs was used as vaccine for tularemia in united states governmental research institutes and the results showed good efficacy. during this time, many experiments using mice model suggested f. tularensis lvs has good efficacy for prevention of tularemia. first, f. tularensis lvs can not completely prevent pneumonic infection of type a bacteria. second, the exact mechanism of attenuation in f. tularensis lvs is not fixed yet. these characters can raise concern about safety, the most important consideration for vaccine, and f. tularensis lvs is not approved by us food and drug administration nowadays. recently, with great advance of genetic engineering technique, exact gene mapping or targeted mutagenesis are possible in f. tularensis biology. it allowed mutated f. tularensis with deletion of specific virulrence factor, and many tests using those bacteria as live vaccine have reported (table 2). swedish group and national institutes of health (nih) group developed various mutant strains with deleted or changed genes for virulence factors on the fpi for live vaccine with higher safety which are not confirmed simultaneously conserving the high protection efficacy yet ; mainly omp gene for capsule formation was deleted, or mglab and pmra that are responsible for transcriptional regulation during bacterial replication were mutated. canadian research group and another united states research group tried to develop new types of vaccine with mixing some of these attenuated strains. as a new candidate for live vaccine development, f. novicida was also tesed after mutated for antigen - mimicking. however, the trials with the mutants of f. novicida did not produce enough protection efficacies for the challenge using type a strain. until now, lvs strains attenuated from f. tularensis subsp. otherwise some tularemia scientists think the subspecies tularensis (experimentally confirmed type a strains such as schu s4) rather than other subspecies should be the best candidate for live vaccine if safety can be guaranteed. it was reported that the immune responses during lvs infection was much different from the responses to the infection of type a strains, especially about the responses against the respiratory infection by bacteria. the difference may imply that the development of tularemia vaccine would be focused on the type a strains for vaccine materials instead of genetically different type b strains. at the early time of subunit vaccine development, research was focused on the protective proteins or components of the bacteria surface, and lipopolysaccharides (lps) is one of the major targets for subunit vaccine because f. tularensis is gram - negative bacteria containing lps on the outer membranes. foshay 's split vaccine trial also was continued for a while even though the results were not very clear. while in vitro research on the cell level indicated increase of proteins activating t - cell - mediated immune responses were secreted by lps injection into the animals or human, the split vaccine components boosting these immune responses did not generate enough protection efficacy on the whole organism level comparing to lvs injection (table 3). other antigenic proteins in addition to the lps did not show remarked efficacy against the type a strains while they induced somewhat protective capacity against lower pathogenic type b strains. fopa is major component of outer - membrane protein in f. tularensis, however the results from the efficacy test using the protein still did not get expected efficacy. antigens such as groel, katg, and tul4 did not suggest any successful results of vaccination efficacies either, and the continuous failure of subunit vaccine trials from foshay 's split vaccine raises the assumption ; it should be required that host system could recognize the multiple antigens to induce combined immune responses including cellular immunity. based on this idea, combined vaccination of lvs and lps is currently tested for the generation of higher efficacy. types of recombinant vaccine using subunits as immunogens can guarantee the safety comparing currently developed lvs, also they can maximize the immunogenicity mixing multiple antigens from different variants. f. tularensis is intracellular bacteria, and its pathogenesis raises both antibody - mediated and cell - mediated immunity. not only antibody generation but also induction of t - cell response would be required to obtain sufficient vaccine efficacies. to induce the combined immune response, research considering cellular immunity f. tularensis is one of the most dangerous pathogens having potential to generate the nationwide disaster through deliberate use because there are no experts in clinical fields who can rapidly find a way to diagnose or treat the infected patients due to very few cases of natural infection in korea while the bacteria keep heavy pathological traits causing severe symptoms in infected human body. furthermore the high fatality of these bacteria, leading victims to death with only 10 multiplicity of infection, can cause the embarrassing results from delayed treatment following improper diagnostics. vaccination should be the most efficient and practical way to prepare the real situation of this bio - threat. however, there is no vaccine officially approved until now. development of tularemia vaccine with undoubted safety and rapid protection efficacy should be an essential part of national preparedness even though lvs or lps could be used for real dangerous situation in spite of the safety issues and less efficacy. strategic support by government should be urgently required for the development and stockpile of reliable vaccine to reach this final destination of tularemia preparedness. | tularemia is a high - risk infectious disease caused by gram - negative bacterium francisella tularensis. due to its high fatality at very low colony - forming units (less than 10), f. tularensis is considered as a powerful potential bioterrorism agent. vaccine could be the most efficient way to prevent the citizen from infection of f. tularensis when the bioterrorism happens, but officially approved vaccine with both efficacy and safety is not developed yet. research for the development of tularemia vaccine has been focusing on the live attenuated vaccine strain (lvs) for long history, still there are no lvs confirmed for the safety which should be an essential factor for general vaccination program. furthermore the lvs did not show protection efficacy against high - risk subspecies tularensis (type a) as high as the level against subspecies holarctica (type b) in human. though the subunit or recombinant vaccine candidates have been considered for better safety, any results did not show better prevention efficacy than the lvs candidate against f. tularensis infection. currently there are some more trials to develop vaccine using mutant strains or nonpathogenic f. novicida strain, but it did not reveal effective candidates overwhelming the lvs either. difference in the protection efficacy of lvs against type a strain in human and the low level protection of many subunit or recombinant vaccine candidates lead the scientists to consider the live vaccine development using type a strain could be ultimate answer for the tularemia vaccine development. |
roughly 40 years have passed since permanent pacemakers (pms) became available in clinical medicine. more recently, implantable cardioverter - defibrillators (icds) and cardiac resynchronization therapy (crt) have been introduced. the rate of device implantation is increasing with the aging of the general population and the indications are expanding. similar to other prosthetic materials, infections complicate a small proportion of patients with these devices. with the increase in device implantation we introduced the excimer laser system in 2009 for the transvenous removal of the implanted leads. however, there have been few reports,, concerning the management of cardiac device infections. the purpose of this study was to review our single center experience and to clarify the current status of cardiac implantable electronic device (cied) infections in japan. all 183 patients with cied infections who underwent a device and transvenous lead removal using an excimer laser system in kokura memorial hospital from july 2009 through march 2014 were reviewed. briefly, a pocket infection was defined as the presence of local warmth, erythema, swelling, edema, pain, or discharge from the device pocket, or an erosion or impending erosion of the device. blood cultures were obtained from all patients on the day of admission ; cultures were also obtained from the generator and the tip of the lead at the time of device removal. the indications for a lead extraction were decided based on the heart rhythm society expert consensus statement. the baseline clinical characteristics, pathogens, results of the lead extraction procedures, and follow - up results were analyzed. the procedures were performed in the cardiac catheterization laboratory or operation room under general or venous anesthesia according to the patient s condition. careful monitoring with surface electrocardiograms, invasive arterial blood pressure monitoring, and transesophageal or intracardiac echocardiography were performed in all patients. briefly, the lead was prepared by inserting a locking stylet into the inner coil lumen when possible. a laser application was performed at binding sites and the laser sheath was gradually advanced from one binding site to another until the tip of the lead was reached. once abutting the myocardium, a combination of traction and counter - traction was performed and the lead was freed. complete procedural success was defined as the removal of all targeted leads and all lead material from the vascular space, with the absence of any permanently disabling complications or procedure - related deaths. clinical success was defined as the removal of all targeted leads and lead material from the vascular space, or retention of a small portion of the lead that did not negatively impact the outcome goals of the procedure. failure was defined as the inability to achieve either complete procedural or clinical success, or the development of any permanently disabling complications or procedure - related deaths. major complications were defined as any of the outcomes related to the procedure that were life threatening or resulted in death, and in addition, any unexpected events that caused a persistent or significant disability, or any events that required a significant surgical intervention to prevent any of the outcomes listed above. minor complications were defined as any undesired events related to the procedure that required a medical intervention or minor procedural intervention to remedy, and did not persistently or significantly limit the patient s function, nor threaten their life or cause death. the continuous variables are expressed as the meansd and were compared using a student s t - test. all 183 patients with cied infections who underwent a device and transvenous lead removal using an excimer laser system in kokura memorial hospital from july 2009 through march 2014 were reviewed. briefly, a pocket infection was defined as the presence of local warmth, erythema, swelling, edema, pain, or discharge from the device pocket, or an erosion or impending erosion of the device. blood cultures were obtained from all patients on the day of admission ; cultures were also obtained from the generator and the tip of the lead at the time of device removal. the indications for a lead extraction were decided based on the heart rhythm society expert consensus statement. the baseline clinical characteristics, pathogens, results of the lead extraction procedures, and follow - up results were analyzed. the procedures were performed in the cardiac catheterization laboratory or operation room under general or venous anesthesia according to the patient s condition. careful monitoring with surface electrocardiograms, invasive arterial blood pressure monitoring, and transesophageal or intracardiac echocardiography were performed in all patients. briefly, the lead was prepared by inserting a locking stylet into the inner coil lumen when possible. a laser application was performed at binding sites and the laser sheath was gradually advanced from one binding site to another until the tip of the lead was reached. once abutting the myocardium, a combination of traction and counter - traction was performed and the lead was freed. complete procedural success was defined as the removal of all targeted leads and all lead material from the vascular space, with the absence of any permanently disabling complications or procedure - related deaths. clinical success was defined as the removal of all targeted leads and lead material from the vascular space, or retention of a small portion of the lead that did not negatively impact the outcome goals of the procedure. failure was defined as the inability to achieve either complete procedural or clinical success, or the development of any permanently disabling complications or procedure - related deaths. major complications were defined as any of the outcomes related to the procedure that were life threatening or resulted in death, and in addition, any unexpected events that caused a persistent or significant disability, or any events that required a significant surgical intervention to prevent any of the outcomes listed above. minor complications were defined as any undesired events related to the procedure that required a medical intervention or minor procedural intervention to remedy, and did not persistently or significantly limit the patient s function, nor threaten their life or cause death. the continuous variables are expressed as the meansd and were compared using a student s t - test. two hundred twenty - two lead extraction procedures were performed between july 2009 and march 2014. one hundred eighty - three patients (mean 72.214.3 years old, 131 males) had explantations of the devices, leads, or both due to infection complications. one hundred twenty patients (65.6%) presented with signs and symptoms of an infection involving the device pocket without the presentation of an endovascular infection. twenty - six of 63 patients were diagnosed with infectious endocarditis according to duke s criteria. among this cohort, 136 patients (74.3%) had a permanent pm, 45 (24.6%) had an icd, and 19 (10.4%) had a biventricular pm with or without an icd. the mean duration of the device implant and device explantation ranged from 2 to 417 months (91.983.7 months). the mean duration of the implantation or last device replacement and device explantation was 30.536.2 months. twenty - seven patients (14.8%) had an early explantation (12 months). eighty (43.7%) patients underwent a device explantation due to a late infection more than 24 months after the device - related procedure. eighty - seven patients (47.5%) had a previous surgical intervention without full removal of all the hardware. twenty - two patients received a device implantation on the ipsilateral side even though the infection was active in the pm pocket. one to five leads were implanted in each patient, and a total of 450 leads were extracted. twenty - five leads were extracted by manual traction ; the remaining leads were extracted using an excimer laser sheath. the summary data of the extracted leads are shown in table 2. among the 450 leads extracted, the positions of the leads were the right atrium (n=170, 37.8%), coronary sinus (n=20, 4.4%), and right ventricle (n=260, 57.8%), and included 79 icd leads. the mean implant duration was 88.577.6 months in total, with 92.376.4 months in the right atrium, 34.927.6 months in the coronary sinus, and 102.590.1 months in the right ventricle ; 62.4 35.5 were icd leads table 3. complete procedural success was achieved with 437 leads (97.1%), while partial removal in nine (2.0%), and failure with four leads (0.9%) occurred. the mean implant duration of complete and partial removals was 86.175.9 months and 162.0101.0 months, respectively (p 12 months). eighty (43.7%) patients underwent a device explantation due to a late infection more than 24 months after the device - related procedure. eighty - seven patients (47.5%) had a previous surgical intervention without full removal of all the hardware. twenty - two patients received a device implantation on the ipsilateral side even though the infection was active in the pm pocket. one to five leads were implanted in each patient, and a total of 450 leads were extracted. twenty - five leads were extracted by manual traction ; the remaining leads were extracted using an excimer laser sheath. the summary data of the extracted leads are shown in table 2. among the 450 leads extracted, the positions of the leads were the right atrium (n=170, 37.8%), coronary sinus (n=20, 4.4%), and right ventricle (n=260, 57.8%), and included 79 icd leads. the mean implant duration was 88.577.6 months in total, with 92.376.4 months in the right atrium, 34.927.6 months in the coronary sinus, and 102.590.1 months in the right ventricle ; 62.4 35.5 were icd leads table 3. complete procedural success was achieved with 437 leads (97.1%), while partial removal in nine (2.0%), and failure with four leads (0.9%) occurred. the mean implant duration of complete and partial removals was 86.175.9 months and 162.0101.0 months, respectively (p<0.01). in all cases of partial removal, only the tip of the lead remained in the myocardium without any complications and the desired clinical outcomes could be achieved. in two patients, open heart surgery the proximal portion of the leads was extracted using an excimer laser sheath from the pm pocket. with another four leads in two patients, the leads were also removed during open heart surgery due to cardiac tamponade encountered during the lead extraction procedure. major complications directly related to the lead extraction procedure occurred in five patients (2.7%, cardiac tamponade in four, and death within 24 h after the procedure due to uncontrollable bleeding from the vein in one patient). further, minor adverse events occurred in seven more patients (3.8%, pneumothorax in two, blood transfusion in four, and pulmonary embolism in one patient). two patients died because the systemic infection could not be controlled even after the removal of all implanted devices. one patient died during the hospital stay because of a cerebral infarction not related to the extraction procedure. fifteen of 183 patients (8.2%) did not require further device therapy, and devices were implanted in the remaining patients. eight of these patients were transferred to another hospital for the re - implantation procedure. two patients had recurrences of infection within one year after the explantation of the devices. 2) and staphylococcus aureus (37.1%) were the most common causes of cied infections followed by the corynebacterium species (eight patients). gram - negative bacilli including pseudomonas aeruginosa (three patients), escherichia coli (two patients), and proteus mirabilis were the pathogens in 3.8%. seven patients had an anaerobic gram - positive bacillus species and two patients had a fungal (candida albicans) infection. thirty - three (18%) patients had localized inflammatory signs in the generator pocket or an erosion of the device / lead, but the cultures were negative. the rate of device implantation is increasing with the aging of the general population and the expanding indications. voigt. reported that the rate of hospitalization for cied infections have increased faster than the rate of cied implants. this disproportionate increase is consistent with the findings of cabell and colleagues who demonstrated accelerating rates of cardiac device infections (including cieds, prosthetic valves, and ventricular assist devices) among medicare beneficiaries from 1990 to 1999. first, while the age has remained relatively constant, there has been an increase in the prevalence of a coexistent morbidity in cied recipients. uslan. have shown that population - adjusted pm implantation incidences have increased and that there has been an age - independent rise in comorbidities in pm recipients. second, voigt. speculated that a widespread and potentially indiscriminate cied utilization for primary prevention of sudden cardiac death and the treatment of heart failure might play a role, primarily due to the disadvantaged health status and prevalent comorbidities of such recipients. particularly, given the adverse impact of such comorbidities on the crt response, a move to a more judicious application of cieds, in general, may be warranted. interestingly a dramatic rise in cied infections occurred beginning in 2001 and 2002, when the positive results of the primary prevention defibrillator trials, were accepted by the medical community, followed by the adoption and increased rate of crt device implantations. reported a case of a thoracoscopy - guided lead extraction with an excimer laser sheath and okada. reported a case of a transjugular extraction using a snare technique our current study consisted of 183 patients, the largest cohort in japan, and, therefore, this study can clarify the status of device infections and lead extractions in japan. reported that device - related infections occurred in 0.68% of patients within the first year after the de novo implantation or replacement. no data is available concerning the prevalence of cied infections in japan, however at kokura memorial hospital, 1855 cieds were implanted between 2008 and 2012 (de novo implantations in 1,174 and replacements or upgrades in 681 patients). this data suggests that the prevalence of cied infections seems to be similar in japan. as for the management of cied infections, the recommendations for a complete extraction of the device, route of administration, duration of antimicrobial therapy, and the timing of the placement of a new device are based on observational data and clinical experience. observations from several medical centers universally support the complete removal of the device to cure the infection and reduce morbidity and mortality,,. the relapse rate when a complete device removal is performed is 04.2%. on the contrary, when a partial removal or antibiotics are chosen, the relapse rate increases to 50100%,,,,,. of the 183 cied infection patients, 27 (14.8%) had an early infection (<3 months after the procedure), 45 (24.6%) had a late infection (412 months after the device - related procedure), and 111 (60.7%) had a delayed infection (more than 12 months after the procedure). of interest, 60 (43.7%) patients presented with an infection after an interval of more than 24 months after the device - related procedure. as previously mentioned, almost half of the patients in this study had a previous surgical intervention without a full removal of all hardware and one - fourth of the patients underwent device implantation on the ipsilateral side even though the infection was active in the pm pocket. this might suggest a considerable number of patients were undertreated in japan and at a risk of recurring infections, endocarditis, or fatal results. complete procedure success was achieved in 97.1% of the lead extractions in our study while partial removal occurred in 2.0%, and failure in 0.9%. okamura. reported the success rate of a complete removal was 97.1%. these two results were almost equally beneficial with those of the lexicon study (complete removal : 96.5%). the mean implant duration of the partially removed leads was 162101 months (longest 338 months). roux. reported that a longer time from the implantation independently predicted a procedural failure. in this patient group, the mean implant duration of complete and partial removals was 86.175.9 months and 162.0101.0 months, respectively (p<0.01). in our study, culture results were positive for staphylococcus aureus in 37.1% of the patients, cns in 30.1%, and other bacterial species in 14.8%. tarakji. reported their pathogens of cied infections were cns in 44.4% of the patients, methicillin - sensitive s. aureus (mssa) in 20.1%, and methicillin - resistant s. aureus (mrsa) in 15.8%. our data and previously published data suggest that the staphylococcus species continues to represent the most common pathogen of cied infections, with 510% being methicillin resistant. the usefulness of the prophylactic use of antibiotics at the time of a device implantation was reported by de oliveira. therefore many institutions continue to use beta - lactam antimicrobial agents at the time of implantation ; however, this is not effective against methicillin - resistant organisms. a single dose of vancomycin before the implantation might be better than that of beta - lactam antibiotics to prevent cied infections in the selected patients such as mrsa carrier. two patients in this patient group (1.1%) had relapses within the first year. one patient had a dual chamber icd due to ventricular tachycardia caused by a remote myocardial infarction. the pocket infection reappeared after re - implantation of an icd on the ipsilateral side at a previous hospital. the pathogen of the second infection was pseudomonas aeruginosa ; indicating that the second pocket infection might not have been a recurrence of the initial pocket infection. the other patient had allergic dermatitis on the body and a vdd pm was implanted due to complete atrioventricular block. that patient was referred to our hospital under a diagnosis of device - related endocarditis. the device and all lead materials were completely removed and the patient underwent successful re - implantation of a device on the ipsilateral anterior chest after an intravenous antibiotic prescription for three weeks. this patient, however, was readmitted due to bacteremia after re - implantation of the device. in our study, 8.1% of the patients no longer required device implantation or had reasonable alternatives after their devices were removed. thus, the need for re - implantation in patients with an infected device should be carefully evaluated. ninety percent of our patients were initially treated by other institutions, and 47.5% had previously failed surgical attempts without a full removal. further investigation with a larger patient group is required to clear up the present circumstances of cied infections in japan. ninety percent of our patients were initially treated by other institutions, and 47.5% had previously failed surgical attempts without a full removal. however, this report was a single center experience. further investigation with a larger patient group is required to clear up the present circumstances of cied infections in japan. the current clinical status of cied infections seems to be similar in japan to that in foreign countries. the optimal treatment of infected pm and implantable defibrillator devices involves the complete explantation of all hardware, followed by antibiotic therapy. the excimer laser appeared to be safe and effective for extracting chronically implanted leads in japanese patients. | backgroundlead extraction using laser sheaths is performed mainly for cardiac implantable electronic device (cied) infections. however, there are few reports concerning the management of cied infections in japan.methods and resultslead extraction procedures were performed in 183 patients targeting 450 leads (atrial leads : 170, ventricular : 181, implantable cardioverter - defibrillators (icds) : 79, and coronary sinus : 20). one hundred twenty patients (65.6%) presented with pocket infections without the presentation of an endovascular infection. blood cultures were positive at least once in 63 patients (34.4%). complete procedure success was achieved for 437 leads (97.1%) while partial removal occurred in nine, and failure in four leads. major complications directly related to the procedure occurred in five patients (2.7%). two of the four patients with a cardiac tamponade required a surgical repair. all patients received intravenous antibiotics, at least, one week after the procedure. pocket or systemic infections were successfully controlled in 181 patients (98.9%). coagulase - negative staphylococci (30.1%) and staphylococcus aureus (37.1%) were the most common causes of cied infections.conclusionthe current status of cied infections in japan seems to be similar to that previously reported from foreign countries. the optimal treatment of cied infections involves the complete explantation of all hardware, followed by antibiotic therapy. |
this prospective study followed the principles of the declaration of helsinki and was approved by the hospital ethical committee, and all participants signed a standard informed consent form reporting the potential risks and benefits of the procedure and subsequent management. all patients were confirmed by the ophthalmology department of beijing hospital for a detailed examination including best - corrected visual acuity (bcva) and iop, slit - lamp biomicroscopy, auto refractometry, gonioscopy, and dilated fundoscopic examinations of both eyes. the study included 352 eyes from 352 patients (1 eye per patient) diagnosed exudative amd, diabetic macular edema (dme), retinal vein occlusion (rvo), pathologic myopia (pm), idiopathic choroidal neovascularization (icnv), or cystoid macular edema (cme) who were treated with ranibizumab (0.5 mg/0.05 ml) ivi between april 2013 and june 2014. exclusion criteria included younger than 18 old years, ocular hypertension before injection, family history or a prior diagnosis of glaucoma, prior vitrectomy or phacoemulsification surgery, use of iop - lowering agents, allergy to sulfur / sulphonamide - containing drugs. all the patients were divided randomly into two groups. in control group, 203 patients (mean standard deviation [sd ], 64.21 14.10 years) only received the ranibizumab ivi while in case group, 149 patients (mean sd, 61.54 14.60 years) received one drop of prophylactic intraocular brinzolamide before ranibizumab ivi. the iop was measured by noncontact topcon tonometry (japan) during the study. for the operation procedure, injection of ranibizumab was performed at the upper pars plana with a sharp 30-gauge needle after complete sterile draping and rinsing with topical povidone - iodine under topical anesthesia with bupivacaine hydrochloride 0.75% eye drops. the injection site was located 3.5 mm posterior to the limbus. both before and after ivi no eye received iop - lowering medications or anterior chamber paracentesis before or after ivi. the iop were measured before injection, at 10, 30, 120 min and 1 day after injection in a sitting position. one drop of brinzolamide was used after the baseline iop measurement approximately 2 h (mean sd, 112.05 26.61 min) before injection. statistical analysis was performed using spss (version 17.0) software (spss inc., smirnov test of nonparametric analysis and chi - square test were used to analyze the data of two groups. we set p 0.05) between two groups. baseline characteristics outcome data collected included iop measurements of baseline and immediately at 10, 30, 120 min and 1 day after ivis. the mean iop measured before injection, at 10, 30, 120 min and 1 day after injection individually were 15.79 2.21 mmhg, 19.33 4.86 mmhg, 16.64 2.93 mmhg, 16.17 3.13 mmhg, and 15.07 2.55 mmhg in case group and were 15.82 2.57 mmhg, 21.34 5.88 mmhg, 18.17 4.06 mmhg, 17.59 4.42 mmhg, and 15.48 2.92 mmhg in control group. the tendency in both curves is a sharp increase on iop a few minutes after injection, with a gradual decline over the next hours. maximum iop elevation occurred at the time point of 10 min after injection in both groups. at time points of 10, 30, and 120 min postinjection, mean iop was significantly higher in control group when compared with the case group (p 0.05) between two groups. outcome data collected included iop measurements of baseline and immediately at 10, 30, 120 min and 1 day after ivis. the mean iop measured before injection, at 10, 30, 120 min and 1 day after injection individually were 15.79 2.21 mmhg, 19.33 4.86 mmhg, 16.64 2.93 mmhg, 16.17 3.13 mmhg, and 15.07 2.55 mmhg in case group and were 15.82 2.57 mmhg, 21.34 5.88 mmhg, 18.17 4.06 mmhg, 17.59 4.42 mmhg, and 15.48 2.92 mmhg in control group. the tendency in both curves is a sharp increase on iop a few minutes after injection, with a gradual decline over the next hours. maximum iop elevation occurred at the time point of 10 min after injection in both groups. at time points of 10, 30, and 120 min postinjection, mean iop was significantly higher in control group when compared with the case group (p < 0.05) ; however, the differences were not significant at baseline and after 1 day (p = 0.463). the mean postinjection iop for control group at the time points of 10, 30, and 120 min was statistically different when compared with baseline iop (p < 0.05). on the contrary, in case group, this difference was observed only for the time point of 10 min (p < 0.05). mean intraocular pressure (mmhg) at before intravitreal and after intravitreal injection comparing two groups by chi - square test, it indicated that the ratio of iop 21 mmhg and the elevation of iop 5 mmhg within 2 h after injection had significant difference as tables 3 and 4 showed (p < 0.05). comparison of intraocular pressure 21 mmhg after injection comparison of elevation of intraocular pressure 5 mmhg after injection we found that the iop increase after ivi in the control group of our study persisted for short period. until 2 h after ivi, similarly, gismondi. reported that transient iop increases within 30 min after ivi, however, there were no significant differences after half an hour. other published studies regarding ivi have reported transient iop increases after 3060 min, and stabilizing at baseline values after 1 day. the little differences could be related to differences in the population race studied and/or iop measurement techniques. about the reasons of acute elevation of iop after ivis, the volume change of the vitreous cavity may be the main reason of immediate iop increasing after ranibizumab ivis. the volume of the vitreous cavity in human eye is 4 ml approximately, and the volume of ranibizumab injected into the vitreous is 0.05 ml. therefore, the increase in fluid volume of the vitreous cavity is 1.25% approximately, which may cause immediate iop elevation. the block hypothesis for the potential mechanisms inducing an iop elevation after ivi is that medications may block the immediate aqueous humor cycle channels, including the trabecular meshwork or schlemm 's canal outflow pathways by an unclear mechanism for several weeks or months. although these hypotheses are related to the sustained increased iop, they may indicated that the ocular structure of patients with ivi have changed and are sensitive to the changes of the volume of the vitreous cavity. in addition, the daily fluctuation of the iop may also play a role in the elevation of the iop. we also found that in case group of our study, patients use prophylactic iop - lowering medication (brinzolamide) before injection have statistically significance only at 10 min after ivi. when comparing with the control group, the significant difference was observed up to 2 h. no matter the ratio of iop 21 mmhg or elevation of iop 5 mmhg, the results are similar within 2 h. frenkel. reported that the iop reduced to < 30 mmhg in all patients within 20 min. by contrast, hariprasad. suggested that 13% of eyes with 30 mmhg or greater 30 min after injection. however, they all indicated that prophylactic medication may not be necessary to prevent postinjection iop spikes. however, theoulakis. reported that brimonidine / timolol - fixed combination may be effective to prevent the short - term iop increase after ivi. kim. also indicated that the prophylactic administration of antiglaucomatic drugs before intravitreal anti - vegf injection effectively reduced the early iop elevation. similarly, in our study, under prophylactically using brinzolamide, the significant difference of iop after injection between two groups is obvious within 2 h which may indicate that brinzolamide can successfully suspend the iop rise. considering the contrast to the previous studies, the time interval of using the prophylactic iop - lowering medication before injection may an explanation. in this study, the time is approximately 2 h which is also the peak of iop - lowering medication (brinzolamide) effectiveness ; however, in frenkel 's study, the time interval was 5 min before injection which may be ineffective to decrease immediate iop postoperation. furthermore, another explanation as kim. demonstrated that a lower iop elevation with 27-gauge needles than with 30- or 32-gauge needles which may suggested that iop rise may was more related to the needle diameter than to the volume injected. supposed that a vitreous reflux occurs higher with a larger needle bore size which led to underestimate the probability of iop rise. giving topical pressure - lowering medications can reduce iop, brinzolamide was used in this study to suppress the produce of aqueous humor mildly because of little side effects clinically comparing to other pressure - lowering medications. as is known that prostaglandin analogs are major pressure - lowering medications with great effect, but it is not suitable for patients after ivis. in addition, blocker drugs have side effect for patients with some heart diseases. because the explanation of how high of ocular hypertension and how long high iop lasting will damage the vision is unclear, we also can not conclude that whether prophylactic iop - lowering medication on iop after ranibizumab ivis is necessary. while as is known that patients should receive more injections to delay the loss of vision, every iop rises even short iop fluctuations in glaucoma or high - iop patients, even at lower amplitudes, are a recognized risk factor for injury of optic nerve. asrani. indicated that a diurnal variation of 5.4 mmhg in iop was associated with a 5.7-fold increase in the progression of visual field loss. addition, murray. suggested that prophylactic treatment may be considered as an option to minimize neuroretinal rim damage in high - risk glaucoma patients who are most vulnerable to iop spikes and undergoing repeated ivis of ranibizumab. therefore, this iop elevation may be harmless in normal patients but should be taken into account on patients with ocular hypertension or glaucoma. first, the most important limitation is that the noncontact tonometer is not golden standard tool like goldmann tonometer though we had rectified it before measurement every time. because we thought goldmann tonometer may increase the risk of inflammation for the contact measure method and we focused on the changes of iop and it may affect little relatively. second, iop elevation is much important to ocular hypertension or glaucoma patients ; we did not investigate these patients which are another limitation of our study and we will make found research about this in further study. third, the central corneal thickness and axial length as important factors for volume changes of ocular were not measured in this study, and the effect would be investigated in advanced study. in addition, this study just focused on short - term effect, but the long - term effect may affect optic nerve seriously which would be investigated in the further study. although these limitations including population race, small sample size, ocular disease distribution, iop measurement tool, unmeasured axial lengths, and operation procedure in this study may impact the result, we still insist that this study may help to illustrate the effect of prophylactic iop - lowering medication on iop after ranibizumab ivis and whether the iop - lowering therapy is necessary after ivis. ivi of ranibizumab causes a considerable short - term transient rise on iop in most patients. the effect of prophylactic iop - lowering medication (brinzolamide) on iop after ivis can be statistically significant from 10 min to 2 h after ivis. this work was supported by beijing natural science foundation, bnsf grant 7152123 and beijing municipal science and technology commission, the capital characteristic clinic project z151100004015147. this work was supported by beijing natural science foundation, bnsf grant 7152123 and beijing municipal science and technology commission, the capital characteristic clinic project z151100004015147. | purpose : to observe the effect of prophylactic intraocular pressure (iop)-lowering medication (brinzolamide) on iop after ranibizumab intravitreal injections (ivis).materials and methods : this prospective case control study included 352 eyes from 352 patients (1 eye per patient) who were treated with ranibizumab intravitreal injection and divided randomly into two groups. two hundred and three patients in control group only received the ranibizumab ivi, but 149 patients in case group received one drop of prophylactic intraocular brinzolamide preinjection. the iop was measured by noncontact tonometer before injection, at 10, 30, 120 min and 1 day after injection in a sitting position.results:the mean iop measured before injection, at 10, 30, 120 min and 1 day after injection individually were 15.79 2.21 mmhg, 19.33 4.86 mmhg, 16.64 2.93 mmhg, 16.17 3.13 mmhg, and 15.07 2.55 mmhg in case group and were 15.82 2.57 mmhg, 21.34 5.88 mmhg, 18.17 4.06 mmhg, 17.59 4.42 mmhg, and15.48 2.92 mmhg in control group. comparing two groups, the mean increase on iop was statistically significant at 10, 30, 120 min postinjection (p < 0.05).conclusions : ivi of ranibizumab causes a considerable short - term transient rise on iop in most patients. the effect of prophylactic iop - lowering medication on iop after ivis can be statistically significant from 10 min to 2 h after ivis. |
etiological factors for spinal epidural hematoma are tumors, anticoagulant therapy, coagulopathy, hypertension, arteriovenous malformations, and trauma. most common presentation is sudden back or neck pain followed by a motor or sensory dysfunction with or without urinary retention. complete neurological recovery is possible with early diagnosis and urgent surgery while delay in the treatment of this condition causes permanent neurological deficit. a previously healthy 14-year - old boy was admitted with a 15-day history of back pain, urinary retention, and weakness of both lower limbs. the patient noted a rapid increase of his symptoms after 2 days with complete loss of power and sensation in lower limbs. the patient was admitted in some local hospital and came to us after 2 weeks of onset of symptoms. there was no history of spinal trauma, systemic disease, anticoagulant usage, or coagulopathy. neurological examination revealed bilateral sensory disturbance of all modalities below the level of fourth dorsal vertebrae level and spastic paraplegia. t1- and t2-weighted magnetic resonance (mr) images revealed a mass lesion suggesting an epidural hematoma, extending from dorsal vertebrae d1 to dorsal vertebrae d6, localized in the ventral spinal epidural space. fourth and fifth dorsal laminectomy with left partial facetectomy was performed, and an epidural clot was totally removed using microneurosurgical technique. during surgery, no vascular abnormality was observed, but no dural pulsation was evident at the end of the procedure. an mr sean of the spine was performed 10 days after the operation which showed complete removal of hematoma [figure 2 ]. (a) t1-weighted magnetic resonance imaging dorsal spine sagittal cuts, (b) t2-weighted sagittal cuts, (c and d) t2-weighted axial cuts showing a ventral spinal epidural hematoma extending from d1 to d6 vertebra and displacing and compressing the cord posteriorly (a and b) t2-weighted postoperative magnetic resonance imaging dorsal spine sagittal cuts, (c and d) t2-weighted axial cuts showing laminectomy defect with complete evacuation of ventral epidural hematoma ventral sseh, as our case, is even rarer with only four previous cases reported in the literature. the most common clinical presentation is sudden back or neck pain followed by motor and sensory dysfunction with or without urinary retention. the neurologic deficits are dependent to the localization of hematoma, horner or brown - sequard syndromes may be noted. the progression of the symptomatology and clinical signs is usually very rapid, as in our case, although slower progression over a few days has also been reported. most common causes of spinal epidural hematoma are the use of anticoagulants, coagulopathies (hemophilia and leukemia), and procedures such as a spinal tap or epidural anesthesia. most authors have contended that sseh arises from epidural venous plexus in the spinal epidural space. because of fluctuations in intrathoracic and intra - abdominal pressures after exercise or other maneuvers, reversal of blood flow may induce rupture of a delicate vein in the valveless epidural plexus. ct findings are similar to intracranial epidural hematoma showing hyperdense lenticular collection. mr imaging is the best examination for diagnosis and follow - up. the dura mater is visualized as curvilinear low signal, separating the hematoma from the cord. the hematoma is isointense or slightly hyperintense on t1-weighted images and heterogeneous on t2-weighted images within 24 h of onset. later, hematoma gives a high signal on both t1- and t2-weighted sequences. in the absence of any signs suggestive of vascular malformation on mr imaging, preoperative angiography is not essential and need not delay the surgical procedure, because the timing of the surgery, together with the preoperative clinical state, determines the quality of the clinical result. posteriorly placed hematoma can be easily removed by doing laminectomy but anteriorly placed hematoma needs good exposure and microneurosurgical techniques to prevent retraction injury to the cord. partial facetectomy can be done in dorsal spine to enhance exposure, and lateral corridor can be used to prevent thecal sac retraction. complete and partial neurological recovery has been described in approximately 50% and 44% of the patients, respectively. recovery was significantly better when decompression was performed within < 36 h of the onset of the neurological deficit. in the majority of cases with sseh, the mainstay of treatment will remain surgical decompression of the neural structures and removal of the hematoma. the decision for conservative treatment has to be based on the severity of the neurological deficit and on the clinical course. prognosis of the patient with sseh depends on the severity of the neurologic deficit on admission and interval from onset of initial symptom to surgery. early diagnosis and prompt surgery are crucial to achieve the best neurological outcome and delay in the diagnosis and treatment may cause permanent neurologic deficit | spontaneous spinal epidural hematoma is very uncommon cause of spinal cord compression. it is extremely rare in children and is mostly located in dorsal epidural space. ventral spontaneous spinal epidural hematoma (sseh) is even rarer, with only four previous reports in childrens. we are reporting fifth such case in a 14 year old male child. he presented with history of sudden onset weakness and sensory loss in both lower limbs with bladder bowel involvment since 15 days. there was no history of trauma or bleeding diasthesis. on clinical examination he had spastic paraplegia. magnetic resonance imaging (mri) of dorsal spine was suggestive of ventral spinal epidural hematoma extending from first to sixth dorsal vertebrae. laminectomy of fourth and fifth dorsal vertebrae and complete evacuation of hematoma was done on the same day of admission. postoperatively the neurological status was same. |
glaucoma comprises a number of different pathomechanisms leading to a specific degeneration of the retinal ganglion cells and changes in the optic nerve head. primary open angle glaucoma is the most common form and affects about 1% of the western population. to date, the only defined risk factors for the development of primary open angle glaucoma (poag) are age and elevated intraocular pressure (iop). patho - morphological correlations discussed for the elevated iop are the appearance of plaque - like extracellular material in the human trabecular meshwork [1, 2 ], empty spaces / giant vacuoles in the juxtacanalicular region next to schlemm 's canal [35 ], and the size of schlemm 's canal itself [6, 7 ]. the active role of tm cells in this regulative process was considered due to their contractile properties [8, 9 ] and due to intracellular volume regulation [1012 ]. interestingly, the increased production of aqueous humour alone seems not to be responsible for elevated intraocular pressure, although a number of therapies modify this input. in recent years, a broader understanding of intracellular volume regulation was gained by the description and investigation of specific ion channels and their molecular regulation. in this context early investigations of steroid hormone function in the eye were lead by clinical observations of iop elevation in one - third of the population after topical cortisone treatment [15, 16 ]. persisting ocular hypertension can lead to a specific type of open angle glaucoma, the cortisone - induced glaucoma [17, 18 ] with a typical morphological appearance [19, 20 ]. interestingly, systemic elevation of cortisone can slightly increase iop but does not lead to a higher risk of glaucoma development. therefore, local mechanisms seem to play a crucial role. one of them is the 11-hydroxysteroid dehydrogenase (hsd) consisting of two isozymes with distinct different functions. hsd1 is the key enzyme for activation of cortisone ; hsd2 leads to inactivation of cortisone in specific tissues with aldosterone receptors which could also be activated by cortisone. to postulate an effect of cortisone, hsd1 for activation and the glucocorticoid receptor (gr) were described originally, but subsequent studies only confirmed the presence of gr [23, 24 ]. from a functional point of view, glucocorticoids lead to an intracellular volume increase in trabecular meshwork cells [2527 ] and modify the extracellular matrix production [28, 29 ]. most surprisingly, one of the extracellular matrix proteins affected is elevated in all human glaucomatous donor eyes [30, 31 ], but physiological studies recently questioned its role for elevation of trabecular meshwork resistance and iop [32, 33 ]. thus the cellular volume increase effect of cortisone has the best evidence to be of pathophysiologic relevance for iop increase at present. for a long time, a second group of corticoid hormones, the mineralocorticoids, were not considered to play any significant function in the eye. however, early investigations mentioned that the aldosterone - antagonist spironolacton led to a decrease of intraocular pressure in glaucomatous patients. mirshahi and coworkers were the first to describe mineralocorticoid hormone receptors (mr) in the retina and all epithelial cells of the eye [36, 37 ]. to consider specific aldosterone function, the presence of hsd2 next to the mr is necessary. the presence of mr and hsd2 in the trabecular meshwork is described controversially [2224 ]. mineralocorticoid effects are thought to be mediated by epithelial sodium channels (enac) [38, 39 ], which are also present at numerous places in the anterior eye segment [4043 ]. these channels might serve two different functions : one is fluid secretion from the ciliary epithelium (increase of aqueous humour formation), and the other is regulation of the trabecular meshwork resistance by volume regulation of the trabecular meshwork cells. the first is the most widely suggested mechanism for aldosterone since a consistent presence of mr and a strong evidence for the presence of hsd2 are reported in ciliary epithelium cells [2224 ]. if the trabecular meshwork is also a target tissue for aldosteron remains to be determined. a number of mouse models were established to study mineralocorticoid effects but no data exists about the eyes of these animals. the existing genetically altered mice show either an overexpression of the mr [4446 ], a knockout of hsd2, or alterations of the enac ion channels (liddle 's syndrome) [48, 49 ]. unfortunately, there is no data in mouse eyes for the presence and distribution of mr and hsd2. personal investigations on the distribution of enac in the mouse anterior eye segment showed intense staining for - and -enac, but no staining for -enac in the trabecular meshwork, while the ciliary epithelium showed only a weak staining reaction (figure 1)., mr overexpression led to massive changes in the anterior eye chamber due to epidermal atrophy in these nonviable puppets. unfortunately, these animals have a dba/2j background leading to changes in the chamber angle beginning at 3 months of age. personal investigations on the eyes of 6-months - old transgenic animals (p1.hmr and p2.hmr from [44, 45 ]) show massive synechiae of the iris, atrophy of the ciliary body, strong pigmentation of the chamber angle, and loss of retinal ganglion cells. knockout of hsd2 was performed in c57/bl6 mice. the mouse model established for liddle 's syndrome there seems to be no effect of aldosteron on the -enac subunit in these animals. schlemm 's canal was widely open, trabecular meshwork cells were not swollen, abnormalities in the anterior and posterior eye segments could not be detected. since the normal mouse eye does not express the -enac subunit in the trabecular meshwork and inner eye surfaces, these results are not surprising. concluding, at this stage of research mouse models do not help to answer questions related to the role of mineralocorticoids in the eye. a number of conditions are known in the human associated with mineralocorticoid dysfunction. a relation to ocular pathologies was tested. hyperaldosteronism is a common, but rarely diagnosed condition (estimated 1.53.5% of the entire population in germany). the induced high blood pressure can affect the eye but not in a glaucoma - specific way. apparent mineralocorticoid excess is a condition with lack of hsd2 and subsequently increased activation of mr. there is no report in the literature that any of the diagnosed patients suffered from either elevated iop or glaucoma. pseudohypoaldosteronism type 1 is related to a reduction of alpha enac function [56, 57 ]. a communication with prof. hanukoglu (tel aviv) revealed that these persons do not complain of any specific eye symptoms. an extended ocular examination of his oldest patient at that time (19 years old) showed normal intraocular pressure (17 mmhg), a normal anterior chamber including the chamber angle, and a normal oct of the nerve fibers in the retina. if this finding is related to a reduced function of the corneal endothelium remains to be determined. systemic application of mineralocorticoids to glaucoma patients shows no changes in the iop in most of the cases [58, 59 ]. however, single individuals react with a high increase in intraocular pressure. unfortunately, these mineralocorticoid - sensitive persons are not further characterized. they could constitute a new subgroup of ocular hypertension or glaucoma patients, but more clinical data has to be collected to define these persons. the lack of general agonist effects combined with the mild iop decrease of antagonists points to a possible role of mineralocorticoids for glaucoma therapy but not for general glaucoma pathophysiology. the narrowed role is also supported by the negative findings in the animal models and the various human conditions described above. the therapeutic effect of mineralocorticoid antagonists seems mainly mediated by a decrease of aqueous humour formation. one additional aspect of mineralocorticoid function is venoconstriction and thus an increase in postcapillary pressure [60, 61 ]. venoconstriction could also be of relevance for elevated intraocular pressure as known from rat glaucoma models.. a different venous sensitivity could be a criterion for the above hypothesized mineralocorticoid - sensitive subgroup of humans. the proposed mechanisms by which mineralocorticoids play a role in glaucoma are summarized in figure 2. while there is some evidence that the ciliary epithelium is affected by mineralocorticoids, the role of the trabecular meshwork cells and of the limbal veins remains to be determined. hopefully this paper attracts more scientists and clinicians for further research in the area of mineralocorticoids with respect to the pathogenesis of glaucoma. | since the pathomechanisms of primary open angle glaucoma are still not defined, different aspects related to this topic have to be discussed and further investigated. possible candidates are the mineralocorticoids, which are known to lower intraocular pressure. a data search and personal investigations assume a limited role of mineralocorticoids for the development of glaucoma. specific experiments for a final conclusion are, however, not yet performed. |
schwannoma is a benign tumor arising from neuroectodermal schwann cell of cranial, intraspinal, peripheral and autonomic nerve sheaths. lesions in head and neck region account for 25 - 45% of extra - cranial schwannomas. however, it rarely involves infra - orbital nerve and are uncommon in the maxillary and buccal space. these tumors usually present as slow growing nodular mass and can mimic any benign growth in the head and neck. the treatment of extra - cranial head and neck schwannomas is surgical and the approach depends on the location and extent of the tumor and the nerve involved. this paper describes a case of schwannoma arising from infra - orbital nerve, presenting as slow growing nodular swelling in the buccal space. the lesion was approached through skin incision and completely resected. at the time of exploration, the lesion was observed to emanate from the nerve trunk of peripheral branch of infra - orbital nerve, which was dissected and preserved. only nine cases of infra - orbital nerve schwannoma are previously reported, which we discuss in relation to our experience. a 40-year - old male patient presented with a chief complaint of painless swelling over left side of face. the patient first noticed the swelling about a year back, which had grown gradually to its present size. patient also complained of mid paresthesia over left cheek since past few months. on examination, a localized, nontender, firm, round swelling of about 4 cm was evident over left side of mid - face, extending from below the infra - orbital margin to upper lip [figure 1 ]. the skin was pinch - able over the lesion, except at the center of the lesion. the overall clinical presentation was suggestive of a benign sub - cutaneous lesion and differential diagnosis included lipoma, fibroma, dermoid cyst, epidermoid cyst and neurogenic tumor. lesion presenting as large sub - cutaneous nodular swelling ultrasonography was advised to study nature, size and extent of the lesion. the ultrasonogram showed well - defined, heterogeneous oval mass of 45 mm 30 mm 23 mm within the buccal sub - cutaneous tissue with no orbital, maxillary sinus or underlying bone involvement [figure 2 ]. aspiration biopsy of the lesion showed spindle shaped cells predominantly arranged in antoni a pattern around verocay bodies, with less organized antoni b tissue in few places [figure 3 ]. diagnosis of schwannoma, probably arising from terminal branch of infra - orbital nerve was established. ultrasonogram showing a heterogeneous oval mass within the sub - cutaneous tissue h and e stained section of aspirate showing verocay bodies (arrow head) surrounded by spindle shaped cells arranged in orderly parallel fashion (arrows) surgical excision of the lesion was planned and executed under local anesthesia. after attaining adequate anesthesia, linear incision of about 3 cm the lesion was well - encapsulated, and freed from surrounding tissue by blunt dissection [figure 4a ]. portion of skin overlying the center of the lesion appeared thinned and was included in the excised nodule. at the time of exploration, the lesion was found to emanate from the nerve trunk of peripheral branch of infra - orbital nerve, which was dissected and preserved [figure 4b ]. (a) exposure of the lesion through skin incision ; (b) dissected tumor attached to the nerve trunk of peripleral branch of infra - orbital nerve (block arrow) h and e stained section (10) of excised specimen showing spindle shaped cells predominantly arranged in antoni a pattern around verocay bodies (black arrow), with less organized antoni b tissue in few places (red arrow). vessel in schwannoma having wide lumen, fibrotic wall and adjacent hyalinization (blue arrow) schwannoma (neurinoma, peripheral glioma, perineural fibriblastoma and neurilemmoma) is ectodermal benign neoplasm, which originates from schwann cell of cranial, intraspinal, peripheral and autonomic nerve sheaths. they are common in head and neck region, accounting for 25 - 45% of extra - cranial schwannomas and 1 - 8% of all head and neck tumors. it can involve any of the 12 cranial nerves, except the olfactory and optic nerves since they lack schwann cells in their sheaths. schwannoma arising from infra - orbital nerve (branch of maxillary division of fifth cranial nerve) is extremely rare. to best of our knowledge, only nine cases of infra - orbital schwannoma has been previously described in english literature. depending on the site of nerve trunk or branch from which it arises, it may be located within the lower part of orbit, maxillary sinus or present as sub - cutaneous mass in infra - orbital region [table 1 ]. intraorbital schwannoma generally develop from supraorbital or supratrochlear nerves, and less frequently from infra - orbital nerves. lesions arising from infra - orbital nerve grow to involve surrounding structures such as maxillary sinus, nasal cavity, infratemporal fossa and pterygopalatine fossa. however, it is rare to find schwannoma in the paranasal sinus, especially in the maxillary sinus. swelling in the cheek, nasal atresia, downward transversion of the palate and pain, rarely accompanying exophthalmosis are common symptoms. however in our case, a well - localized painless infra - orbital sub - cutaneous swelling was observed without any ocular lesions or involvement of underlying osseous skeleton. review of previously reported cases of schwannoma involving ion rarely schwannomas may present with numbness in the distribution of involved nerve or with pain. our patient reported paresthesia localized over area of distribution of superior labial branch of infra - orbital nerve, which was probably due to pressure or direct involvement of the peripheral branch of the nerve. however during the surgical dissection, lesion was found to emanate from the nerve trunk of peripheral branch of infra - orbital nerve, which was dissected and preserved. none of the previous cases of infra - orbital schwannoma reported preoperative paresthesia [table 1 ]. fine - needle aspiration cytology (fnac), ultrasonography, magnetic resonance imaging and computed tomography image are diagnostic tools for schwannoma. diagnosis using fnac is characterized by the presence of spindle cells and has only 17.6% accuracy. aspiration of our lesion yielded blood tinged aspirate, which showed spindle shaped cells predominantly arranged in antoni a pattern around verocay bodies, with less organized antoni b tissue in few places confirming the diagnosis of schwanomma. the treatment of schwannomas is exclusively surgical and the appropriate approach is dictated by the extent and location of the tumor. in our case, the tumor originated from the peripheral branch of infra - orbital nerve and presented as nodular swelling immediately underlying the skin. incision placed in the natural skin crease of naso - labial fold was used to approach and completely remove the lesion. other approaches used include intra - oral vestibular incision for smaller lesions localized in the buccal space, caldwell - luc approach for tumor within the maxillary sinus, subcilliary or eyelid crease incision for intraorbital schwannoma. larger lesion involving greater areas of mid - face are treated by extra - oral weber - furgusson incision combined with ostectomy [table 1 ]. although malignant transformation and recurrence of schwannoma is very low, a case of recurrent infra - orbital schwannoma attributed to incomplete removal of the lesion through a limited skin incision was reported. schwannoma arising from the infra - orbital nerve is rare, making the clinical diagnosis often difficult. although uncommon, schwannoma involving the peripheral branch of infra - orbital nerve may present as localized mid - face swelling and should be included in differential diagnosis of benign sub - cutaneous swelling in infra - orbital region. | extra - cranial schwannomas although common in head and neck region are very rarely seen originating from the infra - orbital nerve. we report a case of schwannoma arising from infra - orbital nerve in a 40-year - old male patient. the case presented as an isolated, asymptomatic, slow growing sub - cutaneous nodular swelling over left side of mid - face. on ultrasonography, a localized lesion within the sub - cutaneous tissue of cheek was observed, without involvement of orbital, maxillary sinus or underlying bone. aspiration biopsy of the lesion showed spindle shaped cells predominantly arranged in antoni a pattern around verocay bodies, with less organized antoni b tissue in few places. diagnosis of schwannoma, probably arising from terminal branch of infra - orbital nerve was established. the tumor was approached through skin incision. at the time of exploration, the lesion was found to emanate from the nerve trunk of peripheral branch of infra - orbital nerve, which was dissected and preserved. we correlate our experience with previously reported cases of infra - orbital nerve schwannoma. |
thyroid cancer is a common endocrine malignancy that has rapidly increased in global incidence in recent decades. in the united states, the 6.6% average annual increase in thyroid cancer incidence between 2000 and 2009 is the highest among all cancers. although the death rate of thyroid cancer is relatively low, the rate of disease recurrence or persistence is high, which is associated with increased incurability, morbidity, and mortality the prevalence of obesity has dramatically increased in the last 2 decades. the diagnosis of obesity is often based on body mass index (bmi), calculated as weight in kilograms divided by height in meters squared (kg / m). obesity has long been recognized as a trigger for many diseases, such as hypertension, hypercholesterolemia, diabetes, and insulin resistance. additionally, during the last decades obesity has been consistently related to the development and progression of different types of cancers. an extensive review published a few years ago estimated that 20% of all cancers might be caused by obesity. the relationship between obesity and risk of thyroid cancer has been studied for more than 10 years. several studies found obesity to be a risk factor in thyroid cancer, but other studies showed no association between obesity and risk of thyroid cancer. two meta - analyses investigated the association between obesity and thyroid cancer risk, reporting that obesity was associated with thyroid cancer risk. therefore, in this study we conducted a meta - analysis to assess the association between obesity and thyroid cancer risk. we searched pubmed, embase, springer link, ovid, chinese wanfang data knowledge service platform, chinese national knowledge infrastructure (cnki), and chinese biology medicine (cbm) databases up to 10 august 2014. body mass index. we included articles if they the following criteria : (1) evaluation of obesity and thyroid cancer risk, (2) using a case - control or cohort design, (3) adjusted risk ratios (rrs), hazard ratios (hrs), or odds ratios (ors) with 95% confidence intervals (cis) were reported. if they encountered conflicting evaluations, agreement was reached following a discussion ; if they could not reached agreement, another author was consulted to resolve the debate. the following information was extracted from each study : first author, year of publication, study type, ethnicity, age, sex, years of follow - up, sample size, number of cases, covariates, adjusted or / hr / or, and the corresponding 95% ci of thyroid cancer risk. for thyroid cancer risk, we calculated summary rrs and 95% cis for obesity versus normal weight. statistical heterogeneity among studies was evaluated using the q and i statistics. for the i metric, we considered low, moderate, and high i values to be 25%, 50%, and 75%, respectively. we did subgroup analyses according to study type, sex, race, pneumonia type, age, smoking status, and histology. sensitivity analysis was conducted by excluding 1 study at a time to explore whether the results were driven by 1 large study or by a study with an extreme result. all statistical analyses were performed with stata software (version 12.0, stata corporation, college station, tx, usa). a threshold of p<0.1 was used to decide whether heterogeneity was present. in other cases, we searched pubmed, embase, springer link, ovid, chinese wanfang data knowledge service platform, chinese national knowledge infrastructure (cnki), and chinese biology medicine (cbm) databases up to 10 august 2014. we included articles if they the following criteria : (1) evaluation of obesity and thyroid cancer risk, (2) using a case - control or cohort design, (3) adjusted risk ratios (rrs), hazard ratios (hrs), or odds ratios (ors) with 95% confidence intervals (cis) were reported. if they encountered conflicting evaluations, agreement was reached following a discussion ; if they could not reached agreement, another author was consulted to resolve the debate. the following information was extracted from each study : first author, year of publication, study type, ethnicity, age, sex, years of follow - up, sample size, number of cases, covariates, adjusted or / hr / or, and the corresponding 95% ci of thyroid cancer risk. for thyroid cancer risk, we calculated summary rrs and 95% cis for obesity versus normal weight. for the i metric, we considered low, moderate, and high i values to be 25%, 50%, and 75%, respectively. we did subgroup analyses according to study type, sex, race, pneumonia type, age, smoking status, and histology. sensitivity analysis was conducted by excluding 1 study at a time to explore whether the results were driven by 1 large study or by a study with an extreme result. all statistical analyses were performed with stata software (version 12.0, stata corporation, college station, tx, usa). a threshold of p<0.1 was used to decide whether heterogeneity was present. in other cases, after detailed evaluations, 21 studies were selected for final meta - analysis [626 ]. a manual search of reference lists from these studies did not yield any new eligible study. eleven studies reported 2 cohorts, and finally 32 studies (n=12 620 676) were included in this meta - analysis. the evaluations of the association between obesity and thyroid cancer risk are summarized in table 2. obesity was associated with a significantly increased risk of thyroid cancer when compared with normal weight (adjusted rr=1.33 ; 95% ci, 1.241.42 ; i=25% ; figure 2). in the subgroup analysis by study type, increased risk of thyroid cancer was found in cohort studies (rr=1.29 ; 95% ci, 1.201.37 ; i=21%) and case - control studies (or=1.76 ; 95% ci, 1.362.28 ; i=0%), respectively. in the subgroup analysis according to sex, both obese men (rr=1.26 ; 95% ci, 1.131.40 ; i=9%) and women (rr=1.43 ; 95% ci, 1.251.64 ; i=33%) were significantly at risk of thyroid cancer. when stratified by ethnicity, significantly elevated risk was observed in caucasians (rr=1.26 ; 95% ci, 1.181.33 ; i=9%) and in asians (rr=1.54 ; 95% ci, 1.271.86 ; i=16%). in the age subgroup analysis, both young (rr=1.23 ; 95% ci, 1.131.34 ; i=0%) and old populations (rr=1.28 ; 95% ci, 1.111.46 ; i=32%) showed increased thyroid cancer risk. subgroup analysis on smoking status showed that increased thyroid cancer risks were found in smokers (rr=1.10 ; 95% ci, 1.021.20 ; i=0%) and in non - smokers (rr=1.20 ; 95% ci, 1.111.28 ; i=0%). in the histology subgroup analyses, increased risks of papillary thyroid cancer (rr=1.26 ; 95% ci, 1.151.39 ; i=35%), follicular thyroid cancer (rr=1.29 ; 95% ci, 1.081.53 ; i=33%), and anaplastic thyroid cancer (rr=1.93 ; 95% ci, 1.233.03 ; i=0%) were observed. however, obesity was associated with decreased risk of medullary thyroid cancer (rr=0.50 ; 95% ci, 0.270.97 ; i=1%). as shown in figure 3, the results showed that the pooled ors tended to be stable. a single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors, and the corresponding pooled ors were not materially altered (figure 4). after detailed evaluations, 21 studies were selected for final meta - analysis [626 ]. a manual search of reference lists from these studies did not yield any new eligible study. eleven studies reported 2 cohorts, and finally 32 studies (n=12 620 676) were included in this meta - analysis. the evaluations of the association between obesity and thyroid cancer risk are summarized in table 2. obesity was associated with a significantly increased risk of thyroid cancer when compared with normal weight (adjusted rr=1.33 ; 95% ci, 1.241.42 ; i=25% ; figure 2). in the subgroup analysis by study type, increased risk of thyroid cancer was found in cohort studies (rr=1.29 ; 95% ci, 1.201.37 ; i=21%) and case - control studies (or=1.76 ; 95% ci, 1.362.28 ; i=0%), respectively. in the subgroup analysis according to sex, both obese men (rr=1.26 ; 95% ci, 1.131.40 ; i=9%) and women (rr=1.43 ; 95% ci, 1.251.64 ; i=33%) were significantly at risk of thyroid cancer. when stratified by ethnicity, significantly elevated risk was observed in caucasians (rr=1.26 ; 95% ci, 1.181.33 ; i=9%) and in asians (rr=1.54 ; 95% ci, 1.271.86 ; i=16%). in the age subgroup analysis, both young (rr=1.23 ; 95% ci, 1.131.34 ; i=0%) and old populations (rr=1.28 ; 95% ci, 1.111.46 ; i=32%) showed increased thyroid cancer risk. subgroup analysis on smoking status showed that increased thyroid cancer risks were found in smokers (rr=1.10 ; 95% ci, 1.021.20 ; i=0%) and in non - smokers (rr=1.20 ; 95% ci, 1.111.28 ; i=0%). in the histology subgroup analyses, increased risks of papillary thyroid cancer (rr=1.26 ; 95% ci, 1.151.39 ; i=35%), follicular thyroid cancer (rr=1.29 ; 95% ci, 1.081.53 ; i=33%), and anaplastic thyroid cancer (rr=1.93 ; 95% ci, 1.233.03 ; i=0%) were observed. however, obesity was associated with decreased risk of medullary thyroid cancer (rr=0.50 ; 95% ci, 0.270.97 ; i=1%). as shown in figure 3, the results showed that the pooled ors tended to be stable. a single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors, and the corresponding pooled ors were not materially altered (figure 4). the present meta - analysis, including 12 620 676 subjects from 32 observational studies, explored the association between obesity and thyroid cancer risk. this result remained significant in various types of studies, such as cohort studies and case - control studies. in addition, obesity was significantly associated with thyroid cancer risk in males and females. subgroup analyses stratified by ethnicity showed that obese asians had higher thyroid cancer risk than caucasians, but it is possible that random error may account for this difference. in fact, only 6 studies investigated the association between obesity and thyroid cancer risk in asians.. found that dynamic patterns of change for thyroid hormones were not different in asian and western caucasian women. in the subgroup analysis by age, we found obesity exhibited increased thyroid cancer risk in young and old subjects. actually, when we limited the meta - analysis to studies that controlled for age, a significant association between obesity and thyroid cancer risk remained (rr=1.30 ; 95% ci, 1.221.40 ; i=22%). cigarette smoking is a pro - inflammatory stimulus and an important risk factor for cancer. our results showed that both smokers and non - smokers had increased thyroid cancer risk. obese subjects showed increased risks of papillary thyroid cancer, follicular thyroid cancer, and anaplastic thyroid cancer. this result indicates that obesity may have a different effect on the pathogenesis and occurrence of thyroid cancer in different histologies. however, why obesity could influence the different histological types of thyroid cancer is still uncertain. clearly, more studies are needed to elucidate the differential effect of obesity in the various thyroid cancer types. there were several potential explanations for why obese individuals may have higher risk of thyroid cancer. first, there is a clinical association between higher serum thyroid - stimulating hormone (tsh) levels and increased risk of malignancy in human thyroid nodules and advanced stage of the disease. some cross - sectional studies in euthyroid subjects demonstrated a positive association between serum tsh and bmi. second, leptin levels were higher in thyroid cancer patients compared to healthy subjects in a case - control study. third, insulin resistance, a common metabolic perturbation in obesity, may play a role in thyroid tumor growth, with insulin directly binding to insulin receptors or stimulating insulin - like growth factor, estrogen, or other hormones, such as tsh, to enhance the proliferation of thyroid cancer cells. studies on the positive association between obesity and thyroid cancer will have important implications in the future, because obesity is a modifiable risk factor [4145 ]. future studies on the effects of weight gain or weight loss on altering risk for thyroid cancer are essential. first, it was the first study of interactions between age, histology, and smoking status specificities and obesity. second, the methodological issues for meta - analyses, such as one - way sensitivity analysis and cumulative meta - analysis, were well investigated. third, this meta - analysis included 32 studies (n=12 620 676) and thus was more conclusive and more powerful than previous studies. results from one - way sensitivity analysis and cumulative meta - analysis suggest the high stability and reliability of our results. heterogeneity and publication bias can be important influences on the results of meta - analyses. in our study additionally, funnel plots and egger s tests were used to find potential publication bias. thus, our results should be interpreted with caution and more studies are needed to confirm the effect of obesity on thyroid cancer risk. first, the number of published studies was not sufficient for a comprehensive analysis, particularly for africans. second, all the studies included in this meta - analysis used a case - control or cohort design, which are susceptible to recall and selection biases. third, because this meta - analysis investigated only obesity, we can not exclude the possibility that the observed associations may be confounded by other lifestyle factors, such as lower physical activity or dietary factors. this meta - analysis found a significant association between obesity and thyroid cancer risk, except medullary thyroid cancer. further studies in more ethnic groups, especially african, are warranted to validate this result. | backgroundseveral studies have evaluated the association between obesity and thyroid cancer risk. however, the results remain uncertain. in this study, we conducted a meta - analysis to assess the association between obesity and thyroid cancer risk.material/methodspublished literature from pubmed, embase, springer link, ovid, chinese wanfang data knowledge service platform, chinese national knowledge infrastructure (cnki), and chinese biology medicine (cbm) were retrieved before 10 august 2014. we included all studies that reported adjusted risk ratios (rrs), hazard ratios (hrs) or odds ratios (ors), and 95% confidence intervals (cis) of thyroid cancer risk.resultsthirty-two studies (n=12 620 676) were included in this meta - analysis. obesity was associated with a significantly increased risk of thyroid cancer (adjusted rr=1.33 ; 95% ci, 1.241.42 ; i2=25%). in the subgroup analysis by study type, increased risk of thyroid cancer was found in cohort studies and case - control studies. in subgroup analysis by sex, both obese men and women were at significantly greater risk of thyroid cancer than non - obese subjects. when stratified by ethnicity, significantly elevated risk was observed in caucasians and in asians. in the age subgroup analysis, both young and old populations showed increased thyroid cancer risk. subgroup analysis on smoking status showed that increased thyroid cancer risks were found in smokers and in non - smokers. in the histology subgroup analyses, increased risks of papillary thyroid cancer, follicular thyroid cancer, and anaplastic thyroid cancer were observed. however, obesity was associated with decreased risk of medullary thyroid cancer.conclusionsour results indicate that obesity is associated with an increased thyroid cancer risk, except medullary thyroid cancer. |
systemic lupus erythematosus (sle) is a severe systemic autoimmune disease and, as such, is characterized by a loss of self - tolerance. the etiology of sle is not well defined, but genetic, hormonal, and environmental factors, as well as immune disorders, are likely implicated. during sle, inflammation leads to damage of various tissues, including the joints, skin, kidneys, heart, lungs, blood vessels, and brain. dysregulation of various components of the immune system can be observed at different stages of disease development, but hyperactivity of b cells, leading to excessive production of multiple autoantibodies (autoab), is one of the major immunological stigmata of sle. indeed, sle is characterized by the production of antinuclear autoab (e.g., autoab specific for chromatin) and by the formation of immune complexes, which contribute to tissue damage. deposits of immune complexes in organs such as kidneys lead to subsequent inflammation through the activation of the complement system and the recruitment of inflammatory cells. the presence of autoab is an absolute prerequisite for the development of lupus nephritis and, interestingly, we demonstrated that pathogenic autoab can be locally produced by plasma cells, which have homed to inflamed kidneys of lupus mice. b cells and derivatives (plasma cells) are thus considered at the center of sle pathogenesis and this is supported by the observation of a high frequency of plasma cell precursors in the blood of children with sle. furthermore, an increase of circulating plasma cells in lupus patients is correlated with disease activity. the generation of ab can occur via the extrafollicular or the germinal center (gc) responses. the extrafollicular response leads to short - lived plasma cells, which do not go through the affinity maturation process. in contrast, the gc is the theater of intense cell collaboration between gc b cells and follicular helper t cells (tfh) leading to the differentiation of long - lived plasma cells harboring high antigen - specificity. interestingly, lupus autoab are high affinity, somatically mutated, and class - switched immunoglobulin (ig)g indicating t and b cell collaboration and intense gc activity. therefore, it is likely that a dysfunction in b cell differentiation mechanisms occurs in lupus, leading to excessive numbers of autoreactive plasma cells. it is particularly attracting and plausible to envisage that a dysregulation of tfh could be the underlying key factor. in this review, we succinctly expose recent understanding in tfh biology (described in detail elsewhere ; see for review), in order to introduce important molecular factors involved in tfh differentiation, regulation, and function. we then give an overview of the aberrant expression and/or function of such key players in lupus patients, and we highlight their potential as therapeutic targets. the generation of high affinity ab requires t / b interactions that mainly occur in gc. tfh cells represent a distinct subset of cd4 t cells involved in gc formation and specialized in providing help to b cells to differentiate into plasma cells or memory b cells. tfh express high levels of cxc chemokine receptor type 5 (cxcr5), pd-1 (programmed death-1), icos (inducible t cell co - stimulator), and the regulator transcription factor bcl6 (b cell lymphoma 6), which provide excellent markers for their identification. moreover, secretion of high levels of il-21 is a critical characteristic of tfh cells. tfh are generated after immunization or infection following the interaction of naive cd4 t cells with dendritic cells (dc) within the t cell zone of secondary lymphoid organs (slo). signals provided by dc induce the expression of a myriad of proteins (transcription factors, surface molecules, and cytokines) that are essential for tfh generation, migration, and function. in fact, tfh differentiation is a multistage process (figure 1), which can be sequentially defined as follows : (i) naive cd4 t cells are activated by dc (thanks to the mhc - peptide complex / tcr interaction) in the t cell zone and become immature tfh (also called pre - tfh) ; (ii) newly generated pre - tfh then migrate to the interfollicular zone, where cognate interactions with b cells allow the final maturation step ; (iii) these mature tfh reach the gc in which tfh - gc b cell interactions will favor isotype class switch, somatic hypermutations, and affinity maturation. the initial priming of cd4 t cells requires cognate interactions and costimulatory signals delivered by dc through cd40, cd80/86, icosl, and ox40l (table 1). cd28 (that binds cd80/86) was shown to be essential to tfh development as mice deficient for cd28 display cd4 t cells that fail to upregulate cxcr5 and ox40, leading to disrupted gc formation. in addition, upregulation of ox40l on dc following cd40-induced maturation allows cxcr5 expression by ox40 t cells. moreover, icos signaling leads to an increased expression of the transcription factors bcl6 and ascl2 (achaete - scute homologue-2). the latter promote both the reciprocal cxcr5 upregulation and ccr7 downregulation on activated cd4 t cells, which then become pre - tfh [12, 13 ]. in turn, bcl6 induces the expression of icos, pd1, cd40l, and sap (slam- (signaling lymphocytic activation molecule-) associated protein ; critical for t - b interaction). cytokines secreted by dc also play a pivotal role in pre - tfh development (table 1). il-6, a dc - derived proinflammatory cytokine, has been demonstrated to be the main soluble factor driving tfh differentiation in mice. in humans, il-12 has been shown to be the key cytokine that promotes tfh - like cell differentiation [15, 16 ]. if, in the initial work, neither il-6 nor il-21 were described as being able to promote tfh differentiation, a recent study suggests that human plasmablasts produce il-6, which is responsible for the subsequent differentiation of naive cd4 t cells into b cell helpers cxcr5icosbcl6il-21 t cells. il-21 is required for tfh function but it is also an important factor for tfh generation and, interestingly, both il-6 and il-12 are potent inducers of il-21 expression in mice and humans, respectively. as il-21 is an autocrine cytokine for pre - tfh generation, further studies are required to better clarify individual cytokine contributions. cytokine signaling involves the subsequent activation of janus kinase - stat (signal transducer and activator of transcription) signaling pathway. stat3 is a major signaling molecule for il-6 and il-21 [20, 21 ], whereas il-12 signaling occurs through stat4 activation. however, il-12-induced expression of il-21 by human cd4 t cells is compromised in patients with functional stat3 deficiency, suggesting that il-12 ability to promote il-21-producing cd4 t cells is predominantly stat3 dependent. moreover, altogether, these data suggest that the stat3 signaling pathway plays an important role in tfh differentiation and subsequent b cell help. during this first step of the tfh differentiation process, both cell surface interactions and cytokine signaling play a crucial role in bcl6 induction. bcl6 requirement for tfh development was reported in 2009 by 3 independent groups [2426 ]. indeed, bcl6 is a master regulator for tfh lineage commitment as its expression can inhibit th1, th2, and th17 differentiation. bcl6 expression is influenced by il-6 and il-21 via stat1 and stat3 signaling and by icos - pi3k (phosphoinositide 3-kinase) signaling. moreover, bcl6 expression is controlled by a complex regulatory network of activating factors (see for detailed review) such as basic leucine zipper transcriptional factor atf like (batf ;), transcription factor 1 (tcf-1 ;), lymphoid enhancer - binding factor (lef-1 ;), and b cell oct - binding protein 1 (bob1 ;), while forkhead box protein o1 (foxo1 ;) negatively regulates bcl6 expression. thanks to cxcr5 expression enhancement and ccr7 downregulation (table 2), pre - tfh cells migrate to the b cell follicle in response to a cxcl13 gradient and their interaction with antigen - specific b cells at the t - b border contributes to final tfh differentiation. indeed, the lower frequency of tfh cells in b cell - deficient mice suggests that b cells are also important for the generation of tfh cells. at this stage, b cells act as the major antigen - presenting cells (apc) for primed - tfh that will then fully differentiate into gc tfh cells. mature tfh and b cells that have formed stable t - b conjugates move together into the follicle to form gc. stable t - b conjugate formation requires interaction between icos on tfh and icosl expressed by b cells, as well as slam interactions (table 2). sap, which is the adaptor signaling protein downstream of slam, was demonstrated to be important for stabilizing cognate t - b interactions. indeed, sap - deficient cd4 t cells have an impaired capacity to stably interact with cognate b cells, resulting in a failure to induce b cell clonal expansion. moreover, patients with x - linked lymphoproliferative disease (xlp), an immunodeficiency resulting from mutations in the sh2d1a gene which encodes sap, harbor humoral defects characterized by hypogammaglobulinemia and reduced numbers of tfh. b cells thus play a key role in the tfh maturation step by both acting as apc and stabilizing tfh - gc b cell interactions through icosl and slam. the major function of tfh is to enhance high affinity memory ab responses following migration to gc. in the follicles, tfh - gc b cell crosstalk involves cd40l, il-21, pd-1, and baff (b cell activating factor) (table 3). the signal delivered through interaction between pd-1 on tfh and pd - l1 expressed by gc b cells is crucial for gc b cell survival. il-21 production by tfh directly regulates b cell proliferation and class - switch, and the il-21 pathway has been identified as a critical component of the memory b cell response as secondary antigen - specific igg responses are impaired in il-21r - knockout mice. baff is a cytokine that belongs to the tumor necrosis factor (tnf) ligand family and its receptors are bcma (b cell maturation antigen), taci (transmembrane activator and calcium modulator and cyclophilin ligand interactor), and baff receptor 3 (br3). baff is produced by stromal cells in the slo and involved during gc development by influencing icosl expression on b cells and thus regulating the ability of gc b cells to promote tfh expansion. moreover, baff production by tfh is critical for the survival of high affinity b cell clones. in summary, molecules that have been described to play a key role in tfh biology do not display equivalent functions. some are necessary for tfh migration from the t cell zone to the gc, others are absolutely required for their development or function, and finally some of them are essential for tfh maintenance and survival (tables 13). considering the important role of tfh cells in humoral immunity, a balance between stimulatory and inhibitory mechanisms regulating their function is required for immune homeostasis. however, while signals important for tfh development are clearly defined nowadays, little is known about mechanisms involved in their regulation. the coinhibitory pd-1/pd - l1 pathway can limit tfh expansion and consequently the humoral ig response. similarly, it was demonstrated that the inhibitory receptor b and t lymphocyte attenuator (btla) suppresses gc b cell development and subsequent igg responses by inhibiting il-21 production by tfh cells (table 3). recently, the existence of regulatory t cells (treg) able to inhibit gc responses was described. this subset of regulatory t cells of thymic origin was first identified in mice and named tfr (follicular regulatory t cells). they express typical markers of both tfh cells (bcl6, cxcr5, pd-1, and icos) and classical treg (foxp3) ; they localize in the gc and possess suppressive activity. a cd4 t cell population coexpressing foxp3, bcl6, and cxcr5 was also visualized in human tonsils. indeed, the mir-17~92 cluster was shown to promote tfh differentiation by repressing pten (phosphatase and tensin homolg), phlpp2 (pleckstrin homology domain and leucine - rich repeat protein phosphatase) (phosphatases that inhibit bcl6 expression through interfering with pi3k signaling), and ror (retinoic acid - related orphan receptor) expression [42, 43 ]. on the other hand, mir-10a negatively regulates tfh differentiation by directly inhibiting bcl6 expression. similarly, mir-146a, a microrna that is highly expressed in tfh cells, was recently described as a negative regulator of tfh cell numbers. mir-146a deficiency leads to accumulation of both tfh and gc b cells, likely due to enhanced icosl and icos expression on gc b cells and tfh cells, respectively. finally, il-2 signaling is also an important negative regulator of tfh differentiation by inducing stat5-dependent expression of blimp1, a bcl6 repressor [4648 ]. moreover, high il-2 production by th1 cells induces t - bet, which in turn inhibits bcl6 expression and tfh differentiation. the main function of tfh cells consists in regulating the clonal selection of gc b cells and providing b cells with signals for ig production, isotype switching, and somatic hypermutations. as abnormal activation of b cells and autoab production are central to autoimmune diseases, such as lupus, altered tfh differentiation, function, and regulation were suspected to play a role in lupus pathogenesis. first hypotheses regarding the role of tfh cells in sle development are based on studies using mice deficient for roquin1 (a negative regulator of icos mrna stability) in which an excessive number of tfh cells and gc reactions and high levels of il-21 are associated with a lupus - like phenotype [50, 51 ]. other evidences come from studies on il-21, the main cytokine produced by tfh, in lupus mice. high il-21mrna as well as elevated il-21 serum levels were described in bxsb.yaa mice, which develop an sle - like disease. the use of a fusion protein consisting in the il-21r linked to the fc domain of a mouse igg2a (il-21r.fc, which therefore binds to il-21 and prevents activation of its receptor) revealed a complex biphasic role of il-21 in this mouse model as it increases or diminishes the disease severity depending of the stage of the disease at the time of il-21 neutralization (at early or late stages). this could be related to the action of il-21 on b cells but also on t cell responses. in lupus mrl / lpr mice, activated cd4 t cells secrete 10 times more il-21 than control mice and il-21r deficiency leads to reduced numbers of tfh cells. in addition, abundant tfh - like cells are located outside the gc where they support extrafollicular b cell differentiation and plasmablast maturation in bxsb - yaa and mrl - fas lupus mice [56, 57 ]. in the latter and contrary to what was expected, the extrafollicular pathway was shown to be the most important way to generate hypermutated autoabs. however, there is no evidence to date supporting the involvement of such extrafollicular response in human sle. tfh cells are located in slo ; therefore the major problem encountered in studies of human tfh is that lymphoid tissues of lupus patients can not be easily accessed, making it difficult to identify tfh cells and to determine whether the generation or function of these cells is dysregulated. first studies were based on the enumeration of cd4cxcr5 in peripheral blood as gc tfh counterparts. using this strategy, it was shown in human sle that circulating tfh cells (ctfh) defined as cd4cxcr5pd-1 and/or icos t lymphocytes are expanded in lupus patients and their presence correlates with a more severe disease phenotype [5964 ]. morita. have described a circulating population in healthy donors that shares common phenotypic and functional characteristics with tfh cells from gc. moreover, they distinguished three subclasses, that is, tfh17, tfh2, and tfh1, defined according to the expression of the ccr6 and cxcr3 chemokine receptors : tfh17 cells are cxcr3ccr6 cells whereas tfh2 cells are cxcr3ccr6 cells and tfh1 cells are cxcr3ccr6 cells. tfh17 and tfh2 cells were identified as able to provide help to b cells via il-21 production, resulting in igm and igg secretion, whereas tfh1 have limited helper functions. however, icos expressing tfh1 are able to help memory b cells (but not naive b cells) to produce ab following influenza vaccination. moreover, morita and colleagues showed that patients with juvenile dermatomyositis displayed a profound skewing of ctfh cells towards tfh2 and tfh17 cells that correlated with disease activity, suggesting that an altered balance of tfh subtypes contributes to human autoimmunity. recently, the differential expression of icos, pd-1, and ccr7 interestingly allowed distinguishing three memory ctfh subsets defined as activated cells (icospd1ccr7) or quiescent cells (icospd1ccr7 and icospd1ccr7) [67, 68 ]. in sle patients, the ccr7pd1 subset is indicative of active tfh differentiation and its overrepresentation is associated with elevated autoab titers and high disease activity. by analyzing cxcr3 and ccr6 expression, we also interestingly described an altered phenotype of ctfh cells characterized by the enhanced frequency of b cell helper tfh2-like cxcr3ccr6 cells and a decreased frequency of cxcr3ccr6tfh1-like cells (not able to provide b cell help) in lupus patients with an active disease. aberrant expression and/or function of tfh - related molecules are associated with lupus - like disease in mice [54, 70 ]. similarly, in lupus patients, numbers of molecules involved in tfh generation and/or regulation cd40/cd40l pathway plays an essential role in the initial phase of tfh development (t - dc interaction in the t cell zone ; figure 1,) and function (tfh - gc b cell crosstalk in the gc ;). interestingly, cd40l was found to be constitutively expressed at abnormally high levels on t cells (but also on b cells and monocytes) from lupus patients [72, 73 ]. furthermore, cd4 t cells from female lupus patients, which overexpressed cd40l mrna, were able to promote autologous b cell stimulation and autoab production. icos - mediated pi3k signaling is absolutely required for tfh differentiation, for tfh migration into the follicle, and also for tfh maintenance. pten acts as a negative regulator of the pi3k signaling pathway, leading to the inhibition of bcl6 expression and tfh differentiation. interestingly, pten expression is significantly decreased in sle b cells ; however, to the best of our knowledge, its expression in lupus cd4 t cells (especially tfh) has not been investigated yet. icos expression has been found to be enhanced in cd4 t cells from lupus patients compared to healthy donors [78, 79 ] and icos levels were higher in patients with nephritis than in those without nephritis. moreover, infiltrated icos t cells were shown to be in close contact with b cells in lupus kidneys. interaction between ox40l (on dc) and ox40 (on activated cd4 t cells) is also important for tfh development. ox40 expression by lupus peripheral blood cells was found to be predominantly restricted to memory cd45ro cd4 t cells and its levels correlated with disease activity. moreover, ox40 has also been found to be highly expressed in kidneys of patients with lupus nephritis. importantly, the upstream region of the ox40 gene contains a single risk haplotype for sle, which is correlated with increased expression of ox40 mrna and protein. finally, it was recently shown that ox40 signal promotes, ex vivo, the generation of tfh - like cells that are functional b cell helpers. elevated levels of il-6 have been found in the serum and in the urine of active sle patients [8587 ]. the increased frequency of il-6-producing peripheral blood mononuclear cells (pbmc) correlates with disease severity / activity and treatment response. raised expression of gp130 (one of the two subunits of the il-6 receptor) has been found on cd4 t cells and b cells from patients with active sle, while an important reduction in the gp130 expression by b lymphocytes was observed upon immunosuppressive treatment leading to milder disease activity. factors responsible for the constitutive expression of il-6 in sle have not been elucidated yet. serum il-21 levels were found to be elevated in patients with sle [69, 90 ], especially in patients with lupus nephritis, and to correlate with disease severity. the real - time pcr analysis of skin biopsies taken from 3 lupus patients also revealed that il-21 transcripts were significantly increased compared to control individuals. furthermore, the percentages of cd4 t cells producing il-21 are significantly enhanced in lupus patients. finally, polymorphisms within the il-21r and the il-21 genes have been reported and may confer risk for sle : a polymorphism in il-21r (namely, rs3093301) was found to associate with lupus in 2 independent cohorts, a genetic association of two snps located in intronic regions of the il-21 gene (rs2221903 and rs907715) was described, and the variant allele rs2055979a of the il-21 gene was recently found to be associated with increased il-21 levels. regarding baff, lupus sera have been shown to contain elevated levels of this cytokine and those levels correlate with both anti - dsdna titers [9698 ] and disease activity. finally, it has been reported that il-2 production (which inhibits tfh differentiation) upon tcr stimulation is impaired in sle t lymphocytes [100, 101 ]. this lower il-2 production could be explained by imbalanced expression between the transcription factors camp response element (cre) binding protein (creb) and the cre - modulator (crem), which, respectively, enhance and suppress the il-2 gene transcription. stat3, which is activated by cytokines such as il-6 and il-21, binds to the bcl6 promoter leading to high levels of bcl6 expression and is thus important for tfh differentiation. t cells from patients with sle display increased levels of total and phosphorylated stat3 [103, 104 ]. reduced expression of mir-146a (a negative regulator of tfh development) has been reported in pbmc from sle patients and seems to correlate with disease activity. moreover, a genome - wide association study has highlighted a variant, that is, rs2431697, in an intergenic region between pttg1 (pituitary tumor - transforming 1) and mir-146a, associated with lupus susceptibility. interestingly, the risk allele of this snp correlates with a diminution of mir-146a levels. to date, the analysis of frequency and/or functionality of tfr cells in an autoimmune context has not been reported. however, although there may be some discrepancies due to variations in phenotype analysis, peripheral regulatory t cells (cd4cd25 t cells) seem to play a role in human lupus pathogenesis. several studies reported that a decreased number of treg might contribute to the pathogenesis [108111 ], but there were conflicting data regarding treg function in lupus patients. the in vitro suppressive activity of these cells was found to be defective in some reports [111, 112 ] but other studies showed that the suppressive activity of highly purified treg from lupus patients is not altered. it has been proposed that defective suppression in lupus could be attributed either to a higher sensitivity of treg to fas - mediated apoptosis in an sle context or to a lower susceptibility of effector t cells to treg suppression. finally, it has been shown that ifn- production by lupus apcs might be responsible for altered treg functionality. data obtained from various lupus mouse models have already highlighted how blockade of signaling pathways involved in tfh generation could lead to disease improvement. the administration of a blocking icos - l specific monoclonal ab (mab) to lupus nzb / w mice interrupted tfh cell development leading to a decrease of autoab levels and glomerulonephritis [115, 116 ]. similar results were obtained in mrl / lpr lupus mice displaying a genetic deletion of icos. blockade of the cd40l - cd40 signaling pathway also led to the reduction of lupus symptoms in different mouse models [117, 118 ]. treatment of mrl / lpr lupus mice with a neutralizing anti - il-6r mab has favorable effects on renal function and leads to a reduction of anti - dsdna ab levels. in nzb / w mice, chronic administration of anti - il-6 or anti - il-6r mab improves survival and reduces the progression of proteinuria and anti - dsdna levels [120, 121 ]. in lupus - prone nzb / w and mrl / lpr mice, raised levels of baff are detected at the onset of the disease and treatment with either taci - ig or br3-ig is effective at preventing clinical disease and ameliorating renal injury. regarding il-21, its neutralization using il-21r.fc showed an improvement of biological and clinical signs of the disease in mrl / lpr lupus mice and bxsb - yaa mice [53, 54 ]. moreover, the administration of ab specific for the il-21r to mrl / lpr mice significantly reduced anti - dsdna ab titers and igg deposits in the kidneys when compared to control mice. in nzb / w mice, such il-21r blocking even allowed reversing nephritis and halting disease progression in mice with preexisting lupus. by using a mirna - delivery approach via bacteriophage ms2 virus - like particles, pan and colleagues recently showed that restoring the loss of mir-146a was effective in abolishing autoab production and delaying sle progression in lupus - prone mice. interestingly also, treatment with the small molecule called stattic (an inhibitor initially reported to block the phosphorylation, dimerization, and nuclear translocation of stat3 in tumor cells) delayed the onset of proteinuria and reduced both anti - dsdna autoab and inflammatory cytokine levels in mrl / lpr lupus mice. several therapeutic tools targeting tfh biology already exist and even if their direct effect on tfh development has not been evaluated, some of them were shown to improve the disease. tocilizumab, a humanized mab specific for the -chain of the il-6 receptor (which prevents il-6 from binding to membrane bound and soluble il-6 receptors), has been recently tested in sle patients with promising results. interestingly, tocilizumab therapy in rheumatoid arthritis patients leads to a significant reduction in circulating tfh cell numbers and il-21 production. belimumab, a human mab that binds soluble baff, therefore inhibiting recognition by baff specific receptors has been tested in patients and results from phase iii clinical trials have demonstrated the safety profile and efficacy in controlling lupus in a broad range of patients. belimumab is the first biologic to meet its primary endpoint in a phase iii clinical trial for lupus patients and it was approved by the us food and drug administration in 2011. among other potential therapeutic candidates, are those targeting t - b interactions, such as idec-131 (anti - cd40l ab), amg 557 (anti - icosl ab), abatacept (ctla4-ig), or targeting cytokines such as atr-07 (anti - il-21r ab), nnc0114 - 0006 (anti - il-21 ab), atacicept (taci - ig), and small molecules inhibiting cytokine signaling pathways (tofacitinib, a jak - stat inhibitor) (figure 2). although prognosis in sle has improved markedly in the last 40 years, a better knowledge of the disease remains of prime importance to develop more potent and specific treatments. new targeted therapies designed to block pathways involved in disease pathogenesis are on the horizon. one promising option could be to specifically target factors involved in the generation of plasma cells responsible for the production of pathogenic autoab in lupus. tfh play a critical role in b cell activation and differentiation, and recent data have evidenced their involvement in lupus pathogenesis. signals required for tfh development may thus represent interesting targets in order to reduce tfh numbers (and/or to correct the altered proportion of tfh subsets) or to qualitatively and/or quantitatively modulate their function. another exciting therapeutic option consists in enhancing the negative molecular and cellular regulators of tfh, such as mirna or tfr. | systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by b cell hyperactivity leading to the production of autoantibodies, some of which having a deleterious effect. reducing autoantibody production thus represents a way of controlling lupus pathogenesis, and a better understanding of the molecular and cellular factors involved in the differentiation of b cells into plasma cells could allow identifying new therapeutic targets. follicular helper t cells (tfh) represent a distinct subset of cd4 + t cells specialized in providing help to b cells. they are required for the formation of germinal centers and the generation of long - lived serological memory and, as such, are suspected to play a central role in sle. recent advances in the field of tfh biology have allowed the identification of important molecular factors involved in tfh differentiation, regulation, and function. interestingly, some of these tfh - related molecules have been described to be dysregulated in lupus patients. in the present review, we give an overview of the aberrant expression and/or function of such key players in lupus, and we highlight their potential as therapeutic targets. |
hypertension is a multifactorial condition characterized by high and sustained levels of blood pressure (bp). it is the most common condition in primary care and often associated with functional and/or structural changes in target organs and metabolic disorders, increasing the risk of fatal and nonfatal cardiovascular events [1, 2 ]. among the risk factors for mortality from cardiovascular disease (cvd), hypertension explains 40% and 25% of deaths from stroke and coronary artery disease (cad), respectively. endothelial cells of the vascular system are responsible for many biochemical reactions that maintain vascular homeostasis and consequently the bp levels. endothelium modulates vascular tone, not only by producing vasodilator substances but also by releasing vasoconstrictive substances through prostanoid of endothelin generation, as well as through conversion of angiotensin i (ai) in angiotensin ii (aii) on the endothelial surface. these vasoconstrictor agents not only act mainly locally but also present some systemic effects, playing an important role in regulating the vascular function and remodeling the arterial wall. in healthy individuals, there is a balance among these substances, tending to vasodilatation when endothelial function is normal. changes in endothelial function precede morphological changes of blood vessels and contribute to the development of clinical complications of cardiovascular diseases. thus, the beginning and the clinical course of adverse cardiovascular events depend directly on changes in vascular biology. the limited no bioavailability is the main mechanism involved in endothelial dysfunction, which is crucial for the development of cvd. in fact, endothelial dysfunction in peripheral and coronary vessels is an independent predictor of cardiovascular events and represents an early stage of cad. as endothelial dysfunction is reversible, early detection and intervention could have critical therapeutic and prognostic implications for patients with risk for, or even with established, cvd [7, 8 ]. therefore, an improvement in the no bioavailability can have a major effect on endothelial function and, consequently, on cvd prevention and treatment [9, 10 ]. metabolic syndrome can be considered a clinical and biochemical expression of insulin resistance, representing a clustering of central obesity, hypertension, hyperglycemia, and dyslipidemia [19, 20 ]. recently, experimental models and clinical studies demonstrated that reductions in the no bioavailability play a central role in the pathophysiology of metabolic dysfunction. endothelial nitric oxide synthase- (enos-) deficient mice were able to develop high bp and metabolic dysfunction, and both might be the result of insulin resistance [21, 22 ]. in the same experimental model, the amount of dietary nitrate used for this effect was comparable to those derived from enos under normal conditions, which corresponds to a rich intake of vegetables for humans. besides, dietary nitrate was able to increase tissue and plasma levels of bioactive nitrogen oxides. lastly, chronic nitrate supplementation prevented the prediabetic phenotype in these animals by reducing visceral fat accumulation and circulating levels of triglycerides. in humans, enos polymorphisms have been associated with insulin resistance, type 2 diabetes mellitus, and metabolic syndrome. furthermore, recent evidences have shown that obese subjects present a reduced ability to produce no [10, 24 ]. therefore, the dietary nitrate has been widely studied in clinical trials as an alternative form of the classical pathway of l - arginine to no production. poor eating habits may be considered as risk factors for cvd. in fact, high intake of foods rich in cholesterol, lipids, and saturated fatty acids and low consumption of fiber sources are related to dyslipidemia, obesity, diabetes mellitus, and hypertension. thus, nutritional interventions associated with changes in lifestyle are recognized as important strategies for primary prevention of hypertension and are auxiliary to pharmacological therapies to reduce cardiovascular risk. epidemiological evidences suggest that vegetable consumption reduces bp and risk of cvd [2729 ]. dash (dietary approach to stop hypertension) eating plan is one of the major effective strategies for prevention and nonpharmacological management of hypertension. this eating proposal highlights the importance of increasing fruit and vegetables intake, and recent research suggests that the beneficial effects of dash plan on bp are related to high inorganic nitrate content of food included in this eating plan (e.g., green leaves and root vegetables) [32, 33 ]. beetroots, lettuce, chard, arugula, and spinach are the vegetables containing the highest amount of nitrate, > 250 mg nitrate/100 g. beetroot is a vegetable, particularly rich in inorganic nitrate, which contains an average of 2056 mg of nitrate in a traditional cultivation. there are some studies using beetroot to test the effects of inorganic nitrate intake on bp. nitrate (no3) and nitrite (no2), present in beetroot and in other food sources, were recently related to cardiovascular benefits. however, they were previously considered as toxic compounds due to the development of malignancies such as gastric cancer. therefore, strict rules regarding these inorganic anions are regulated in food and in drinking water. beetroot and other vegetables sources of nitrate contain approximately 6080% of the daily nitrate intake in the western population. nitrate content in vegetables may be influenced by factors related to the plant itself, such as variety, species, and maturity, and to the environment, such as temperature, light intensity, lack of some nutrients, and fertilizer use. international organizations indicate that the consumption of dietary nitrate is about 31 to 185 mg / day in europe and 40 to 100 mg / day in usa, and the oral bioavailability of dietary nitrate is 100%. in 1962, world health organization (who) set an upper limit of nitrate consumption in food. an acceptable daily intake is 3.7 mg no3/body weight (kg), which is the same value adopted by the european authority for food safety. this amount is equivalent to 300 mg / day for an adult weighing 80 kg. after intake, dietary nitrate quickly increases in plasma, in about 30 minutes, reaching its peak in 90 minutes. in contrast, nitrite levels are considerably slower in circulation, reaching their peak in 2.5 to 3 hours. most of inorganic nitrate, about 75% of absorbed nitrate, is excreted in urine and 25% of plasma nitrate is excreted in saliva [18, 39 ]. the exact mechanism of salivary concentration is unknown. as a result, there is supply of substrate for nitrate reductase expressed by bacteria that colonize the dorsal surface of the tongue, resulting in reduction of nitrate to nitrite. after nitrite is then swallowed, the stomach and the acid environment reduce it to no. no and nitrite continue through the systemic circulation, and the remaining nitrite is reduced to no in high resistance vessels, promoting vasodilatation and consequently lowering bp (figure 1). both no3 and no2 from diet and via l - arginine participate in the no synthesis lately, there is a growing body of interest on the role of these two anions in biological function. the improvement in vascular dysfunction and in bp levels after dietary nitrate seems to be mediated by effects on oxidative stress and inflammation [42, 43 ]. after an acute intake of beetroot juice (500 ml), it is possible to observe reduction of 10 mmhg in systolic bp (after 2.5 h) and reduction of 8 mmhg in diastolic bp (after 3 h) in healthy individuals. the decrease in bp was sustained after 24 h of juice intake. the highest reduction in bp liu. evaluated the effects of a meal rich in nitrate (based on spinach consumption) on bp and arterial stiffness in healthy individuals. two hours after a nitrate - rich meal consumption (220 mg nitrate), there was a larger artery elasticity index, lower pulse pressure, and lower systolic bp compared to the values after a standard meal, low in nitrate. recent experimental studies [23, 45, 46 ] and clinical trials have shown that nitrate dietary intakes from beetroot juice [18, 47 ], beet - enriched bread, or inorganic nitrate supplements have a protective effect against cvd because of reducing bp, platelet aggregation inhibition, and prevention of endothelial dysfunction. recently, bondonno. have shown that chronic ingestion of beetroot juice (one week, 420 mg nitrate / day) did not improve the bp control in treated hypertensive patients. in another study with overweight elderly subjects, jajja. showed reduction of 7 mmhg in systolic bp, after three weeks of beetroot juice intake (350 mg nitrate / day). however, when bp was evaluated for 24 hours by ambulatory blood pressure monitoring (abpm), no significant changes were shown in bp levels. table 2 shows some clinical trials that evaluated the effects of nitrate intake on bp and vascular function. in fact, there is no consensus about the effects of dietary inorganic nitrates on bp and endothelial function, and their effects on cardiovascular health, despite studies with positive results. since the initial investigations in healthy volunteers, studies using inorganic nitrate and formulations with nitrate salt showed promising results reducing bp, with nitrate doses ranging from 155 to 1484 mg / day, between 1 and 15 days, with reductions of 4 mmhg in systolic bp and of 1 mmhg in diastolic bp [33, 50 ]. in hypertensive patients, systolic bp remained significantly reduced in approximately 8 mmhg over 24 hours after the intervention, which is similar to the reduction provided by drug therapy (9 mmhg). this is important because the ingestion of dietary nitrate in a single dose per day may be sufficient to achieve benefits in lowering bp [50, 51 ]. increasing nitrite levels by nitrate intake appears to have beneficial effects in many physiologic and clinical settings. several clinical trials are being conducted to determine the great therapeutic potential of increasing the bioavailability of nitrite in human health and disease, including studies related to vascular aging. nevertheless, there are many limitations in nitrate studies, such as the type of population enrolled in each trial and the dependent effect of the baseline bp. therefore, the effects are unlikely to be the same among healthy and hypertensive individuals. in addition, when evaluating treated hypertensive patients, use of medications, such as calcium channel antagonists, may affect endothelial function and hence can interfere in some vascular parameters. sample size is also a limiting factor considering the fact that it is small in most of nitrate studies. indeed, large clinical trials are necessary to confirm the potential beneficial effects of inorganic nitrate in patients with cvd. even with these considerations, dietary nitrate seems to represent an inexpensive and a promising complementary therapy to support hypertension treatment with benefits for cardiovascular health. | poor eating habits may represent cardiovascular risk factors since high intake of fat and saturated fatty acids contributes to dyslipidemia, obesity, diabetes mellitus, and hypertension. thus, nutritional interventions are recognized as important strategies for primary prevention of hypertension and as adjuvants to pharmacological therapies to reduce cardiovascular risk. the dash (dietary approach to stop hypertension) plan is one of the most effective strategies for the prevention and nonpharmacological management of hypertension. the beneficial effects of dash diet on blood pressure might be related to the high inorganic nitrate content of some food products included in this meal plan. the beetroot and other food plants considered as nitrate sources account for approximately 6080% of the daily nitrate exposure in the western population. the increased levels of nitrite by nitrate intake seem to have beneficial effects in many of the physiological and clinical settings. several clinical trials are being conducted to determine the broad therapeutic potential of increasing the bioavailability of nitrite in human health and disease, including studies related to vascular aging. in conclusion, the dietary inorganic nitrate seems to represent a promising complementary therapy to support hypertension treatment with benefits for cardiovascular health. |
the determinants of physical activity (pa) in patients with copd are poorly understood. a recent systematic review identified several clinical, functional, sociodemographic, and lifestyle factors associated with pa in these patients, but the quality of evidence was low to very low, mainly due to the use of a cross - sectional design and lack of control for confounders. this lack of information is in contrast with the importance of pa in copd, as regular pa has been consistently related to a reduced risk of hospitalizations and death.1 among the potential determinants of pa that can be modified at the clinical setting, anxiety and depression are relevant because they are highly prevalent in copd patients and affect copd prognosis.24 anxiety has been reported in ~40% of copd patients.5 it can lead to tachypnea, which may worsen lung hyperinflation, leading to increased exertional dyspnea, reduced exercise capacity, and poor quality of life.6 depression, in turn, has been identified in ~25% of copd patients5 and is also associated with poorer exercise capacity and worse health status.2 previous studies have assessed the association between anxiety and depression symptoms with pa.715 however, they have a cross - sectional design so the directionality of the association (eg, whether anxiety / depression reduces pa or pa reduces anxious / depressive symptoms) could not be established. moreover, prior analyses have not considered the role of potentially relevant confounders, like exercise capacity9,11,12,15,16 or smoking status.711,13 hence, it remains unclear if anxiety or depression exerts an independent effect on pa levels alongside other factors commonly influencing these levels. finally, most studies mentioned79,12,14,15 used indirect measures of pa such as questionnaires, so there is potential for misclassification of the primary outcome of interest (ie, pa).17 we therefore aimed to assess the effect of anxiety and depression symptoms on pa in patients with copd, overcoming the limitations of previous studies by using a prospective design, measuring pa with an accelerometer and controlling for a wide range of potential confounders. we hypothesized that symptoms of anxiety and depression are inversely related with future pa in copd patients. this study is part of the physical activity as a crucial patient reported outcome in copd (proactive) project. see complete version in the supplementary material. prospective study, with repeated measures at baseline, 6 months, and 12 months. clinical trial registration : www.clinicaltrials.gov ; number : nct01388218. between july 12th and november 18th, 2011, we recruited a consecutive sample of 236 patients with copd defined by spirometry (post - bronchodilator forced expiratory volume in 1 second to forced vital capacity ratio [fev1/fvc ] 10% change in coefficient) the estimates for the remaining variables and ii) pa at t. as a final step, we built a parsimonious model using a backward stepwise elimination process from the saturated model removing variables (one at a time) if wald s p - value > 0.15.28 we tested goodness - of - fit of the final models (supplementary material). to identify potential effect modification, we stratified main analyses according to sex, health - related quality of life, copd spirometric severity, smoking status, comorbidities, exercise capacity, socio - economic status, and bmi (supplementary material). to test the potential role of copd exacerbations during follow - up as mediators of the effect of anxiety or depression on pa, we repeated the analyses : i) including copd exacerbations during follow - up as a covariate in multivariable models, and ii) excluding patients who had at least one copd exacerbation during the follow - up period. we performed several secondary analyses (supplementary material) to assess the sensitivity of our estimates to our assumptions regarding biases, as well as to test for model misspecification. briefly, we repeated analyses : i) using pa at 12 months only (ignoring measures at 6 months), ii) additionally adjusting for season of pa assessment, and iii) using complete case dataset. the analyses were performed using stata 12.1 (stata statistical software : release 12 ; statacorp lp, college station, tx, usa) and r 3.0.1 (r foundation for statistical computing, vienna, austria). prospective study, with repeated measures at baseline, 6 months, and 12 months. between july 12th and november 18th, 2011, we recruited a consecutive sample of 236 patients with copd defined by spirometry (post - bronchodilator forced expiratory volume in 1 second to forced vital capacity ratio [fev1/fvc ] 10% change in coefficient) the estimates for the remaining variables and ii) pa at t. as a final step parsimonious model using a backward stepwise elimination process from the saturated model removing variables (one at a time) if wald s p - value > 0.15.28 we tested goodness - of - fit of the final models (supplementary material). to identify potential effect modification, we stratified main analyses according to sex, health - related quality of life, copd spirometric severity, smoking status, comorbidities, exercise capacity, socio - economic status, and bmi (supplementary material). to test the potential role of copd exacerbations during follow - up as mediators of the effect of anxiety or depression on pa, we repeated the analyses : i) including copd exacerbations during follow - up as a covariate in multivariable models, and ii) excluding patients who had at least one copd exacerbation during the follow - up period. we performed several secondary analyses (supplementary material) to assess the sensitivity of our estimates to our assumptions regarding biases, as well as to test for model misspecification. briefly, we repeated analyses : i) using pa at 12 months only (ignoring measures at 6 months), ii) additionally adjusting for season of pa assessment, and iii) using complete case dataset. the analyses were performed using stata 12.1 (stata statistical software : release 12 ; statacorp lp, college station, tx, usa) and r 3.0.1 (r foundation for statistical computing, vienna, austria). a total of 220 patients were included, distributed similarly across study sites (48 [21.8% ] from athens, 41 [18.6% ] from edinburgh, 48 [21.8% ] from leuven, 48 [21.8% ] from london, and 35 [15.9% ] from groningen). a majority of patients were men (68%), with a mean (sd) age of 67 (8) years, fev1 57 (20)% predicted, rv / tlc 51 (11)%, bmi 27.1 (5.5) kg / m, and a moderate quality of life (table 1). the baseline prevalence of suggested and probable anxiety was 19% and 10% respectively, and the corresponding prevalence for depression was 16% and 5%. all followed patients had valid (ie, at least 3 days with at least 10 hours wearing time) data from the accelerometer. the mean (sd) dynaport wearing time was 880 (120) min / day, the mean number of steps was 4,812 (3,147) and mean time in locomotion was 60 (36) min / day. during follow - up, 22 (10%) patients were hospitalized at least once for a copd exacerbation. in bivariate analysis, depression symptoms at t were associated with a lower amount of pa at t+1 (6 months later) in a linear dose - response shape : 4,752 (2,334), 3,837 (1,987), and 3,446 (2,144) steps / day in patients with no symptoms, suggested and probable depression, respectively, p - trend = 0.02 (figure 2 and table s3). corresponding time in locomotion was 59.2 (37.3), 49.1 (31.5), and 39.8 (44.1) min / day, p - trend = 0.01. the differences of pa across hads - a categories were not statistically significant. in the multivariable saturated model (adjusted for age, 6mwd, comorbidities, fev1, and steps at t), patients walked 70 steps / day less (p=0.05) for each extra point on the hads - d score (table 2). this association persisted in the parsimonious model (=-81 steps / day, p=0.02). hads - d score was also significantly associated with less time in locomotion (table s4). hads - a score was not statistically associated with any pa outcome in multivariable models. the effect of hads - d on steps did not change after stratifying by potential effect modifiers, with two exceptions : i) a stronger association among patients with moderate cat scores (=132, p=0.03) compared to patients with lower or higher cat scores (=19, p=0.79 and =-26, p=0.73, respectively), and ii) a stronger association among people with high socio - economic status compared to those with low socio - economic status (=88, p=0.02, vs 2, p=0.98, respectively) (table s5). the inclusion of copd exacerbations during follow - up in the model did not modify the estimate of the association between depression and steps either (=70 steps / day per each hads - d point, p=0.05) (table s6). similarly, the exclusion of patients with any copd exacerbation during follow up (n=101, 46%) did not modify such estimate (=81, p=0.15) (table s6). sensitivity analyses yielded very similar results (tables s7s9). our study shows that depression symptoms, as measured by the hads, are prospectively associated with fewer steps per day and less time in locomotion in copd patients after 6 months previous studies reported a cross - sectional association between depression and pa levels in copd patients.712 our findings move beyond these preliminary findings by testing the directionality of this association. indeed, presence of depressive symptomatology very quickly seems to negatively impact pa (ie, over a time period of 6 months already), even after adjusting for baseline pa levels. furthermore, in contrast to previous studies, our study for the first time controlled for a comprehensive set of potential confounders such as lung function, exercise capacity or comorbidities. moreover, while previous studies focused on the role of depressive disorder, we demonstrated that presence of depressive symptoms impacts pa, a finding that is of major clinical importance. as each extra point in the hads - d score was associated with a reduction of 81 steps, our results support that copd management includes the assessment and treatment of mood disorders, even at subclinical stages. we would suggest that further research includes randomized controlled trials of drug and non - drug interventions to reduce depression symptoms, since they may be valuable also to improve pa. several mechanisms have been proposed that support the plausibility of depression symptoms affecting pa in both copd and healthy individuals. first, a recent systematic review found a clear association between depression and hospital admissions caused by copd exacerbations29 and an effect of exacerbations on pa levels has also been reported.30 however, we could not confirm that the effect of depression on pa is mediated by copd exacerbations. second, hormonal changes that appear during depression, including over - activity of the hypothalamo - pituitary - adrenocortical axis31 or dysregulation of the autonomous nervous system,32,33 have been associated with reduced pa in experimental studies34,35 and deserve further scrutiny in copd patients. finally, symptoms of depression may exacerbate the perception of dyspnea,36 which in turn could reduce pa levels. the lack of a measurable confounding role of exercise capacity in the association between depression and pa in our study, together with the large body of evidence showing a weak to moderate correlation between exercise capacity and daily pa,37 suggests that the clinical effects of depression on copd patients may be underestimated if only controlled assessment of exercise capacity is performed at the clinical or research setting. further, we found a stronger effect of depression on pa among people with moderate impact on quality of life than in those with low or high impact on quality of life. it could be speculated that patients with a moderate quality of life are those who experience the bigger impact of depressive symptoms on health outcomes, because in milder stages the depression symptoms are lower while in worsened quality of life, patients have reached the acceptance of the disease together with a higher understanding of their limitations. as previous research has reported a strong relationship between depression symptoms and quality of life scores in copd patients,38,39 the interaction between these factors on copd outcomes should be considered both in clinical practice and research settings. the role of anxiety in pa levels is unclear because a previous study has found an inverse association (ie, anxiety reduces pa level),12 while another study found a positive association (ie, anxiety increases pa level).10 yet, these studies suffer from methodological limitations as previously discussed, including a cross - sectional design, indirect measures of pa, and lack of control for confounders. our study, overcoming all the aforementioned limitations, found no significant association between anxiety symptoms and pa level in copd patients. although several studies have postulated common characteristics between anxiety and depression, both at molecular40 and clinical levels,41 other studies support significant differences between the two. clinically, levels of psychomotor hyperactivity are increased in anxiety42 but reduced in depression,43 which could lead to differential effects on pa. although elucidating these mechanisms is beyond the scope of the present study, our data support the need to further research the pathophysiological differences between the two entities44 in order to avoid attributing, a priori, common effects. the strengths of our study include the exhaustive control for potential confounders, the use of direct methods to assess pa, and the longitudinal design. limitations of our study are the following. information bias in the measure of anxiety and depression is possible because the questionnaire is subject to potential mis - classification. however, hads has been demonstrated to have good psychometric properties.45,46 it could be argued that the proportion of patients with hads anxiety and hads depression 11 was low (10% and 5%, respectively) in comparison with other studies.24,4749 this can be due to the fact that we excluded patients with a diagnosis and/or treatment for clinical depression and/or anxiety (to avoid any effect of therapies on pa level). consequently, it is likely that the association between depression and pa level is higher in the whole copd population than in our sample. in any case, confirmation of our results in studies using other validated instruments to assess anxiety and depression is warranted. secondly, it could be argued that our population does not represent the full copd spectrum and so the results can not be generalized to all patients. however, it is worth noting that this risk was mitigated by including patients recruited in diverse settings (primary care, hospital, and rehabilitation) and a range of mild to very severe airflow limitation. anyhow, any difference in the prevalence of depression, anxiety or pa levels with respect to the full copd population would not have affected the association between them. third, reverse causation, ie, that pa affects depression symptoms, could have been an alternative interpretation if the study design was cross - sectional. however, our study had a longitudinal prospective design, with anxiety and depression being measured at baseline, pa measured 6 months later, and baseline pa having been included as a covariate in the regression models to account for pa history and potential reverse causation. finally, although the possibility of residual confounding can never be ruled out, to the authors knowledge, all potential confounders were collected and tested. in conclusion, symptoms of depression are prospectively associated with a measurable reduction in pa 6 months later in copd patients. whereupon, and since the current management guidelines for patients with copd include the treatment of comorbidities, our study suggests that the management of symptoms of depression need to be considered, including in subclinical states, to prevent decline in pa over time. | backgroundthe role of anxiety and depression in the physical activity (pa) of patients with copd is controversial. we prospectively assessed the effect of symptoms of anxiety and depression on pa in copd patients.methodswe evaluated anxiety and depression (hospital anxiety and depression scale [hads ]), pa (dynaport accelerometer), and other relevant characteristics in 220 copd patients from five european countries at baseline and at 6 and 12 months of follow - up. hads score was categorized as : no symptoms (score 07), suggested (810), and probable (> 11) anxiety or depression. we estimated the association between anxiety and depression at t (baseline and 6 months) and pa at t+1 (6 and 12 months) using regression models with a repeated measures approach.resultspatients had a mean (standard deviation) age of 67 (8) years, forced expiratory volume in 1 second 57 (20)% predicted. at baseline, the prevalence of probable anxiety and depression was 10% and 5%, respectively. in multivariable models adjusted by confounders and previous pa, patients performed 81 fewer steps / day (95% confidence interval, 149 to 12, p=0.02) per extra point in hads - depression score. hads - anxiety symptoms were not associated with pa.conclusionin copd patients, symptoms of depression are prospectively associated with a measurable reduction in pa 6 months later. |
human immunodeficiency virus (hiv) infection and acquired immune deficiency syndrome (aids) affect all aspects of human endeavors as they cut across age, gender, race, socio - political status, education, relation, ethnic, culture, and barriers of any kind. as in 2005, about 43 million have been infected with the dreaded virus while over 25 million have died of the infection world - wide. it has also been reported that over 60% of the global hiv / aids cases are concentrated in africa. in nigeria, it has been reported that at least 10,000 youths are infected monthly, while the hiv prevalence rate as in 2008 was 5.0%. similarly, in the same year, the prevalence rate in adamawa state was 4.6%. the infection has now assumed an astronomical dimension world - wide, hence described as pandemic rather than being an epidemic. ordinarily, students are expected to be among the low - risk groups. however, because of the considerable complacency among them about hiv and its mode of infection as earlier documented,[79 ] the group is now tending toward high risk. the risky behaviors of some students in some parts of the world have been reported in many studies.[1018 ] this, therefore, calls for a serious attention to ascertain the extent of the infection among them. in view of devastating impact of hiv infection on african economy, it was recommended that for proper resource allocation, the epidemiological study of the infection is very essential, especially with particular reference to youths and demographic influence. youth, a group ranging from 15 to 24 years, occupies a prominent position in the economy of any nation as they are often regarded as future leaders. many of the members in this group are either in the institutions of learning or undergoing vocational training. some reports have shown that immoral and risky sexual behaviors are common among sexually mature and active students. this study reports the prevalence of hiv infection among students in some tertiary and secondary institutions in nigeria. a total of 1978 apparently healthy students aged 1135 years composed of 981 males and 997 females were randomly sampled in three geographical zones (north, south, and central) of adamawa state in north - eastern nigeria between september 2008 and august 2009. the subjects were those attending federal medical center, specialist hospital, general hospitals in ganye, mubi, garkida, and numan. others were subjects attending kowa hospital, peace hospital, adamawa hospital, yola biomedics laboratories, and health centers in tola, pella and fufore. they were apparently healthy seeking medical check - up for job recruitment, admissions, and blood donation. prior to the commencement of the study approval from research and ethics committee of the state ministry of health was obtained in accordance with the helsinki declaration guidelines. also, informed consent of the participants was sought and obtained in writing before volunteers were enrolled in the study. after proper sterilization of the antecubital fossa of the arm with swab of methylated spirit, 5 ml of blood was collected from each of the students by vein puncture and put in a plain tube, held at room temperature for about 20 minutes and spun to remove the serum needed for hiv antibodies tying. a capillus hiv kit (trinity biotech, uk) was used for the hiv antibodies detection while a determine (abbot, japan) hiv kit was used to confirm initial - positive cases. both positive and negative control specimens were included and run concurrently with the test samples to ascertain the reliability of the screening kits used. in - depth interview was also conducted with each student for information on educational institutions, settlement, and age. data obtained from the tests were entered and analyzed using spss version 16 software of computer (spss inc. the test of significance was performed using chi - square for hiv sero - prevalence with respect to educational institution category, settlement of the institutions, age, and gender of the subjects. a total of 1978 apparently healthy students aged 1135 years composed of 981 males and 997 females were randomly sampled in three geographical zones (north, south, and central) of adamawa state in north - eastern nigeria between september 2008 and august 2009. the subjects were those attending federal medical center, specialist hospital, general hospitals in ganye, mubi, garkida, and numan. others were subjects attending kowa hospital, peace hospital, adamawa hospital, yola biomedics laboratories, and health centers in tola, pella and fufore. they were apparently healthy seeking medical check - up for job recruitment, admissions, and blood donation. prior to the commencement of the study approval from research and ethics committee of the state ministry of health was obtained in accordance with the helsinki declaration guidelines. also, informed consent of the participants was sought and obtained in writing before volunteers were enrolled in the study after proper sterilization of the antecubital fossa of the arm with swab of methylated spirit, 5 ml of blood was collected from each of the students by vein puncture and put in a plain tube, held at room temperature for about 20 minutes and spun to remove the serum needed for hiv antibodies tying. a capillus hiv kit (trinity biotech, uk) was used for the hiv antibodies detection while a determine (abbot, japan) hiv kit was used to confirm initial - positive cases. both positive and negative control specimens were included and run concurrently with the test samples to ascertain the reliability of the screening kits used. in - depth interview was also conducted with each student for information on educational institutions, settlement, and age. data obtained from the tests were entered and analyzed using spss version 16 software of computer (spss inc. the test of significance was performed using chi - square for hiv sero - prevalence with respect to educational institution category, settlement of the institutions, age, and gender of the subjects. overall, the sero - prevalence rate of 13.7% was recorded consisting 9.9% in the tertiary and 3.8% in secondary institutions. the distribution of the infection showed no significant difference by age (=1.07, p>0.05) and by gender (=0.85, p>0.05). also, the prevalence had no significant association with the settlement of students (=0.96, p>0.05) and the status of educational institutions (=1.42, p>0.05). the findings from the study were expressed in percentages and presented in tables 13 as shown below. geographical distribution of hiv infection among students in tertiary and secondary institutions prevalence of hiv infection among students by educational settlement prevalence of hiv infection among students in relation to age and gender students are often regarded as future leaders of any nation considering their pivotal position in manpower development, socioeconomic, political, and technological advancement of a nation. however, many students and indeed some citizens of african countries are still reluctant to acknowledge the spread of hiv pandemic owing to their social and cultural norms. hiv infection is no more a news all over the world, what seems new now judging from this present study is that students are tending toward being among the high risk groups. the findings in this study revealed an amazing prevalence rate of 13.7% consisting of 9.9% among the students of tertiary institutions and 3.8% from those in secondary schools. meanwhile, there is a possibility that the hiv prevalence could still be higher than these findings because some subjects may be in the window period. in addition to some risky sexual behaviors, unhealthy and unhygienic living in the hostels which include sharing of shaving blade, clippers, and toothbrushes could also contribute to the high prevalence of the virus among students. similarly keeping multiple sex partners, youthful exuberance, complacency, economic hardship, and some immoral social activities on the campus such as gala night, beauty contests, disco parties, and watching pornographic films, all could enhance risky sexual behaviors and consequently resulting to high rate of infection. students that are involved in taking hard drugs have higher risk of being infected than nonusers. these social vices are found among many students of higher learning especially in tertiary institutions and they could explain why the infection rate in this study is higher in tertiary institutions (9.9%) than in secondary schools (3.8%). however, statistical analysis by chi - square showed no significant difference in the prevalence of the infection in secondary and tertiary institutions (=1.42, p>0.05). judging the prevalence of the infection by the settlement of students, a higher rate (16.5%) was recorded in the urban centers than those in rural settlement with 9.8%. statistically, there was no significant difference in the distribution of the infected students with respect to institutions settlement (=0.96, p>0.05). however, the slight variation in findings among students from rural and urban institutions could be attributed to nonuniformity in the number examined. age - related prevalence of hiv infections among students examined showed a highest rate (22.5%) within the 2125 years age bracket while the least (2.3%) was recorded within 3135 years age group. progression from adolescent to early adulthood is usually accompanied by hormonal, emotional, and physical changes which usually result to high sexual activities in early years of puberty. this could explain the high rate of infection within the age group 2125 years. also, younger and matured females of this age bracket are often preferred to older ones by male sex partners. consequently, the group is more exposed and more vulnerable to sexually transmitted infection than the older group. statistical analysis by the chi - square test however showed no significant difference in the prevalence of the infection by age (=1.07, p>0.05). similarly, by gender consideration, the females recorded a higher rate (10.3%) than the male counterpart (9.4%). this variation in findings could be attributed to difference in the anatomical structure of the female genital tract that makes them more vulnerable to genital infection. poverty and violence, against young females, are also some factors that could probably predispose them to infection. this result agrees with the findings from similar studies that also reported a higher hiv rates among females than males but disagrees with report of center for disease control which documented higher rate in males than females. from the present findings, it is obvious that the hiv prevalence rate among students in this part of the globe is still high, and if allowed to spread further, it could be a serious threat to the nation 's economy in the nearest future considering the impact of skilled manpower in national development. a situation whereby over 50% nigerian girls have had sex even before their first menstruation should be discouraged in totality. in the past, attempts to provide sex education for young people were hindered by cultural and religious insinuations. however, with the increasing rate of the infection among youths it has become very necessary to legislate the development of new academic curriculum that will take into consideration sex education at all levels in nigeria, africa, and even the world at large. adequate consultation with religious, traditional, and community leaders is very essential for the success of this arrangement. hiv screening should be made mandatory before admission is given by any educational institution. finally, since several studies[2830 ] have associated high rate of hiv with other common sexually transmitted infections, epidemiological studies of such infections should be carried out among students as part of control measures toward reducing the spread of the dreaded virus among them. | background : students are pivotal to manpower development and technological advancement of any nation. nigerian nation was recently ranked third human immunodeficiency virus (hiv) most endemic nation in the worldaim : the study was designed to determine the frequency of hiv infection among nigerian tertiary and secondary institution students.materials and methods : a hiv screening test was conducted on 1,978 apparently healthy students composed of 981 males and 997 females aged 1135 years, randomly selected from some nigerian tertiary and secondary institutionsresults : overall, the sero - prevalence rate of 13.7% was recorded consisting 9.9% in the tertiary and 3.8% in secondary institutions. the distribution of the infection showed no significant difference by age (2=1.07, p>0.05) and by gender (2=0.85, p>0.05). also, the prevalence had no significant association with the settlement of students (2=0.96, p>0.05) and the status of educational institutions (2=1.42, p>0.05).conclusion : the findings indicate a high hiv prevalence rate among students in this part of the globe. general behavioral changes about sex among the students are suggested. |
it has been reported that over 400 million people are obese worldwide, and the number is projected to reach 700 million by 2015.1 according to the reports from the international association for the study of obesity, approximately one - fourth of european men and women are obese and approximately one - half of european men and one - third of european women are overweight.2 in the united states, where adult obesity is 30%35%, the obesity epidemic also poses threats to public health.36 the escalation of obesity and overweight has become a global problem in the past decade. accumulating evidence indicates that the obese state shares some characteristics with chronic low - grade inflammation, which deliberates various diseases, particularly cardiovascular disease,7,8 chronic kidney disease,911 dyslipidemia,12 hypertension,13,14 liver disease,1517 type 2 diabetes,18 as well as a number of tumors.1921 many tumors, including gynecologic tumors (breast, ovarian, cervical, uterine cancer), digestive system tumors (esophageal, stomach, colon or rectal, liver, gall bladder, pancreatic), and hematologic tumors (multiple myeloma and non - hodgkin lymphoma), as well as others, such as kidney and glioma, are found to be correlated with obesity.19,20,2224 it is estimated that being overweight or obese contributes to 20% of cancer deaths in the united states.25 although obesity has been considered as an increased risk for many cancers, the molecular mechanisms by which obesity affects cancer incidence is still unclear. obesity - associated inflammatory, metabolic, and endocrine mediators, as well as the functioning of the gut microbiota, are suspected to contribute to tumorigenesis. among obese people, proinflammatory cytokines / chemokines including tumor necrosis factor - alpha (tnf-), interleukin (il)-1, il-6, insulin and insulin - like growth factors (igfs), adipokines, plasminogen activator inhibitor-1, adiponectin, and leptin are found to play a crucial role in the initiation and development of cancer.2630 the gut microbiota, including altered microbial metabolism, is able to contribute to the generation of procarcinogenic toxic metabolites ; increased extraction of energy and nutrient availability leading to metabolic dysregulation contributes to tumor initiation and progression.3133 among the above molecules, leptin is the most abundant adipokine. since it was first cloned in 1994,34 this cytokine - like hormone, controlling adipocyte mass and energy balance by binding to the leptin receptor (ob - r), has been the subject of intensive studies in cancer development. approximately three - quarters of total liver cancer worldwide are associated with hcc, which is the major histological subtype of liver cancer burden worldwide, and complicating cirrhosis due to chronic viral infection or toxic injury remains the third leading cause of cancer death in the world. a systematic review and meta - analysis, along with other evidences, linked obesity to increased risk of common and less common malignancies, such as hcc.35 a number of epidemiological studies have reported that overweight and/or obesity are associated with a greater risk of hcc compared to the general population.3639 a significant increase in serum leptin levels and a positive correlation between the serum levels of leptin and -fetoprotein in cirrhotic hcc group were also observed in hcc patients.40 the serum leptin levels were also found to be considerably higher in patients with hcc than in normal healthy controls in another study.41 in light of the increasingly reported role of leptin in several types of cancer,4247 this review is focused on the updated knowledge on the oncogenic role of leptin signaling in the occurrence and development of hcc, clinical significance, and development of specific drug targets in hcc. additionally, leptin - induced angiogenic ability and molecular mechanisms in hcc cells are also discussed. the stringent binding affinity of leptin / ob - r, the overexpression of leptin / ob - r, and its targets in cancer cells make leptin a unique drug target for the prevention and treatment of hcc, particularly in obese patients. leptin, coded by the lep gene, is a small, 167-amino acid, nonglycosylated protein. the biological function of leptin in energy homeostasis was determined by normalization of hyperphagy and obese phenotypes using recombinant leptin administration in rodents and humans.48,49 leptin also plays critical roles in the regulation of immune response, growth, reproduction, glucose homeostasis, and angiogenesis.5053 the n - terminal region (94 amino acids) in leptin protein is essential for both its biological and receptor binding activities.54 the binding of leptin to ob - r is capable of inducing the extracellular domains of ob - r to form a homodimer, which constitutes the functional unit responsible for leptin - mediated signals. ob - r belongs to a member of the class i cytokine receptor superfamily.55 this superfamily of receptors needs auxiliary kinases for activation because they lack autophosphorylation capabilities. so far, six leptin receptor isoforms generated by mrna alternative splicing have been discovered56 : shorter isoforms with less biological activity (ob - rs) and the long isoform (ob - rl or ob - rb) with full intracellular signaling capabilities.47,55 all ob - r forms have the common large extracellular domain of ob - r (816 amino acids).47 in contrast, all ob - r forms have variable lengths of cytoplasmatic tail (300 amino acid residues).57,58 ob - r binding to leptin induces its conformational changes that recruit janus kinases (jaks), which in turn phosphorylate ob - r and activate signal transducers and activators of transcription (stats).47 in addition to the jak2/stats signaling pathway, leptin binding to ob - r also induces canonical (phosphoinositide 3-kinase [pi-3k]/protein kinase b [akt ], mitogen - activated protein kinase [mapk]/extracellular regulated kinase 1 and 2 [erk 1/2 ]), and noncanonical signaling pathways (ampk, jnk, pkc, and p38 mapk) in diverse cell types. the long form (ob - rb) has a long intracellular domain which is essential for intracellular signal transduction. only ob - rb in the leptin receptor isoforms contains an intact intracellular domain and has the ability to activate the intracellular jak / stat pathway on ligand binding.47,59 importantly, leptin - mediated stat3 (signal transducer and activator of transcription 3) signaling needs tyr-1138 of ob - rb for its action.6062 in addition, leptin - induced signals occur in normal peripheral tissues, but the high level of leptin in obesity could amplify leptin signaling, thereby finally inducing the development of obesity - associated cancers. wang examined, using immunohistochemical staining, leptin expression in 36 cases of adjacent nontumorous liver tissues (36/36, 100%) with moderate (+ +) to strong (+ + +) intensity and in 72.22% (26/36) of hcc with weaker (+) intensity (p<0.05). however, they suggested that further studies were needed to determine the inhibitory and/or activating role of leptin in the etiology, carcinogenesis, and progress of human hcc.63 in another report,64 high leptin expression was demonstrated in 60.3% of patients with hcc and was not correlated to ki-67 expression, but it is significantly correlated to intratumor microvessel density (high vs low ; 59.2 [standard deviation 3.2 ] vs 44.2 [19.5 ], p=0.004). however, leptin expression was determined as a predictor for improved overall survival of patients with hcc (odds ratio [or ] 0.16 ; 95% confidence interval [ci ] 0.030.87 ; p=0.033) using a multivariate cox s proportional hazards model. interestingly, high ob - r expression was detected in 53% of hcc patients and was also significantly correlated to intratumor microvessel density (high vs low ; 59.4 [3.2 ] vs 44.7 [3.7 ] ; p=0.004).64 in addition, high ob - r expression was associated with a better overall survival (p=0.027) using the kaplan meier survival curve.64 multivariate analysis also showed that ob - r expression was a significant determinant for hcc (or 0.02, 95% ci 0.010.85 ; p=0.041).64 in a recent study,65 the overexpression rate of leptin and ob - r in 81 hcc patients was 56.8% and 35.8%, respectively. ob - r overexpression was significantly correlated to the tumor size and tnm stage (p<0.05), but not to age, body mass index, -fetoprotein, hepatitis b surface antigen status, tumor grade, vascular invasion, or liver cirrhosis (p0.05). leptin overexpression showed no significant correlations to the above clinicopathological factors (p0.05). in vitro, leptin and ob - r are simultaneously expressed in the hcc cell line hepg2.66 leptin increased hepg2 cell proliferation in a concentration- and time - dependent manner. the effect of promotion of cell proliferation by leptin is due to the increment of dna synthesis and enhancement of mitotic activity. the existing conflicts among different studies might be due to the use of different tools or different stages of tumor tissues. therefore, leptin may be involved in the occurrence and development of hcc, and the specific role and mechanism needs further research. in the central nervous system, particularly in the hypothalamus, which is a site of high ob - rb mrna expression, many of the effects are attributable to leptin. alternative splicing and proteolytic cleavage events also produce a circulating extracellular domain of ob - r, which may affect the stability of circulating leptin.47,51 the cell - membrane - bound short - form receptors may also have potentially important roles, including the endocytosis and transport of leptin across the blood high levels of leptin in obese patients are not able to suppress feeding and decrease body weight (bw)/adiposity. the proposed mechanisms of leptin resistance include perturbations in developmental programming, alterations in cellular ob - rb signaling, alterations in the transport of leptin across the blood brain barrier, and others.67 in peripheral tissues, high levels of circulating leptin could also overregulate the signaling and expression of active ob - r. these phenomena lead to the deregulation of leptin signaling, thereby significantly contributing to hcc progression through its crosstalk with multiple signaling pathways, as discussed in breast cancer47 or colorectal cancer.68 the pi-3k / akt pathway, an assembly of membrane - localized complexes, plays a central role in a variety of multiple biological processes such as cell motility, proliferation, survival, and angiogenesis in tumor cells including hcc.6972 the pi-3k / akt pathway also plays a major role in tumor growth factor (tgf)--induced epithelial mesenchymal transformation (emt), notably through the regulation of translation and cell invasion during carcinogenesis.73,74 in addition, many of the transforming events in hcc are a result of the enhancement or deregulation of pi3-k / akt pathway.72,75 a great number of studies have already established the central role of leptin - induced regulation of the pi-3k / akt signaling pathway in several types of cancer including hcc (figure 1).47,7685 mounting evidence has shown that the stat3 is a frequent biochemical aberrant in the development, progression, and maintenance of cancer cells.86,87 stat3 regulates a variety of genes involved in the regulation of critical functions, including immune responses, cell proliferation, differentiation, angiogenesis, apoptosis, and metastasis. stat3 can function either as an oncoprotein or a tumor suppressor depending on the specific genetic background or in different contexts of cancer biology.88 however, for many tumors, increased levels of activated stat3 have been associated with worsened prognosis, indicating that stat3 could be an attractive molecular target for the development of novel malignancy therapeutics.89,90 the stat3 pathway mediates leptin actions on food intake, weight gain, glucose metabolism, and neuroendocrine function, but does not influence fertility and glycemic control.91 in malignant cells, the stat3 pathway is involved in leptin actions in cell migration,85,92 proliferation,9398 and anti - apoptosis.82,99,100 leptin - stat3 regulates the genes cyclooxygenase (cox)-2,83 cyclin d1 (kinase and regulator of cell cycle d1),93,95 human telomerase reverse transcriptase (htert),101 vegf,102,103 leptin,102 and survivin.104 stat3 could also regulate nuclear factor kappa - light - chain - enhancer of activated b cells,105,106 il-1, notch,107,108 canonical wnt,109,110 and vegfr-2,107,108 and thereby regulate tumor angiogenesis. leptin could crosstalk with signaling pathways which are involved in the pathogenesis of nonalcoholic fatty liver disease, which is a risk disease of hcc.111113 leptin is able to contribute to the development of insulin resistance, steatosis, proinflammation, and liver fibrosis.46,114 leptin injections have been shown to result in the increased expression of procollagen - i, tgf-1, and smooth muscle actin which is a marker of activated hepatic stellate cells, and eventually to increased liver fibrosis.115 leptin could also crosstalk with signaling pathways which involve in the development of fibrosis. aleffi identified the effect of leptin on fibrogenic cells is the induction of vascular endothelial growth factor (vegf) via oxygen - independent activation of hypoxia - inducible factor 1a, which is a master switch of the angiogenic response.116 their results strongly suggest the fibrogenic role of leptin in the liver. evidence ever more strongly implicates that leptin / ob - r signaling is correlated to many cancer types and point toward new drug targets. moreover, the extracellular activation of ob - r is obtained only upon leptin binding to its extracellular region.47 interestingly, this family of receptors is capable of binding only to leptin or leptin - modified peptides, indicating the potential use of leptin antagonists and/or other inhibitors in blocking ob - r signals.47,117 previous studies have shown that blocking of leptin signaling could cause decreased growth and development of mammary tumors derived from mice and humans.118,119 tumor growth and the expression of vegf - a / vegfr-2 were markedly reduced in orthotopic mouse models using a pegylated leptin peptide receptor antagonist (peg - lpra2).118,119 the bw or appetite of a large number of normal lean (male and female) cd-1 and balb / c mice did not change during a several months of using peg - lpra2. surmacz s group reported similar results in the same orthotropic xenograft model using a different leptin antagonist (allo - aca).120 allo - aca induced 6%10% bw increase, but it significantly extended the mouse survival time for 12 weeks and did not show systemic toxicity when tested for toxicity effects in normal cd-1 mice.120 recently, the same group tested a number of allo - aca analogs. d - ser was a peptidominetic and distributed only in the periphery of experimental animals. this novel peptide d - ser may serve as a prototype to develop new therapeutics because it significantly inhibited leptin - dependent proliferation of ob - r - positive cancer cells in vitro at 1 nm concentration without exhibiting any partial agonistic activity.121 the above results indicate that inhibition of leptin signaling by leptin antagonists may serve as a novel adjuvant for the treatment of hcc. zabeau produced and evaluated a number of neutralizing nanobodies targeting ob - r.122 three classes of neutralizing nanobodies targeting different ob - r subdomains, ie, the ig - like and fibronectin type iii domains and cytokine receptor homology 2, were identified. among them, only nanobodies directed against the cytokine receptor homology 2 domain inhibited leptin binding. ross and strasburger s groups developed monoclonal antibodies (mabs) against human ob - r and verified their antagonistic activity using an lep - signaling bioassay.123,124 9f8, the most promising mab showed dose - dependent antagonist activity using the lep bioassay. however, all the above - mentioned antibodies have not been used in cancer therapy. in summary, although there have already been compounds or antibodies targeting leptin / ob - r that showed significant in vitro or in vivo anticancer effect, they have not been utilized in clinical settings. the major reason might be the low efficiency and specificity of some compounds or antibodies. in addition, the side effects of these drugs are not completely known and require further studies. obesity - associated inflammatory, metabolic, and endocrine mediators are suspected to play a role in tumorigenesis. body fat and adipocyte size are clearly correlated to high leptin levels in obesity and in overweight individuals or populations. high leptin level is a hallmark of obesity, which has been correlated to the incidence and progression of several malignancies including hcc. in vitro studies have clearly demonstrated the role of leptin in hcc proliferation, migration, and angiogenesis. in addition, leptin signaling and its crosstalks with many signaling pathways, such as pi-3k / akt and stat3, play critical roles in hcc cell growth, invasion, angiogenesis, and metastasis. there are still a number of gaps to fill in the field of leptin signaling in hcc, especially further identification of the molecular mechanisms of leptin - signaling - mediated regulation of hcc. although several groups have developed antibodies targeting leptin signaling, all these antibodies have not been used in cancer therapy. novel opportunities could emerge from the discovery of leptin crosstalk with other oncogenic pathways, inflammatory and angiogenic cytokines, and their links to obesity - related cancers. | previous reports indicate that over 13 different tumors, including hepatocellular carcinoma (hcc), are related to obesity. obesity - associated inflammatory, metabolic, and endocrine mediators, as well as the functioning of the gut microbiota, are suspected to contribute to tumorigenesis. in obese people, proinflammatory cytokines / chemokines including tumor necrosis factor - alpha, interleukin (il)-1 and il-6, insulin and insulin - like growth factors, adipokines, plasminogen activator inhibitor-1, adiponectin, and leptin are found to play crucial roles in the initiation and development of cancer. the cytokines induced by leptin in adipose tissue or tumor cells have been intensely studied. leptin - induced signaling pathways are critical for biological functions such as adiposity, energy balance, endocrine function, immune reaction, and angiogenesis as well as oncogenesis. leptin is an activator of cell proliferation and anti - apoptosis in several cell types, and an inducer of cancer stem cells ; its critical roles in tumorigenesis are based on its oncogenic, mitogenic, proinflammatory, and pro - angiogenic actions. this review provides an update of the pathological effects of leptin signaling with special emphasis on potential molecular mechanisms and therapeutic targeting, which could potentially be used in future clinical settings. in addition, leptin - induced angiogenic ability and molecular mechanisms in hcc are discussed. the stringent binding affinity of leptin and its receptor ob - r, as well as the highly upregulated expression of both leptin and ob - r in cancer cells compared to normal cells, makes leptin an ideal drug target for the prevention and treatment of hcc, especially in obese patients. |
early - stage tuberculous (tb) meningitis has no specific symptoms, which may lead to delayed diagnosis and consequently to a worsened prognosis. tb meningitis with dementia as the presenting symptom after intramedullary spinal cord tumor resection without meningitis symptoms or positive tests for tb has not been described. here, we report a fatal case with delayed diagnosis of tb meningitis after intramedullary spinal cord tumor resection. the patient was a 77-year - old man with recurrence of gait disturbance and dysuria. gadolinium - enhanced magnetic resonance imaging (mri) revealed tumor recurrence with syrinx formation (fig. the postoperative course was uneventful and he was discharged after regaining the ability to walk normally. (a) t2 mri and (b) gd - enhanced mri before the initial surgery (nine years ago), showing the presence of an intramedullary tumor. (a) t2 mri after the first surgery, showing total resection of the tumor. (b) t2 mri four years after the first surgery, showing regrowth of the tumor. (c) t2 mri and (d) gd enhanced mri nine years after the first surgery. at 1.5 months after surgery, the patient suffered from dementia with memory loss and diminished motivation and speech in the absence of fever, and was readmitted. tumor recurrence was not seen on mri and blood parameters did not indicate an infection (white blood cell count [wbc ], 7900 /mm ; c - reactive protein level [crp ], 0.1 mg / dl ; erythrocyte sedimentation rate [esr ], 13 mm / h). 2b) and mri revealed slight enlargement of the ventricles (mild hydrocephalus) and the patient was diagnosed with senile dementia by a psychiatrist. however, after two weeks, his symptoms progressively worsened : dementia was more severe, he became disoriented, and his temperature increased to 39c in association with a decrease in consciousness. at that time, the wbc count (12000 /mm), crp level (0.9 mg / dl) and esr (42 mm / h) were elevated, and meropenem (2.0 g / day) was started. after a further one week, he became comatose and a second mri revealed further hydrocephalus - induced ventricular dilatation (fig. 2c). no tuberculosis bacteria in csf were detected in a polymerase chain reaction (pcr) test, -d - glucan in serum was normal, a sputum sample was negative for mycobacterium tuberculosis in a quantiferon - tb gold (qft - g) in - tube test, and a tuberculin skin test (tst) was also negative. despite the absence of findings of tb meningitis, we suspected that this disease was present from the patient s history, and based on the relatively slow progression of symptoms and the increases in adenosine deaminase (ada), cell counts and protein and the decrease in glucose in the csf. we administered a four - drug regimen (isoniazid, 0.3 g ; rifampicin, 0.5 g ; ethambutol, 0.75 g ; streptomycin, 0.5 g), but the patient died 29 days after admission. subsequently, m. tuberculosis was detected in the second csf sample after a 28-day culture. the patient s wife and son gave informed consent to submit this case study for publication. we encountered a relatively rare case of tb meningitis in an elderly patient that manifested as dementia and impaired consciousness. fever, elevation of inflammatory markers, and signs of fever, neck stiffness, and headache, which are characteristic of meningitis, were notably absent. furthermore, -d - glucan in serum was normal, no bacteria were detected in the initial csf, sputum was negative for mycobacterium tuberculosis in the qft - g in - tube test, and the tst was negative. therefore, we initially assumed age - related dementia or depression, rather than fungal or tb meningitis. common initial symptoms of tb meningitis include internuclear ophthalmoplegia and sensorineural hearing loss, but the disease may be asymptomatic. tb meningitis accounts for only 0.5% of all tb cases, but the mortality is as high as 30% because of delayed diagnosis. in previous reports, < 20% of tb meningitis infections were detected in csf cultures ; pcr had low sensitivity (53%), compared with microbiological ziehl - neelsen staining and culture methods (73%) ; and the sensitivities of the qft - g and tst were 80% and 28%, respectively. in our case, these tests were negative and m. tuberculosis was detected in a the second csf sample after long - term culture, although the initial findings were negative. thus, a definitive diagnosis of tb meningitis is difficult. in a review by thwaites the sensitivity and specificity of the " vietnam diagnostic rule " (table 1) for tb meningitis were shown to be 86% and 79%, respectively. this rule can help in diagnosis of tb meningitis using simple clinical and laboratory data, and can be used for tb meningitis with low glucose in csf, particularly in settings with limited microbiological resources. retrospectively, the present case had a total score 4, which is suspicious for tb meningitis. therefore, our case supports the effectiveness of this rule for early diagnosis of tb meningitis, especially for cases without abnormal findings. many secondary cases develop due to a decrease in immunity caused by diabetes or a malignant tumor in the elderly, and many of these cases have no obvious active pulmonary tuberculosis. the patient in the present case had no history of hiv, diabetes, steroid hormone treatment, or malignant tumor, but he had undergone two surgeries for an intramedullary tumor and may have been in a postoperative compromised state. also, in spinal cord tumor surgery, csf leakage may occur after a dura mater procedure, and there is a risk of postoperative meningitis due to long - term drain placement. even if typical symptoms such as headache, fever, and stiff neck are absent, meningitis should be suspected in a patient with deterioration of consciousness and disorientation after intramedullary surgery. the present case indicates that symptoms of dementia after intramedullary spinal cord tumor resection should first be suspected as tb meningitis, even if tests for meningitis are negative. we propose that anti - tuberculosis therapy should be initiated immediately in cases of suspected tb meningitis prior to positive identification in culture. | abstractearly - stage tb meningitis has no specific symptoms in patients, potentially leading to delayed diagnosis and consequently worsening prognosis. the authors present the fatal case with a delayed diagnosis of tuberculous (tb) meningitis with dementia as the presenting symptom after intramedullary spinal cord tumor resection. the medical records, operative reports, and radiographical imaging studies of a single patient were retrospectively reviewed. a 77-year - old man who underwent thoracic intramedullary hemangioblastoma resection for 2 times. the postoperative course was uneventful, but 1.5 months after surgery, the patient suffered from dementia with memory loss and diminished motivation and speech in the absence of a fever. no abnormalities were detected on blood test, brain computed tomography and cerebrospinal fluid (csf) analysis. a sputum sample was negative for mycobacterium tuberculosis in the quantiferon-tb gold (qft - g) in - tube test and the tuberculin skin test was also negative. the patient was diagnosed with senile dementia by a psychiatrist. however, the patient s symptoms progressively worsened. despite the absence of tb meningitis findings, we suspected tb meningitis from the patient s history, and administered a four - drug regimen. however the patient died 29 days after admission, subsequently m. tuberculosis was detected in the csf sample. this case is a rare case of tb meningitis initially mistaken for dementia after intramedullary spinal cord tumor resection. symptoms of dementia after intramedullary spinal cord tumor resection should first be suspected as one of tb meningitis, even if the tests for meningitis are negative. we propose that anti - tuberculosis therapy should be immediately initiated in cases of suspected tb meningitis prior to positive identification on culture. |
when a 72-year - old white male with ms heard the doorbell ring he manually opened the door. a visitor entered the residence and introduced himself as a gal who is a paid state employee appointed by a judge. the ms patient responded that he would not have time to talk to the gal because he had been scheduled at a daycare surgery center for a suprapubic cystostomy. realizing that there would not be adequate time to talk, the gal left an envelope filled with papers that could be read by the individual after his appointment. the gal said that he would return to meet with the ms patient after he returned from the hospital following surgery. because the patient had been developing excessive urine in his bladder that became infected with pseudomonas aeruginosa, his urologist prescribed ciprofloxacin (500 mg po bid), which did not eliminate his fever of 102f.5 suprapubic cystostomy is very popular because it is an easier technique for people with ms who have limited mobility as well as patients with spinal cord injuries. the catheter drains into a sterile collection bag and is irrigated daily with sterile saline. according to torres - salazar and ricardez - espinosa, it is ideal to change the catheter every two weeks.6 the authors also advise that patients with a permanent suprapubic cystostomy have the catheter changed before cultures are taken to reduce the amount of antimicrobials prescribed, and lessen the increase in microbial resistance. after placement of the suprapubic cystostomy, the patient became afebrile and was allowed to return to his residence without further treatment. because he was now afebrile, personal care could be easily provided to him by certified nursing assistants that were familiar with the guldmann ceiling lifts located adjacent from the patient s bed, as well as in the bathroom. using these modern lift systems, he was able to be transferred by his certified nursing assistants who also wear back braces to reduce the chance for potential injury to their backs.7 on his scheduled meeting with the gal, the gal informed the patient that there was a meeting with the judge about the guardian ad litem case. a petition had been given to the ms patient by the gal, in which his two children wanted to monitor and take control of his investment and real estate assets. the gal advised the ms patient that his own attorney should be in the courthouse. the gal indicated that he should select an attorney that focuses primarily on litigation on gal cases. since 1890, the adverse effects of hyperthermia in patients with ms have been recognized.8 while most patients with ms experience reversible worsening of their neurologic deficits, several individuals experience irreversible neurologic deficits. in fact, hyperthermia can be caused through sun exposure, exercise, and infection especially a urinary tract infection. the patient with ms selected a skilled attorney who specializes only in gal cases, and was willing to represent the individual at the hearing and until settlement of the litigation. the patient, now with a suprapubic cystostomy, was transported by a handicap accessible van to the courthouse for the hearing. the judge made it very clear to the patient that he was responsible for paying his attorney fees, the salary of the gal, the attorney for the gal, and the attorney fees for his two children. in addition, he would need an evaluation by a psychologist selected by the gal. the psychologist met with the ms patient and found normal communication skills, unless the consultation room had an elevated temperature. finally, the judge indicated that he should receive personal care assistance 24 hours a day by a nursing home service selected by the gal. the patient seated in the wheelchair explained that he did not need nursing provided people care in his home 24 hours a day because he had trained certified nursing assistants working between 7:00 am and 7:00 pm in his residence. in addition, he had an emergency medical alarm system from adt home monitoring in his residence that was connected to either a wristband or a necklace with a medical alert button that could be pressed to alert emergency medical technicians in an ambulance service to take him to the hospital to treat his life - threatening illness. after the nursing assistant would leave at 7 pm, he was able to contact the individual s cell phone and request any further assistance. in the past, he had never needed the nursing assistant to return for personal care. the judge ignored this suggestion and encouraged the gal to request that the new nursing home service start providing care 24 hours a day. after the judge made this decision, the ms patient now had two personal care assistants working in his residence. the individual with ms was now cared for from 7:00 pm until 7:00 am by relatively untrained licensed personal care assistants. when he found that the new nursing home services had no protective back braces and had never transferred a patient from a wheelchair using an overhead ceiling lift, he realized that he could be dropped inadvertently from the ceiling lift. during the three months of his experience with the appointed nursing home services, he had two challenges with hyperthermia induced by the nursing home services staff in his residence. in one case, the nursing home service closed his bedroom door causing a dramatic increase in the temperature in his bedroom. when he developed life - threatening hyperthermia, he had to call on the telephone one of his own hired nursing assistants to come to his home to reduce his body temperature. she opened the door of the bedroom and then removed the patient s pajamas that were soaked with sweat. she than had to wipe his body with cool water, dry his body, and put new dry clothing back onto the patient. in the next episode, the employee from the nursing home service inadvertently turned off the switch in the bedroom that activated the overhead ceiling fan. when the patient with ms called out for her help, he surprisingly learned that she had a hearing deficit and could not hear his request to turn on the electricity to the overhead fan so he could control it with his remote. because he now could not use his remote control unit for the fan, he had to call his same hired nursing assistant to return to his home to take care of his elevated body temperature. because the hired nursing assistant came to the patient s home to treat him for hyperthermia without contacting the nursing home service, the nursing home service requested that adult protective services file a misconduct or criminal charge against his employee. the attorney for adult protective service s refused to read the scientific publication listed as reference number eight. the attorney expressed no interest in the dangers of hyperthermia in patients with ms and was only interested in processing the litigation case against the employee who saved the patient s life. the patient s attorney requested that he contact another attorney who specializes in financial settlements. this attorney developed settlement agreements with the gal and his attorney, the children s attorney, and the nursing home services. the amount of the final bill for the gal case was us$62,412.52 (table 1). the attorney for the patient gave the list of the expenses that were approved and signed by the judge in written documents. it is important to point out that the two petitioners were not asked to reimburse payment for the gal or the gal s attorney. in this case, the judge made a premature decision in appointing the gal without initially requesting the patient s medical report so that the judge could understand the medical facts and circumstances that led the patient to manage her physical needs through hired certified nursing assistants. after exploring these facts, the judge could have made her determination of mental competency based on a report from a licensed psychiatrist who spoke with the patient. the fact that the patient with ms was able to make his own decision regarding his physical needs implied that he had mental competency. although the patient had significant physical disability, he had managed to arrange for surgery by the urologist. this case has implications for elder abuse, both physical and financial, medical neglect, and the inadequate nursing care rendered in the individual s residence. the physical abuse can be explained by the judge s replacement of the patient s hired personal care assistants with a 24-hour nursing home service that eventually endangered the patient s life by exposing him to hyperthermia, which had to be resolved by one of the patient s hired nursing assistants. the financial abuse can be explained by the judge s order asking the patient to pay all of the expenses that occurred during the litigation. the medical neglect can be attributed to the lack of medical information provided to all parties involved, particularly the children who brought the petition against the father. the petitioners for a gal case should have some monetary responsibility. ideally, the petitioners should be able to pay for the expenses of their own attorney as well as the salary for the gal. if the petitioners have this financial responsibility, it would ensure that he / she reviews the case carefully before making a decision to file a petition. secondly, the gal should be an attorney or a registered nurse who takes training and becomes certified as a gal. | the children of a multiple sclerosis (ms) patient filed a guardian ad litem case to be brought against the patient. the basis for the petition was that the ms patient had a significant reduction is his mental competence. the children were not aware that hyperthermia could adversely affect the brain of ms patients. the patient s urologist recommended he have a suprapubic cystostomy done in a hospital. passage of the two channel foley catheter into his bladder immediately resolved his urinary tract infection, fever, and difficulty in communicating. despite this dramatic improvement in his health from the urologic treatment, he was now faced with resolving his children s petition for a guardian ad litem that would allow them to control his estate including his residence and financial retirement assets. a judge supported this petition by requesting that the patient with ms pay for his children s attorney fees, 24 hour nursing home services that duplicated his own hired personal care assistants, the salary of the guardian ad litem, the attorney fees for the guardian ad litem, and payment for a psychological evaluation. the state law should be changed to require that the petitioner have adequate income to pay for his / her attorney as well as the salary of the guardian ad litem to prevent mismanagement of patients with cognitive disorders. in addition, the guardian ad litem should be an attorney or a registered nurse. the care of disabled individuals subjected to litigation should be coordinated by an attorney or registered nurse. |
through development, the wiring of the cortex is refined to receive and establish both local (callaway, 1998 ; fino and yuste, 2011 ; ktzel., 2011 ; ko., 2011 ; kozloski., 2001 ; yoshimura., 2005) and long - range (berezovskii., 2011 ; salin and bullier, 1995) projections that convey information for multimodal integration (iurilli., 2012 ; mao., 2011) and normal cognitive function (huang., 2014 ; reis marques., 2014 ; zhang., 2014) in many sensory cortical areas, the final organization of the network contains reoccurring features that include dedicated cortico - cortical (cc) versus cortico - thalamic (ct) projection pathways formed by the principal cells found in deep layer six (l6) (kumar and ohana, 2008 ; marx and feldmeyer, 2013 ; pichon., 2012 although the functional importance of these two output pathways is highlighted by their anatomical prominence, their precise physiological role in cortical and cortico - thalamic processing has proven difficult to dissect. one approach to understanding the function of cortical pathways in general terms has been to chart regional projectivity (oh., 2014) with the view that the resultant wiring diagram may be used as a template for understanding the emergent physiological properties of underlying circuits (douglas and martin, 2007 ; reid, 2012). on the other hand, while this approach can provide an overview of connection likelihood and strength both within (petersen and sakmann, 2000) and between (binzegger., 2004 ; feldmeyer., 2013 ; oberlaender., 2012) cortical layers and regions such descriptions are often limited by their cellular and functional resolution (oh., 2014). the difficulty in superimposing precisely the function and connectivity of individual elements within the circuit makes it extremely challenging to accurately attribute potential connectivity rules within a functionally heterogeneous population of neurons and prohibits a detailed understanding of network function. it is well documented that even at a local level, neurons within the same cortical layer can show significant functional heterogeneity (allman., 1985). in visual cortical areas, the diversity of sensory responses of individual neurons is highlighted by their degree of tuning to the orientation (hofer., 2011 ; hubel and wiesel, 1968 ; kerlin., 2010 ; maruyama and ito, 2013 ; nauhaus., 2008 ; niell and stryker, 2008), velocity (priebe., 2006 ; roth., 2012), and direction of the motion of alternating bars of different luminance (gratings) (allman., 1985 ; hubel and wiesel, 1968 ; martin and schrder, 2013 ; roth., 2012). these functionally diverse populations are also thought to project to, and receive connections from, multiple cortical layers (angelucci., 2002 ; bolz and gilbert, 1986 ; olsen., 2012 ; thomson and bannister, 2003) forming interlaminar pathways for integration of both local and long - range input (berezovskii., 2011 ; de pasquale and sherman, 2011, 2013 ; glickfeld., 2013 ; hup., 1998 ; salin and bullier, 1995 ; schwarz and bolz, 1991 ; wang and burkhalter, 2007 ; xu., 2007). understanding the functional heterogeneity of cortical networks therefore requires simultaneous analysis of their cellular composition (helmstaedter., 2013 ; oberlaender., 2012), sensory response properties (niell and stryker, 2008 ; oberlaender., 2012), input connectivity, and output projectivity (briggman., 2011 ; denk., v1 we have undertaken an in vivo single - cell analysis of the sensory response properties and connectivity of the l6 network that is known to contain a functionally heterogeneous population of principal cells (hirsch., 1998 ; niell and stryker, 2008) that comprise cc- and ct - projecting neurons (briggs, 2010 ; katz, 1987 ; kumar and ohana, 2008 ; marx and feldmeyer, 2013 ; pichon., 2012 ; thomson, 2010 ; usrey and fitzpatrick, 1996 ; zhang and deschnes, 1997). by targeting retrograde transsynaptic tracing (marshel., 2010 ; rancz., 2011 ; wickersham., 2007) to individually recorded cells (rancz., 2011) and charting their brain - wide connectivity, we find that cc- versus ct - projecting neurons relay functionally distinct signals and are differentially innervated by higher - order cortical areas. we performed blind in vivo whole - cell recordings (margrie., 2002) in v1 of anesthetized mice at a depth of 600 to 950 m from the pial surface (n = 81 cells). on the basis of their recorded intrinsic properties including the initial action potential (ap) half - width, the mean frequency of firing, and input resistance, we could distinguish fast spiking cells from regular spiking (rs) neurons (figure s1 available online). these criteria were used to identify the rs population of l6 cells (n = 74) expected to mediate the cc and ct pathways under investigation in this study. to begin to explore the functional diversity of l6 principal cells, we first recorded the ap tuning of rs neurons in response to moving sinusoidal gratings (figure 1a). the stimulus - evoked instantaneous firing rate of rs cells extended over a large range (0400 hz) and was found to encompass a broad range of selectivity to the orientation and direction of the gratings (figure 1a). in order to attribute these diverse response properties to specific types of l6 projection neurons, we performed morphological reconstructions (n = 16 cells) and identified two distinct anatomical classes (marx and feldmeyer, 2013 ; thomson, 2010 ; zhang and deschnes, 1997) (figures s2a s2e). the first group (n = 6) exhibited a large dendritic field (convex envelope = 0.0077 0.0011 mm) and an elongated total basal dendritic length (1,884 303 m) with dendrites rarely extending beyond layer 4 (figure s2b ; figure 1b). the dendritic tree of this neuronal class was morphologically diverse, displaying classical upright, but also inverted and tangential projecting apical dendrites (figure s2b). their axonal morphology was strikingly elaborate (total length 14,152 2,666 m ; figure 1b), with an extensive cortical horizontal span (1,070 223 m ; figure s2e) largely contained within layers 5 and 6 (13% 4% and 73% 4% of total length, respectively ; figures s2b and s2e). arborizations often entered the white matter and returned to the cortex and regularly extended into secondary visual areas. these dendritic and axonal morphological properties are consistent with previous anatomical descriptions of cc - projecting l6 cells (figure 1c) (pichon., 2012 ; zhang and deschnes, 1997). the second group contained cells that extended their apical dendrites beyond the l4-l5 border (n = 10) and, despite having a similar total dendritic length (cc : 4,038 1,090 m versus 3,297 738 m [sd ], p > 0.05), exhibited a significantly less elaborate dendritic morphology (figures s2c and s2d ; figure 1b) with a smaller convex envelope (0.0046 0.0004 mm, p 0.9 ; figures 3c and 3d). on the other hand, for both cell types the psp peak amplitude (figures 3a and 3b) displayed a significant preference for grating orientation (osi l6 pspintegral = 0.18 0.01 versus psppeak = 0.28 0.03, p < 0.01) and direction (dsi l6 pspintegral = 0.1 0.02 versus psppeak = 0.23 0.03, p < 0.01). this improved tuning of the psp peak was most striking for ct cells (osi : ct psppeak = 0.33 0.03 versus ct pspintegral = 0.18 0.02, p < 0.01 ; figures 3e and 3f) such that already for gratings presented at 30 from the preferred direction, the average amplitude of the peak depolarization was significantly reduced (psppeak : pref. = 14.6 1.2 mv versus 30 = 9.1 1 mv, n = 28, p < 0.01 ; figure 3e). compared to its integral, the peak amplitude of the psp therefore conveys the most accurate orientation and direction information, whereby the inputs onto ct cells are the most strongly tuned (osi psppeak : cc = 0.2 0.04 versus ct = 0.33 0.03, p < 0.01 ; figure 3f). this indicates that the cc population receives comparatively strong yet broadly tuned synaptic drive, while the ct cells receive a highly tuned orientation signal. although ct cells show sharp synaptic and ap tuning, the orientation selectivity of the input may not necessarily cause the apparent exquisite tuning of ap output. ct cells are extremely sparse firing (cc = 1.2 0.61 hz versus ct = 0.1 0.04 hz, p < 0.05 ; figure 2f). for example, over multiple stimulus repetitions cts may only discharge one or two aps. we therefore looked to establish causality in psp - ap tuning by injecting psp waveforms evoked in both cc and ct cells (figure 4a) back into individual neurons in the absence of visual stimulation (figures 4a and 4b). we found that the injected psps faithfully reproduced the grating - evoked cc and ct cell ap tuning, irrespective of the identity of the injected neuron (figures 4b and 4c). thus, the ap tuning of these two groups (figures 4c and 4d) results directly from the cell - type - specific dynamics of the evoked psp (cc : ccinject median = 0.47, q1 = 0.31, q3 = 0.69 versus ctinject median = 1, q1 = 0.92, q3 = 1, n = 7, p < 0.05 ; ct : ccinject median = 0.44, q1 = 0.4, q3 = 0.56 versus ctinject median = 1, q1 = 0.94, q3 = 1, n = 7, p < 0.05 ; figure 4d). consistent with these data, we also find that the specificity of the grating - evoked ct spiking is a highly reliable indicator of the orientation preference of the underlying synaptic input (figure 4e). synaptic signaling onto l6 principal cells therefore produces two functional distinct distributions of tuning profiles, whereby sparse, highly orientation - selective information is relayed to thalamic target areas. these data show that across the population, different morphological classes of l6 cells relay specific visual information and form unique signaling pathways within and outside the v1 circuit. one might therefore expect these functionally discrete cc and ct populations to be targeted by specific upstream pathways. to begin examining the functional specificity of cc and ct connectivity, we targeted retrograde transsynaptic tracing from individually recorded neurons using a glycoprotein deficient form of the rabies virus encapsulated with the avian sarcoma and leucosis virus envelope protein (rv) (wickersham., 2007). by performing whole - cell recordings with internal solutions containing dna vectors (rancz., 2011), we could drive the expression of the envelope protein receptor (tva) and the rv glycoprotein (rvg) that are required for single - cell targeted infection and monosynaptic retrograde spread of rv (figure 5a). immediately following whole - cell recording with the plasmid - containing internal solution, we injected rv and up to 12 days later performed whole - brain serial two - photon tomography (osten and margrie, 2013 ; ragan., 2012) (figure 5b) to chart the spatial profile of presynaptic cells. to directly assess the regularity of connectivity onto these two classes of l6 neurons, the intrinsic, synaptic, and ap tuning response properties were first recorded (figure 5c). following singe - cell rabies tracing, we found that for cc cells more than 90% of the labeled presynaptic neurons (138 21 labeled cells, n = 3 ; figures 5d and 5e ; movie s3) were located locally within v1. the majority of these presynaptic cells were observed in layers 5 (26% 5.2%) and 6 (38.9% 7.6%) although input from layer 2/3 (17.6% 3.3%) and to a lesser extent layer 4 (9.6% 0.5%) was also apparent (figures 5e and 5f). very few long - range projecting cells were observed, though a small fraction of presynaptic neurons were found in areas including thalamus (0.5% 0.3%), secondary visual (2.9% 0.7%), and retrosplenial (1.4% 0.4%) cortices. single - cell rabies tracing in ct cells revealed almost three times the number of presynaptic cells when compared to cc neurons (383 70 labeled cells, n = 4 ; figures 6a and 6b ; movie s3). in relative terms, ct cells received almost the identical fraction of inputs from layer 2/3 (18.8% 3.7%) and layer 4 (10.7% 1.7%) within v1 as that observed for cc cells (figures 6c and 6d). in contrast, ct neurons received more than 20% of input from secondary visual and retrosplenial cortices (v2 + rsp : cc = 4.2% 0.7% versus ct 20.8% 5.2%, p < 0.05 ; figures 6c and 6d). consequently, ct cells had relatively fewer input cells located in deeper layers 5 and 6 of v1 (l5 = 17.7% 2.4% and l6 = 29.5% 3.2% ; figure 6d). our electrophysiological and input tracing data indicate that the upstream network connectivity of individual l6 neurons is not random but respects the morphological and hence the functional identity of the target neuron. if these single - cell - based connectivity maps highlight general rules of monosynaptic connectivity onto these two classes of neurons, one might expect genetically targeted input tracing onto a specific population to show similar connectivity profiles. to examine the correspondence between our single ct cell - based maps and the broader l6 ct population, we took advantage of the fact that in l6 of mouse v1, ct - projecting cells are known to selectively express neurotensin receptor 1 (ntsr1) (gong., 2007). by using a cre - ntsr1 mouse line, we targeted injections of cre - dependent aav helper viruses to drive expression of the rv glycoprotein and the avian receptor protein across the ct population (n = 4 ; figures 6e and 6f). when comparing to cc cells, presynaptic connectivity in cre - ntsr1 mice was widespread (figure 6 g), with relatively fewer cells in v1 (cc = 92.1% 1.2% versus cre - ntsr1 = 41.3% 2.4%, p < 0.01) and substantially more labeled neurons located in secondary visual and retrosplenial cortices (v2 + rsp cc = 4.2% 0.7% versus cre - ntsr1 = 40.8% 1.5%, p < 0.01, ct = 20.8% 5.2%, p < 0.05 ; figures 6 g and 6h). taken together, despite receiving most of their synaptic drive from neurons located within the v1 circuit, the l6 cc pathway conveys visual motion signals that cover a broad spectrum of orientation selectivity (figure 7). on the other hand, cells relaying motion information to thalamus output exquisitely tuned orientation- and direction - related signals and received substantial widespread innervation from higher - order cortical areas known to convey visual and spatial information (figure 7). this study shows that in v1, the functional diversity of l6 can be attributed to specific populations of output neurons that are embedded in different anatomical microcircuits. we show that the output tuning of cc and ct cells can be directly attributed to the tuning profile of the somatic depolarization, rather than to their difference in intrinsic properties. at least for the stimuli used here, the cc and ct cell populations are wired to receive functionally distinct, direction - related synaptic signals and therefore appear to play distinct roles in visual processing. we reveal that several intrinsic biophysical parameters of l6 neurons recorded in vivo may be used to classify cells according to their morphological identity and projection targets. although we have not directly determined the differential impact of these properties on the integration of signals in the dendrites of cc and ct neurons, it seems likely they will impact psps arriving at the soma (chadderton., 2014). experiments in which we injected previously recorded visually evoked somatic responses back into cc and ct cells indicate that their output tuning can not simply be explained by differential intrinsic membrane properties expressed proximal to the axonal initial segment or soma. for example, on the basis of their dendritic profile, claustrum - projecting neurons in cat have morphological features similar to that of ct - projecting cells described here (katz, 1987). however, previous studies injecting retrograde tracers into the claustrum (carey and neal, 1985) indicate that in rat, claustrum - projecting cells in the visual cortex are confined to the deep layers of secondary visual areas. the axonal projection from our population labeling experiments indicated that cre - ntsr1 l6 cells in mouse v1 do not target the claustrum. also, on the basis of our biocytin reconstructions of cc cell axons, we find no evidence for a direct claustrum projection. however, since cc cells in v1 can extend their axons into deep layers of secondary visual areas, an indirect primary visual cortical - claustrum pathway may exist. our data show that the broad stimulus selectivity in l6 can be attributed specifically to the cc - projecting population. these cells receive more net depolarization during drifting gratings, yet they are, on average, only modestly tuned to stimulus orientation. in contrast, ct neurons were more selective for stimulus orientation and/or direction. since we have not directly determined the functional identity of the presynaptic cells providing visual information, the precise contribution of intra- versus interlaminar connectivity to the subthreshold tuning of cc and ct neurons remains to be established. in v1, subsets of gaba - ergic interneurons also exhibit broad stimulus selectivity (sohya., 2007 ; kerlin., 2010 ; their tuning is believed to arise from them receiving input from many local pyramidal cells tuned to different stimulus orientations (chen., 2013 ; hofer., 2011 ; bock., our data indicate that cc cells receive substantial synaptic input from within l5 and l6 (mercer., 2005 ; zarrinpar and callaway, 2006) and raise the possibility that such rules of local functional convergence are not specific to inhibitory interneurons but may also apply to this principal cell type. it also remains a possibility that multiple subclasses of cc cells differentially tuned to orientation and/or direction receive input from nonoverlapping constellations of presynaptic cells. the paucity of information about the influence of cortex on thalamus arises, at least in part, from the functional and morphological diversity of l6 neurons. compared to cc cells, our data show that synaptic drive onto ct cells is highly selective for stimulus orientation and/or direction and that ct cell firing is extremely sparse. the fact that ct axons project to thalamic structures, including the dorsal lateral geniculate nucleus and the reticular nucleus, indicates that this highly selective feedback signal may be used for stimulus - specific thalamic gain control via excitatory and inhibitory modulation (mease., 2014 ; olsen., 2012). this highly tuned ct output signal is also expected to directly impact activity within layers 5 and 6 and, via ascending polysynaptic pathways, provide modulation of upper cortical layer activity (bortone. the observation that cc and ct cells have a similar total dendritic length (oberlaender., 2012) and that cts have a lower spine density appears at odds with our observation that ct cells receive input from more than double the number of presynaptic cells. this may suggest that the number of contacts per connection is reduced in ct cells such that cts are more densely innervated (in terms of the number of presynaptic cells) compared to cc cells. alternatively, it is possible that cell - specific tropisms may impact or bias the retrograde transmission of the rabies virus in some way. however, the fact that we find presynaptic cells located in distant areas, including the thalamus, indicates that long - range inputs can be labeled in both cell types. the increased connectivity onto ct cells may therefore reflect a dynamic role in integration of information converging from across functionally nonoverlapping upstream networks. dense innervation of ct cells may also be explained by them receiving input from a larger number of inhibitory neurons that ensure the sharpening of ct responses (chadderton., 2014). while our study does not attempt to explain the relative contribution of presynaptic neurons to stimulus selectivity in l6 principal cells, these data do however show that the sparsely encoded information about stimulus direction is processed by a specific subpopulation of l6 neurons that are biased in receiving input from secondary visual and retrosplenial cortices. this l6 pathway may therefore mediate thalamic integration of both cortical visual and egocentric information. integration of self - motion and head - direction signals (clark., 2010) within v1 l6 ct cells could optimize object motion detection (hup., 1998) by providing a contextual influence on thalamic relay neurons. approaches that combine physiological analysis with dense electron microscopy - based reconstruction are elucidating the circuit organization mediating stimulus motion processing at the very early stages of the visual system (briggman. the relation between function and connectivity in large - scale cortical circuits however remains exceedingly challenging (bock. the combination of in vivo single - cell physiology and retrograde monosynaptic tracing enables identification of local and global projections onto individual cells whose subthreshold sensory response properties have been characterized, an approach that permits the generation of cortical wiring diagrams with single - cell and functional resolution. as recently shown in other sensory systems (angelo., 2012), here we find that l6 cc- versus ct - projecting cells have distinct intrinsic and functional properties. furthermore, these two classes of projection cells are embedded within distinct wiring motifs that indicates top - down, targeted innervation of l6 microcircuits may provide contextual modulation during sensory computation. adult c57/bl6 mice (58 weeks old) were anaesthetized with a mixture of fentanyl (0.05 mg / kg), midazolam (5.0 mg / kg), and medetomidin (0.5 mg / kg) in saline solution (0.9% ; intraperitoneal) and supplemented as necessary (20% of initial dose). mice were head fixed using nonpuncture ear bars and a nose clamp (sg-4n, narishige, japan), and their body temperature was maintained at 37c 38c using a rectal probe and a heating blanket (fhc, bowdoinham, me, usa). an incision was made in the scalp and a small craniotomy was drilled above the primary visual area of the cortex using a dental drill (osada electric, japan) and the dura removed. following recordings, the craniotomy was sealed using a silicone sealant (kwik - sil, world precision instruments) and the scalp sutured. anesthesia was reversed by injection of a mixture of naxolon (1.2 mg / kg), flumazenil (0.5 mg / kg), and atipamezol (2.5 mg / kg) in saline solution (0.9%). the wound was infiltrated with lidocaine and an antibiotic (cicatrin, glaxosmithkline, uk) topically applied. during initial recovery, mice were kept in a climate - controlled chamber (harvard apparatus, holliston, ma, usa) for 34 hr under observation. all procedures were approved by the local ethics panel and the uk home office under the animals (scientific procedures) act 1986. in vivo whole - cell recordings data were filtered at 4 khz and digitized at 1020 khz using an itc-18 a / d - d / a interface (instrutech, heka elektronik, germany) and the neuromatic package (http://www.neuromatic.thinkrandom.com) under igor pro 5 (http://www.wavemetrics.com). intracellular solutions for whole - cell recordings were made up in concentrated stock (two times the final concentration) to allow for biocytin or plasmid addition. the final concentrations were (all from sigma - aldrich or vwr international, uk) 110 mm k - methanesulphonate, 40 mm hepes, 6 mm nacl, 0.02 mm cacl2, 3 mm mgcl2, 0.05 mm egta, 2 mm na2atp, 2 mm mgatp, and 0.5 mm na2gtp ; the ph was adjusted to 7.28 using koh. the final osmolarity after adding either 0.5% biocytin or suspended plasmids was adjusted to the range of 280294 mosm. intracellular solutions were filtered through a 0.22 m pore size centrifuge filter (costar spin - x). plasmid concentrations were verified by spectrophotometry (nanodrop 2000, thermo scientific) : 200 ng/l rvg plasmid and 40 ng/l tva plasmid. in some cases an xiap plasmid (40 ng/l) was also included. for individual cc and ct tracing experiments, only one whole - cell recording was performed in each brain within a maximum of three attempts. stimuli were presented on a 56 cm lcd monitor positioned 21 cm from the contralateral eye spanning 72 (in elevation) and 97 (in azimuth) of the animal s visual space. stimuli consisted of sinusoidal gratings (spatial frequencies including 0.01, 0.025, and 0.04 cycles/ [niell and stryker, 2008 ]) drifting in 1 of up to 12 directions at a temporal frequency of 2 cycles / s. in cells where we compared two or more spatial frequencies, we observed no effect on the psp integral, evoked firing rate, or osi. for each trial, gratings were presented in the following manner : stationary (1 s)-moving (2 s)-stationary (1 s). gratings were presented in sequences according to their maximal difference (+ 210). jitter in the onset of the stimulus caused by the refresh rate of the monitor was compensated for by implementing a small photodiode in front of the screen, which allowed for alignment of the onset of the stimulus. we excluded direction - nonspecific onset responses by analyzing the membrane voltage during the second half of the 2 s drift. this analysis time window is expected to include evoked feed - forward and feed - back signals (ringach., however, when the entire stimulus duration is analyzed, there remains a significant difference between cc and ct tuning for both orientation and direction (p < 0.05). the membrane potential responses for each direction were determined by averaging across stimulus repetitions (four to six trials). for analysis of the subthreshold membrane potential, aps were clipped on each side of the peak at the level where the membrane potential variance (vm standard deviation) equaled the mean variance in the absence of spiking. linear interpolation was then used to join the membrane voltage traces. for the analysis of the peak depolarization (psppeak, in mv), the most depolarized membrane potential value was used for directions in which evoked spiking was recorded. the analysis of the integral of the responses (pspintegral, in mv.s) was performed of average traces for each direction. the output response to drifting gratings was calculated by detecting action potentials in each trial and averaging the spiking rate for each direction. for a given cell, the tuning profile was analyzed by first calculating the vector average of the responses (for pspintegral, psppeak, or output) for the 12 directions. the direction closest to the value of the vector average was then defined as the preferred direction. the response to the orthogonal direction was calculated as the average of the two sets of responses recorded for the pref + /2 (ortho1) and pref /2 (ortho2). the null direction response is defined as the response recorded at pref + (null direction). the orientation and direction indexes (osi and dsi, respectively) are defined as follows : osi = (pref ortho)/(pref + ortho) and dsi = (pref null)/(pref + null). for experiments where the evoked synaptic potentials were injected into cells, the last 1 s of the response to four directions was used (pref, ortho1, null, ortho2). the amplitude of the current waveform to be injected was first determined by injecting the same cell type input and adjusting the current amplitude such that at least one spike could be evoked. current - voltage relationships were obtained from each neuron by injecting step currents ranging from 400 to 0 pa (in + 50 pa steps of 600 ms) and depolarizing currents from 0 pa to two times the rheobase (in + 25 pa steps of 600 ms). briefly, the initial instantaneous frequency of the first two action potentials (f1) was extracted from rheobase to two times the rheobase, and the slope of the relationship between the initial instantaneous frequency and the current injected was calculated (f1-i slope). in addition, the initial instantaneous frequency and the instantaneous frequency 200 ms after the onset of the current injection at two times the rheobase were calculated (f12xrb and f2002xrb, respectively) in order to assess the early accommodation index of spiking (eacc index = ((f12xrb f2002xrb)/f12xrb) 100). for quantification of membrane potential sag, hyperpolarizing current steps (400 pa, 600 ms) were injected. to determine the sag potential amplitude, the most negative membrane potential value determined in the first 100 ms (peak) was compared to the average membrane potential recorded during the last 200 ms (steady state) of the current step period. the absolute difference in these two values was used as the sag potential amplitude. when comparing these intrinsic properties for morphologically identified cc (n = 6) and ct cells (n = 10), statistically significant differences were observed for all four parameters (p < 0.05). on average, there was found to be no significant difference in input resistance between cc and ct cells. to classify cells a cluster analysis (cauli., 2000 ; thorndike, 1953 ; ward, 1963) using f12xrb, the f1-i slope, the eacc index and sag potential amplitude was performed. cells sharing similar parameters are expected to be close to one another in multidimensional euclidean space. the number of clusters was defined using the thorndike method (thorndike, 1953), by comparing the within - cluster linkage distances. student s t test and wilcoxon tests were used to determine the significance of normally and nonnormally distributed data, respectively. for tissue processing - related information, see the supplemental information. for neuronal reconstructions, recorded neurons were either (1) filled with biocytin and mice immediately perfused or (2) loaded via the patch pipette with a dna vector to drive expression of gfp. in the latter case, mice were returned to their home cage for up to 72 hr, after which they were anesthetized and transcardially perfused. for tracing of gfp - labeled cells, the cells were first immunostained as described in the supplemental information. cells were reconstructed in 3d using neurolucida (mbf bioscience) under an olympus bx61 at magnifications ranging from 4 to 100. final 3d reconstructions were analyzed in neurolucida explorer (mbf bioscience). the field volume of dendrites and axons was calculated by computing the 3d convex hull, which is the convex volume enclosed between the neuronal process ends. to further characterize neuronal processes, we plotted the number of intersections between processes and concentric spheres of the gradually increased radius (+ 10 m) centered at the cell body (sholl, 1953). cells were first centered according to the location of the soma, then aligned with respect to the pial surface. dendritic and axonal trees were then separately exported as vectorial models from neurolucida to vrml or wavefront obj files. the files were opened in a 3d graphic software (v. 2.68, the blender foundation, http://www.blender.org), and model lines were converted to mesh lines and then to tubes of identical diameter. finally, the meshes were converted to a 3d image stack (grothausmann. stacks were then opened in imagej (fiji, wayne rasband, nih) using the metaimage reader / writer plugin, converted to 32 bits image stacks and low pass filtered. stacks of each cell type were averaged and the result projected as an integral in the coronal plane. finally, images were scaled by the ratio of the integral of the resultant stack and the average total length of processes in the given cell type. the movies of density map rotations were produced with a custom version of the imagej built - in 3d - projector plugin. the plugin was modified to work with 32 bit images and produce projections as sums. for spine counting, either biocytin - filled or gfp - expressing cells were imaged using wide - field (olympus bx61, 100/1.25 numerical aperture [na ]) or confocal (leica sp5, 40/1.3 na) microscopy. for each cell, spines were manually tagged in between three and seven 80-m - long dendritic segments. the length of each dendritic segment was extracted on the basis of either neurolucida reconstructions or using a plugin (simple neurite tracer) in image j. for each cell, we sampled at least one dendritic segment per cortical layer. fixed whole brains were embedded in 4% agar and placed under a two - photon microscope containing an integrated vibrating microtome and a motorized x - y - z stage (osten and margrie, 2013 ; ragan., 2012). coronal images were acquired via three optical pathways (red, green, and blue) as a set of 6 by 9 tiles with a resolution of 1(x) 1(y) m obtained every 5 m (z) using an olympus 10 objective (na = 0.6) mounted on a piezoelectric element (physik instrumente). following acquisition, image tiles were stitched using fiji and custom routines, including a custom version of the fiji stitching plugin (preibisch., 2009) allowing the parallel processing of several image planes for higher throughput. briefly, the illumination profile was computed from the average of all tiles across the brain and used to normalize the individual tiles that were then stitched together using a combination of the readout from the microscope stage and cross - correlations. the coordinates of each marked cell were then used to position markers (red spheres) in the whole - brain image stack. the brain regions were determined using a standard mouse brain atlas (franklin and paxinos, 2008). for cc, ct, and cre - ntsr1 rv - labeling, we counted 138 21, 338 116, and 4,088 945 cells, respectively. in 2 of the 11 data sets, fixed whole brains were immediately sliced using a standard vibratome, antibody - stained, and then imaged using a confocal microscope (1.8(x) 1.8(y) 5(z) m, 10 and 20 objective ; leica sp5). cell counting in these cases were manually performed on individual coronal slices. for combined single - cell physiology and rabies virus tracing experiments from cc neurons, labeled presynaptic cells were identified and counted for nine different brain regions located in the ipsilateral hemisphere and include v1, thalamus (different nuclei pooled), hippocampal formation, cortical associational areas (including temporal associational cortex and parietal cortex) secondary visual cortex (including lateral and medial), retrosplenial cortex, and auditory cortex (primary and secondary). for histograms, all of these regions (excluding v1, thalamus, v2, and rsp) are represented by the others category. in the contralateral hemisphere, cells were only found in the secondary visual cortex. for ct experiments, presynaptic cells were found in additional areas and include the hypothalamus, somatosensory, motor, cingulate cortices, and contralateral v1. for histograms, all of these additional regions were included (excluding v1, thalamus, v2, and rsp) and are represented by the others category. for cre - ntsr1 tracing, cells were found in all of the above ct - related brain areas. in addition, some cells were found in over 30 other regions (not described). all of these regions (excluding thalamus, v2, and rsp) were allocated to the other | summarysensory computations performed in the neocortex involve layer six (l6) cortico - cortical (cc) and cortico - thalamic (ct) signaling pathways. developing an understanding of the physiological role of these circuits requires dissection of the functional specificity and connectivity of the underlying individual projection neurons. by combining whole - cell recording from identified l6 principal cells in the mouse primary visual cortex (v1) with modified rabies virus - based input mapping, we have determined the sensory response properties and upstream monosynaptic connectivity of cells mediating the cc or ct pathway. we show that cc - projecting cells encompass a broad spectrum of selectivity to stimulus orientation and are predominantly innervated by deep layer v1 neurons. in contrast, ct - projecting cells are ultrasparse firing, exquisitely tuned to orientation and direction information, and receive long - range input from higher cortical areas. this segregation in function and connectivity indicates that l6 microcircuits route specific contextual and stimulus - related information within and outside the cortical network. |
the incidence of renal cell carcinoma is consistently increasing, and the increase in use of imaging technologies has resulted in an increase in the incidental detection of renal cell carcinoma, especially of small renal masses. nephron - sparing surgery has become the standard treatment for small renal masses, demonstrating improved overall survival and superior preservation of renal function compared with traditional radical nephrectomy. in addition, laparoscopic partial nephrectomy (lpn) and robot - assisted partial nephrectomy (rpn) have been accepted as treatment options for small renal masses with adequate oncologic outcomes. several factors predict renal functional outcome after partial nephrectomy, including older age, sex, lower preoperative glomerular filtration rate (gfr), single kidney, tumor size, and longer ischemic interval. the limit of 30 minutes in patients with normal preoperative kidney function is currently accepted as a safe warm ischemia time (wit). our previous study demonstrated that patients with a wit>28 minutes had a significantly greater decrease in the gfr of the affected kidney ; in multivariate analysis, wit was an independent predictive factor of functional reduction of the affected kidney. although a wit ranging from 20 to 30 minutes was thought to be safe, some authors have suggested that every minute counts when the renal hilum is clamped. to minimize ischemic renal injury, many surgeons make an effort to decrease the wit to less than 30 minutes during partial nephrectomy under pneumoperitoneum. however, there are few studies on preoperative predictors of prolonged wit during lpn or rpn performed by a single surgeon. in this study, we evaluated patients who underwent lpn or rpn and tumor characteristics predictive of prolonged wit. with the approval of our institutional review board, we retrospectively analyzed the medical records of 317 patients who underwent partial nephrectomy. partial nephrectomy was conducted by laparoscopic or robot - assisted surgeries and was performed by the same surgeon between october 2007 and may 2013. according to the tumor location, we performed transperitoneal approaches for anteriorly or laterally located tumors and retroperitoneal approaches for posteriorly located tumors. for arterial clamping during surgery, we used bulldog clamps (aesculap, center valley, pa, usa) (fig. color doppler laparoscopic ultrasound was used to confirm whether hilar clamping was proper or not. resection of the tumor was completed with scissors, without application of electrosurgical coagulation devices (fig. after tumor excision, surgical bed hemostasis was achieved by oversewing vessels by using 3 - 0 pds or 3 - 0 v - loc sutures with a lapra - ty clip (ethicon, cincinnati, oh, usa) at the terminal end (fig. the renal parenchyma was repaired with 1 - 0 vicryl sutures placed in an contiuous fashion across the defect, and the sliding technique with hem - o - lok clip was used to tighten and secure the sutures (fig. ischemia time was defined as the interval between placement of the first arterial clamp and removal of the last clamp. demographic and patient characteristics were recorded, including age, sex, body mass index, american society of anesthesiologists classification, preoperative estimated gfr, clinical tumor size, and pathologic outcome. clinical tumor size was recorded as the largest diameter seen on radiological images. the nephrometry score (nephrometry score ; r.e.n.a.l. [radius, exophytic / endophytic, nearness to collecting system or sinus, anterior / posterior and location relative to polar lines ] score, padua [preoperative aspects and dimensions used for an anatomical ] score, and c - index) was determined by a retrospective review of images. patients were divided into two groups : group a was defined as prolonged wit (30 minutes) and group b as short wit (20 minutes.. showed a much stronger correlation between nephrometry score and wit, with the c - index system (coefficient, -0.609) and the padua score system (coefficient, 0.735) showing the strongest correlation in the overall analysis, whereas the r.e.n.a.l. in concordance with previous studies, our results indicate that each component of the nephrometry score is correlated with wit. among the variables making up the nephrometry score, our study demonstrated that only the padua score (p=0.032) could predict wit>30 minutes. these findings suggest that risk group stratification using the padua score may improve patient selection for partial nephrectomy, especially for novice surgeons. however, the results of our subgroup analysis showed that using the nephrometry score to predict prolonged wit in the groups divided by surgical type (rpn or lpn) was not effective. previous studies have set the wit cutoff value to 20 minutes, whereas we evaluated the predictors of prolonged wit defined as longer than 30 minutes. regarding surgeon experience, mottrie. demonstrated that surgeon experience significantly correlated with robotic console time (p<0.001) and wit (p<0.0001). when we compared each period, the ratios of group a were 68%, 54%, 30%, 6%, 4%, and 4%, respectively. our data showed that prolonged wit was very rare when surgeon experience was greater than 150 cases of partial nephrectomy under pneumoperitoneum. in further subgroup analysis, surgeon experience was the most important predictor of wit in both the rpn and lpn groups. after 50 cases of each type of surgery were accumulated, the risk of prolonged wit was reduced dramatically compared with that in the first 50 cases (fig. several current studies have shown shorter learning curves for rpn than for lpn. two systematic reviews and meta - analyses of rpn versus lpn reported no significant differences in perioperative outcomes between the two groups, except for a significantly shorter wit. however, type of surgery was not predictive of prolonged wit in the present study. more complex cases were treated with rpn because of its likelihood to greatly mitigate the difficulty of complex partial nephrectomy. surgeon experience, padua score, and tumor size are significant predictors of prolonged wit during partial nephrectomy under pneumoperitoneum. among these predictive factors, accumulating surgical experience is the most important and the only modifiable factor for reducing the risk of prolonged wit. an accompanying video can be found in the ' urology in motion ' section of the journal homepage (www.kjurology.org). the supplementary video clips can also be accessed by scanning a qr code located on the fig. 1 of this article, or be available on youtube (https://youtu.be/fcfhdcib1ue). | purposecurrent clinical data support a safe warm ischemia time (wit) limit of 30 minutes during laparoscopic partial nephrectomy (lpn) or robot - assisted partial nephrectomy (rpn). we evaluated independent factors predicting prolonged wit (more than 30 minutes) after lpn or rpn.materials and methodsa retrospective data review was performed for 317 consecutive patients who underwent lpn or rpn performed by the same surgeon from october 2007 to may 2013. patients were divided into two groups : group a was defined as prolonged wit (30 minutes) and group b as short wit (< 30 minutes). we compared clinical factors between the two groups to evaluate predictors of prolonged wit.resultsamong 317 consecutive patients, 80 were in the prolonged wit group. baseline characteristics were not significantly different between the groups. in the univariable analysis, padua (preoperative aspects and dimensions used for an anatomical) score (p=0.001), approach method (transperitoneal or retroperitoneal approach ; p<0.001), and surgeon experience (p<0.001) were significantly associated with prolonged wit. in the multivariable analysis, padua score (p=0.032), tumor size (25 mm ; odds ratio, 2.98 ; 95% confidence interval, 1.48 - 5.96 ; p=0.002), and surgeon experience (p<0.001) were independent predictors of prolonged wit.conclusionssurgeon experience, tumor size, and padua score predicted prolonged wit after rpn or lpn. among these factors, increasing surgical experience with lpn or rpn is the most important factor for preventing prolonged wit. |
physical activity is very important for health and helps to protect from many chronic diseases. in addition to its contribution to development of motor skills, it is necessary for socialization and self - confidence, especially in childhood and adolescence. many guidelines commonly state that children between the ages of 6 and 17 years should participate in 60 min or more of moderate - to - vigorous intensity physical activity daily. visually impaired subjects are reported to be less active physically when compared to their sighted peers. difficulty in adaptation to a new environment, safety problems, and economic issues are suggested to be responsible of the limited participation in physical activity of visually impaired children [46 ], which can negatively affect motivation and cause social disengagement. although the literature indicates a lack of activity in visually impaired children, some studies report that physical activity levels of many sighted children and adolescents are also insufficient to promote health benefits [79 ]. the reasons for this need to be seriously questioned and strategies must be devised to improve children s and adolescents activity levels. some authors, however, indicated that the recommended 60 min or more of the physical activity can be obtained in a cumulative manner in school during physical education lessons, playtime, recess, intramural activities, and programs before and after school. structured physical education lessons in schools seem to offer an ideal opportunity to provide physical activity for students. but, to the best of our knowledge, there exists no study in the literature investigating the participation of visually impaired adolescents and their sighted peers in physical education and sports lessons, and comparing their condition in terms of problems, attitude, and targets. so, in this study we had 3 objectives : to investigate participation of the visually impaired children and their sighted peers in physical education and sports lessons, in terms of many aspects such as problems, attitude, and targets ; to compare their physical activity status ; to find out if physical education and sports lessons are sufficient enough to meet the major part of the recommended activity level for visually impaired children and their sighted peers. this study included 22 visually impaired adolescents (7 girls and 15 boys) at mehmet akif ersoy secondary school for the visually impaired, tokat (turkey), and 31 sighted counterparts (14 girls and 17 boys) at a government school. group i was composed of visually impaired students and group ii included their sighted peers. the school for the visually impaired in tokat is a boarding school and it is the only one in the black sea region of turkey. the school had a total of 22 students in 5, 6, 7 and 8 grades, so the whole universe was included in this study. the demographic data including age, height, and weight were recorded. in the visually impaired group, information about vision loss the reason of vision loss (congenital / acquired), duration (year), and degree of vision loss (blind / low - vision) was obtained. we used a questionnaire prepared to investigate participation of visually impaired adolescents and their sighted peers in physical education and sports lessons at their school and the problems they encounter while doing sports. (except the 12th question and the second part of the 13th question) : do you participate in physical education and sports lessons in your school ? do you think you are offered a sufficient playing field for you to do sports ? do you think the physical education and sports lessons in your school are sufficient to meet your sports demands ? have you ever participated in a sport organization like sports tourney or inter-/intra- school matches ? have you ever been injured while doing sports ? dou you have any specific target for the future in terms of sports ? (and if so, what is it ?) there are tools which can be used to assess physical activity level of an individual, such as field tests, laboratory tests and questionnaires. in our study we aimed to assess the activity level of the groups by using the turkish version of the ipaq. the international physical activity questionnaire (ipaq) was designed for epidemiological studies by a multinational working group (10). long (31 items) and short (7 items) versions of the ipaq are available and the short version (ipaq - sf) in particular has gained wide acceptance. the short form assesses physical activities like domestic activities, work - related activities, recreationally performed and/or transport - related activities, and sitting. the turkish version of the ipaq - sf was used to evaluate the physical activity level of the subjects in this study. the validity and reliability study of the turkish version was created by saglam and reported that the turkish versions of the ipaq short and long forms were reliable and valid in assessment of physical activity. in the short form, activities that are assessed are walking, moderate - intensity activities, and vigorous - intensity activities. frequency (measured in days per week) and duration (time per day) were recorded for each specific type of activity. because the subjects were students, the separate scores on walking, moderate - intensity activities, and vigorous - intensity activities are combined into a total score to determine overall level of activity. the met values and formulae for computation of met - minutes were derived from the ipaq validity and reliability study. in their international study, craig. demonstrated that reliable and valid physical activity data can be collected by the ipaq instruments in many countries. they also stated that the short form is feasible to administer, and there was no difference between the reliability and validity of the short and long ipaq forms. independent - sample t test was used to compare the continuous normal data between groups. the mann - whitney u test was used to compare ipaq scores between groups, between genders, and between totally blind children and children with low vision. chi - square tests were used to compare the categorical variables between vi and control groups. a p value analyses were performed using spss 19 (ibm spss statistics 19, spss inc., this study included 22 visually impaired adolescents (7 girls and 15 boys) at mehmet akif ersoy secondary school for the visually impaired, tokat (turkey), and 31 sighted counterparts (14 girls and 17 boys) at a government school. group i was composed of visually impaired students and group ii included their sighted peers. the school for the visually impaired in tokat is a boarding school and it is the only one in the black sea region of turkey. the school had a total of 22 students in 5, 6, 7 and 8 grades, so the whole universe was included in this study. the demographic data including age, height, and weight were recorded. in the visually impaired group, information about vision loss the reason of vision loss (congenital / acquired), duration (year), and degree of vision loss (blind / low - vision) was obtained. we used a questionnaire prepared to investigate participation of visually impaired adolescents and their sighted peers in physical education and sports lessons at their school and the problems they encounter while doing sports. (except the 12th question and the second part of the 13th question) : do you participate in physical education and sports lessons in your school ? do you think you are offered a sufficient playing field for you to do sports ? do you think the physical education and sports lessons in your school are sufficient to meet your sports demands ? have you ever participated in a sport organization like sports tourney or inter-/intra- school matches ? have you ever been injured while doing sports ? dou you have any specific target for the future in terms of sports ? (and if so, what is it ?) there are tools which can be used to assess physical activity level of an individual, such as field tests, laboratory tests and questionnaires. in our study we aimed to assess the activity level of the groups by using the turkish version of the ipaq. the international physical activity questionnaire (ipaq) long (31 items) and short (7 items) versions of the ipaq are available and the short version (ipaq - sf) in particular has gained wide acceptance. the short form assesses physical activities like domestic activities, work - related activities, recreationally performed and/or transport - related activities, and sitting. the turkish version of the ipaq - sf was used to evaluate the physical activity level of the subjects in this study. the validity and reliability study of the turkish version was created by saglam and reported that the turkish versions of the ipaq short and long forms were reliable and valid in assessment of physical activity. in the short form, activities that are assessed are walking, moderate - intensity activities, and vigorous - intensity activities. frequency (measured in days per week) and duration (time per day) were recorded for each specific type of activity. because the subjects were students, the separate scores on walking, moderate - intensity activities, and vigorous - intensity activities are combined into a total score to determine overall level of activity. the met values and formulae for computation of met - minutes were derived from the ipaq validity and reliability study. in their international study, craig. demonstrated that reliable and valid physical activity data can be collected by the ipaq instruments in many countries. they also stated that the short form is feasible to administer, and there was no difference between the reliability and validity of the short and long ipaq forms. independent - sample t test was used to compare the continuous normal data between groups. the mann - whitney u test was used to compare ipaq scores between groups, between genders, and between totally blind children and children with low vision. chi - square tests were used to compare the categorical variables between vi and control groups. analyses were performed using spss 19 (ibm spss statistics 19, spss inc., ibm co., somers, ny). mean ages of the visually impaired and the sighted children were 13.591.14 and 13.610.50 years, respectively (p>0.05). in group i, total duration of vision loss was 13.021.94 years (min : 6 years ; max : 16 years) ; 81.8% of the cases (n : 18) were congenitally impaired, 4 cases (18.2%) were totally blind, and the remainder had low vision (n : 18 ; 81.8%). students in both groups reported that they received 1 h per week of physical education and sports lessons in their schools. all of the children in both groups reported that they participated in physical education and sports lessons in their school and all indicated that their participation was willing. most of the children in both groups (in group i n : 20 ; 90.9% and in group ii n : 23, 74.2%) thought that the education they received about sports was sufficient (x : 0.166 ; p : 0.118). all of the visually impaired children stated that the area and the equipment required to perform sports were sufficient. most of the children in group ii (83.9%) also reported that the equipment was sufficient (x : 0.068 ; p : 0.059) ; but that the area was not (n : 13 ; 41.9%) (x : 10.064, p : 0.002). all of the children in group i indicated that the authorities took required precautions for their safety while doing sports. in group ii, most of the children (n : 26 ; 83.9%) also reported that the precautions were sufficient. therefore, no significant difference was observed between the groups (x : 0.068 ; p : 0.059). except for 2 students in the sighted group (6.5%), all of the children in both groups mentioned that their parents were supportive enough of their participation in sports activities (x : 0.505 ; p : 0.337). nine children (40.9%) in the visually impaired group and 11 children (35.5%) in the sighted group thought that the physical education and sports lessons in their school were not sufficient to meet their sports needs (x : 0.013 ; p : 0.909). sighted children were found to participate more in sports organizations (x : 6.325 ; p : 0.012). two children (9.1%) in the visually impaired group reported participation and 14 children (45.2%) in group ii said they participated in a sports organization. the percentage of children who had a sports injury before in the visually impaired group was surprisingly lower than that of the sighted ones 22.7% (n : 5) and 41.9% (n : 13), respectively, but the difference was not statistically significant (x : 1.347 ; p : 0.246). falls were the most common cause of injury in the visually impaired children and contact injuries were the leading cause in the sighted group. four visually impaired children (18.2%) and 12 sighted counterparts (38.7%) indicated that they had some problems while doing sports in the lessons (x : 1.691 ; p : 0.193). when they were asked to define the problems, they stated that fear of injury was their major problem, but in group ii the causes of injury were being in a hurry (n : 1 ; 8.3%), getting tired early (n : 1 ; 8.3%), and fighting with friends (n : 1 ; 8.3%). pain after sports (n : 5 ; 41.7%) was reported, in addition to the fear of injury (n : 4 ; 33.3%) (x : 5.333, p : 0.255). groups were found to be similar in terms of ratio of children having a specific sports target for the future (x : 1.326 ; p : 0.250) ; 15 children (68.2%) in group i and 15 children in group ii (48.4%) indicated that they wanted to engage in sports in the future. the desires of the children were to be a player in a team, to achieve success in the branch of sports performed, to be a professional in sports, to be a sports trainer, and to participate in a different sport. groups were found to be different in terms of sports - related targets (x : 16.944 ; p : 0.002). to play on a team (n : 7 ; 46.7%) and play a different sport (n : 6 ; 40%) were the most desired targets among visually impaired children, but the sighted children wanted to be a professional in sports (n : 8 ; 53.3%) (table 2). physical activity status of the groups were found to be similar according to ipaq scores (p>0.05) (table 3). the level of total physical activity of the visually impaired children examined expressed by arithmetic mean reached the value of 1997 met - min / week. the total activity value was 1596 met - min / week for the sighted children. for all children in both groups, girls were found to be less active physically than boys (u : 495.500, p : 0.004). when the total ipaq scores were analyzed according to gender in each group, no gender difference existed in the visually impaired group (u : 75.500, p : 0.105). in sighted children, girls were less active than boys (u : 183.000, p : 0.011). totally blind students had lower total ipaq scores than the children with low vision (u : 6000, p : 0.011). this study investigated the participation in physical education and sports lessons of the visually impaired adolescents and their sighted peers and compared their physical activity levels using the international physical activity questionnaire short form. although limited to 1 secondary school for the visually impaired, which included just 22 students (as it is the only one in black sea region of turkey), this study, to the best of our knowledge, is the first to focus on the sports lessons of visually impaired adolescents and compare their situation with sighted children. we believe that our study offers many insights that may be inspirational for researchers interested in visually impaired people and sports issues. the result in our study was a surprise because related literature reports that visually impaired children are not as active physically as their sighted peers, and the low physical fitness level and impaired motor skills of visually impaired children are suggested to arise from limited participation in physical activities. although the activity status was similar between the groups in our study, it must be remembered that most of the children in both groups were in 8 grade. in turkey, children in 8 grade take 2 main exams centrally administered by the national education ministry 1 in the middle of autumn semester and 1 in middle of the spring semester. the distribution of students to various high schools is determined based on their total exam score. these exams are important for children and their families, and children spend most of their out - of - school time preparing for these exams by continuing to study at home or by going to after - school courses. while completing the activity questionnaire, many children, especially the girls in group ii, reported that they did too little physical activity, even in sports lessons. they indicated that all they did during several weeks was to sit and study their lessons. there are no specific high schools for visually impaired children in turkey, and visually impaired children also take these exams. but we think there are some differences in the preparation process of the visually impaired children and their sighted peers ; therefore, spending most of the time by sitting and studying lessons in group ii may lead these children to stay sedentary and have activity levels similar to the visually impaired children. in addition, girls were found to be less active physically than boys and had lower total energy expenditure. assessed physical activity levels of polish adolescents aged 1618 years and the factors that conditioned this level. they reported that the participation of schoolchildren in physical exercise classes was a factor that conditioned the level of total physical activity, and that dismissing adolescents from physical exercise classes, especially adolescent girls, was common. however, in their study, students attended sports classes up to 5 h a week, and some students had even more than 5 h a week, so the exercise lessons in their study seem sufficient to increase average total activity level (2387 met - min / week) of the children. the centers for disease control recommends daily quality physical education for all age groups, from kindergarten through grade 12. in our study children received just 1 h of sports lessons a week in both schools and all of the children stated that they willingly participated in physical education lessons, but the ratio of children who felt a need to have more sports lessons was not high. total activity level was 1997 met - min / week for the visually impaired children and 1596 met - min / week for their sighted peers in this study. in our opinion, this level of activity can not be maintained with this frequency and duration of sports lessons. the total activity level in the study, we suppose, was achieved in a cumulative manner in school during physical education, recess, intramural sports, and before- and after - school activities. we suggest more frequent participation in physical education classes, as 1 h of lessons is insufficient to fulfill the recommended level of physical activity. the literature has conflicting results regarding the activity level of totally blind children and children with low vision. atasavun and dulger stated that the motor skills of children with low vision were higher than those of the blind children in their study., on the contrary, found that blind children and adolescents did light activities for longer periods and moderate activities for shorter periods when compared to low - vision children and adolescents, but no difference was found when 1-mile run / walk test durations of low - vision and blind children were analyzed. in another study by atasavun, it was reported that children with normal vision had significantly better test scores than the low - vision and totally blind groups (p<0.05) when their mobility levels were compared by the independent mobility questionnaire. nevertheless, they found no significant differences between low - vision and blind children s mobility levels. in our study, totally blind children had lower total ipaq scores than the children with low vision (p<0.05). visually impaired children in the study seemed to have no marked problems other than fear of injury. equipment and playing field required to perform sports in the lessons were found to be sufficient among visually impaired children, while sighted ones thought that the area was not sufficient. this may arise from the difference in using a smaller playground space due to the lack of visual perception. although they had similar activity levels with the sighted children, unfortunately, very few of the visually impaired children indicated that they took part in a sports organization. we think these children need to be motivated more about playing on a team and participating in sports events such as tourneys or interschool matches. although not significant, the percentage of children who had a previous sports injury in the visually impaired group was surprisingly lower than that of the sighted ones. while falls were found to be the most common injury cause in the visually impaired children, contact injuries were the leading reason in the sighted group. visually impaired children are thought to be well - directed and protected from harmful environmental factors while they were doing sports in the lessons. while fear of injury seemed to be the only problem for group i, various problems such as delayed onset of soreness or early fatigue existed in group ii. the pain and fatigue problem might be due to the differences in activity type. although the total activity scores of the groups were similar, visually impaired children were more likely to perform moderate activity or walking, but the sighted children were more likely to perform moderate or vigorous activities. goalball and torball are the games especially designed for the blind and low - sighted individuals. students in our study reported that they usually played goalball and torball, but if they had the chance, they would like to play different games or do another sport / activity like swimming, dancing, or yoga, or to play on a team. unlike these modest wishes, being a trainer or a professional player are the leading goals among the sighted children. visually impaired children must be encouraged more regarding participation on a team or must be offered appropriate options in terms of physical activity. the sample size of our study was too small to draw conclusions concerning the whole population of visually impaired children. therefore, to understand if the activity level of the visually impaired children and their sighted peers are really similar or if it is a temporary situation specific to the 8 grade students, comparison should be made with larger samples or with students in other grades, for example, in high schools. we also think that children in secondary school, especially the visually impaired ones, need to be more motivated and more encouraged to take part in various sports or physical activities to make them increase their activity level to the recommended levels that promote health benefits. finally, frequency of physical education and sports lessons in a week can be increased, because it appears that 1 h of sports lesson per week is insufficient to fulfill the recommended level of physical activity. | backgroundthis study investigated participation in physical education and sports lessons of visually impaired adolescents and their sighted peers and compared their physical activity levels.material/methodsa total of 22 visually impaired children of mean age 13.591.14 years and 31 sighted children aged 13.610.50 years participated in the study. a questionnaire prepared for this study was used to investigate participation of visually impaired adolescents and their sighted peers in physical education and sports lessons at school and the problems they encounter while doing sports. the turkish version of the international physical activity questionnaire short - form (ipaq - sf) was used to evaluate the physical activity level of the subjects.resultsthe results of our study suggest that physical activity levels of visually impaired children and their sighted children were similar (p>0.05). totally blind children had lower ipaq scores than those with low vision (p<0.05), and girls were less active physically than boys (p<0.05). there were few differences in physical education lessons of the groups, in taking part in sports - related organizations, and future plans.conclusionschildren in secondary school, especially visually impaired children, need to be more motivated and more encouraged to take part in various sports or physical activities. |
caspase-2 is one of the closest mammalian homologues of the caenorhabditis elegans caspase ced-3 and shares significant homology with the drosophila nedd2-like caspase (dronc), both of which are essential for developmentally programmed cell death [1 - 4 ]. although several studies have implicated caspase-2 as a crucial mediator of apoptosis in mammalian cells, its apoptotic function has remained enigmatic, partly due to the fact that casp2 mice are viable and fertile with only minor apoptotic defects in some cell types [2,5 - 7 ]. furthermore, lymphocytes and fibroblasts from mice lacking both initiator caspases, casp9 and casp2, are no more resistant to apoptosis than cells from casp9 mice. together, these findings indicate that the role of caspase-2 in developmental cell death is redundant and can be compensated by other caspases. however, this does not rule out context - dependent and cell - specific caspase-2 functions. for example, one study found an accumulation of oocytes in casp2 mice (although this was not reported in a second casp2 strain) and casp2 mice display premature ageing - related traits. the activation of caspase-2 has been shown to occur both upstream (by the piddosome) and downstream (by caspase-3 or -7) of mitochondrial outer membrane permeabilisation (momp). although this is also controversial since raidd (receptor - interacting protein - associated ich-1/ced-3 homologous protein with a death domain) and pidd (p53-inducible protein with a death domain), the protein components of the piddosome, are dispensable for caspase-2 activation. interestingly, caspase-2 activation can be mediated by caspase-8-induced cleavage following recruitment to the death receptor - inducing signaling complex (disc). however, the importance of the disc as an activation platform is also unclear since caspase-2 dimerisation and self - processing are sufficient for its activation. in addition, cells from casp2 mice are normally sensitive to death receptor - induced apoptosis, indicating that caspase-8-mediated cleavage of caspase-2 is not critical for its activation. while there is limited information on physiologically relevant substrates, caspase-2 can cleave and activate the protein bid, which provides a significant link between caspase-2 and momp. furthermore, a unique feature of caspase-2 is its ability to localise to the nucleus in an importin - mediated fashion [18 - 20 ]. this nuclear localisation of caspase-2 is likely associated with the recently found functions for caspase-2 in cell cycle regulation and cellular dna damage response. in the absence of an overt phenotype in knockout mice, one may speculate that caspase-2 functions under specific contexts, such as under conditions of stress or in the fine - tuning of stress signaling, resulting in relatively minor aberrations in the whole animal physiology. several recent studies showing caspase-2 functions in cell cycle regulation, dna damage response, and tumor suppression seem to be consistent with these predictions. a role for caspase-2 in cell cycle regulation became apparent from observations that casp2-deficient murine embryonic fibroblasts (mefs) proliferate faster than their wild - type counterparts and that transformation of casp2 mefs with e1a / ras exacerbated this proliferative effect. another recent study found that caspase-2 is involved in maintaining a g2/m cell cycle checkpoint in response to ionising radiation (ir)-induced dna damage, with cells lacking casp2 unable to completely arrest in g2/m. in addition, caspase-2 activation has been shown to be inhibited by cyclin - dependent kinase 1 (cdk1)/cyclin - b1-mediated phosphorylation at ser340 during mitosis to allow for the repair of replication - induced dna damage. during mitotic arrest, prolonged activation of spindle assembly checkpoint results in apoptosis by mitotic slippage and prematurely exit mitosis with chromosomal abnormalities, resulting in genomically unstable aneuploid cells. in support of this, casp2 mefs show resistance to cell death induced by microtubule - disrupting drugs and also display increased genomic instability in culture compared with wild - type cells. these findings indicate that deregulation of g2/m checkpoint in casp2 cells may contribute to the accumulation of cells with damaged dna. the study by shi and colleagues found that caspase-2 is involved in dna damage repair through its interaction with a nuclear complex comprising pidd and dna - dependent protein kinase catalytic subunit (dna - pkc). following ir - induced dna damage, this dna - pkc piddosome complex phosphorylates caspase-2 at ser122, leading to its activation. activated caspase-2 is then required for the repair of double - strand dna breaks by non - homologous end - joining (nhej) with cells lacking casp2 unable to efficiently repair dna breaks. although it is unclear how caspase-2 mediates nhej, these important observations establish an additional non - apoptotic nuclear role for caspase-2 in dna damage signaling (figure 1). following double - strand dna breaks (dsbs), the ataxia telangiectasia mutated (atm) and atm - related (atr) kinases are activated and in turn phosphorylate and activate several target proteins, including checkpoint kinase 1 (chk1) and chk2. atr also activates chk1, which can then act in a feedback loop to negatively regulate atr and inhibit further activation of nuclear caspase-2. dna - dependent protein kinase (dna - pk) is also activated by dsbs, presumably by atm / atr, and forms a complex with p53-inducible protein with a death domain (pidd) and caspase-2 (dna - pk piddosome). this complex serves to phosphorylate and activate caspase-2, which is then required for the initiation of non - homologous end - joining (nhej) and dna repair. cytosolic caspase-2 is also activated by other stress signals such as reactive oxygen species (ros), metabolic stress, cytotoxic drugs, heat shock, or endoplasmic reticulum (er) stress. following heat shock, raidd (receptor - interacting protein - associated ich-1/ced-3 homologous protein with a death domain) can activate caspase-2, which is inhibited by hsp90. ca / calmodulin - dependent kinase ii (camkii) acts to inhibit caspase-2 activation and cell death in oocytes. activated cytosolic caspase-2 is able to cleave bid to its truncated form (tbid), which (via bax / bak) can induce mitochondrial outer membrane permeability (momp), activation of caspase-9 and -3, and cell death. sidi and colleagues have described an unexpected nuclear function of caspase-2 in an apparently novel pathway of apoptosis in p53-deficient cells. using zebrafish as a model system, it was found that inhibition or loss of checkpoint kinase 1 (chk1) restores -radiation - induced apoptosis in p53 mutant fish embryos. a similar ataxia telangiectasia mutated (atm)/atm - related (atr)/caspase-2-dependent pathway seems to be present in chk1-inhibited p53-deficient human tumor cells and in mefs following -irradiation. these findings implicate caspase-2 in an apoptosis pathway downstream of atm / atr in response to dna damage, which is independent of p53 (figure 1). since atm / atr can induce phosphorylation and activation of dna - pkc, it would be of interest to investigate whether the regulation of caspase-2 phosphorylation in the nucleus finely tunes its function in either apoptosis or dna damage repair following ir exposure. the role of caspase-2 in specific cell death pathways other than dna damage is also emerging. in oocytes, the apoptotic activity of caspase-2 has been shown to be inhibited by ca / calmodulin - dependent kinase ii (camkii)-mediated phosphorylation at ser135. the binding of 14 - 3 - 3 to phosphorylated caspase-2 prevents ser135 dephosphorylation, thereby promoting oocyte survival. however, under nutrient - depleted conditions, the dephosphorylation at this site leads to caspase-2 activation and oocyte cell death. these findings suggest that caspase-2 is an important player in metabolic regulation of oocyte cell death and that ser135 dephosphorylation is a sensor for caspase-2 activation. caspase-2 has also been implicated in cell death induced by heat shock. a recent study assessed the real - time recruitment of caspase-2 to activation platforms during stress - induced apoptosis, including heat shock, cytoskeletal disruption or dna damage, and found that caspase-2 activation occurred in the cytosol, not the nucleus. furthermore, heat shock - induced activation of caspase-2 occurred upstream of momp and was raidd - dependent and negatively regulated by hsp90. the caspase-2 functions in oxidative stress - induced apoptosis and ageing are also coming to light. caspase-2, along with bid and bak, were reported to be mediators of superoxide - induced cell death in muscle and in primary neurons. in addition, caspase-2 has been shown to be involved in an age - related increase in muscle cell apoptosis in mice. consistent with these findings, zhang and colleagues found that casp2 mice show significantly higher levels of oxidised proteins in liver than wild - type mice. this indicates that lack of casp2 can antagonise apoptosis induced by reactive oxygen species, leading to accumulation of cells with oxidative damage and consequently enhanced ageing phenotypes. the reduced nhej activity in casp2 cells may also contribute to the premature ageing phenotype observed in casp2 mice. our own studies using the e-myc transgenic mouse model of b - cell lymphoma found a potential role for caspase-2 in lymphoma suppression. specifically, the loss of even a single copy of casp2 resulted in increased tumor susceptibility and markedly accelerated tumor formation in e-myc transgenic mice. these studies suggest that caspase-2 can suppress myc - induced lymphomagenesis. while the precise mechanism of caspase-2-induced tumor suppression remains unclear, it is tempting to speculate that its roles in cell cycle checkpoint, dna damage repair, and removal of oxidative damaged cells are important for this function. while the recent observations have shed light on possible physiological functions of caspase-2, it remains entirely speculative how caspase-2 might carry out some of the apparently unrelated functions in apoptotic and non - apoptotic contexts. it is becoming clear that caspase-2 may act as a sensor to protect against cellular stress and that regulation of caspase-2 by phosphorylation or nuclear translocation or both may determine its role in cell death, the cell cycle, or nhej. it will be important to establish whether these functions also contribute to the tumor suppressor mechanism of caspase-2. the major deficiency in caspase-2 research is that the targets of caspase-2, which may mediate its various functions, remain largely unknown. while it remains a technical challenge to find proteins that are specifically cleaved by caspase-2 in specific contexts, identification of these substrates will be the key to unraveling the functional versatility of caspase-2 | caspase-2 is the most evolutionarily conserved of caspase family members, yet its physiological function has remained unclear and is a matter of considerable debate. newly published data now suggest that caspase-2 is required for cell cycle regulation, repair of damaged dna, and in suppressing myc - induced lymphomagenesis. additionally, loss of casp2 in mice leads to features of premature ageing. these findings suggest that caspase-2 has non - apoptotic functions in addition to its context - dependent roles in cell death. |
endothelial keratoplasty (ek) in the forms of descemet stripping automated endothelial keratoplasty (dsaek) and descemet membrane endothelial keratoplasty (dmek) has revolutionised corneal transplantation for the treatment of endothelial dysfunction and is the procedure of choice in such cases, as it carries several advantages such as faster rehabilitation, better refractive outcomes, no suture related problems, smaller risk of traumatic graft dehiscence, and less risk of corneal graft rejection [14 ]. although dmek has gained interest over the last few years due to improved visual acuity results and decreased rejection rates, the associated technical challenges have limited widespread acceptance and dsaek still remains the most common endothelial keratoplasty procedure. in patients with fuchs endothelial corneal dystrophy (fecd) and cataract that require both dsaek and phacoemulsification plus intraocular lens implantation (iol), there is a difference of opinion as to whether the surgery should be performed concurrently or sequentially with the dsaek following shortly after the phacoemulsification plus iol implantation. some authors have advocated that a staged procedure should be preferred while others have presented supporting evidence for the combined new triple procedure [6, 7 ]. in this study, we are presenting the results of a retrospective comparative study performed in a uk tertiary referral corneal unit where every effort has been made to perform a direct comparison of the 2 surgical options by making sure that there are no clinical or donor tissue related confounding factors. case notes review of all patients who had phacoemulsification followed by dsaek within 2 months or phacoemulsification and dsaek performed concurrently in our unit from january 2009 till december 2013 was performed. only cases with documented diagnosis of fecd (with clinically evident stromal oedema and central guttata) and cataract (visually significant cataract or mild cataract with expectation of progression) and that completed at least 6-month follow - up exclusion criteria were other comorbidities like glaucoma, optic nerve or retinal disease age related macular degeneration, and previous ocular surgery. only cases performed by the same senior surgeon (dl) were included. which surgical approach was chosen in each case was dictated by clinical circumstances and the choice of either surgical option was not randomized. data collected and compared included patient demographics, tissue related parameters taken from our eye bank, and clinical data including pre- and postoperative best spectacle - corrected visual acuity (bscva), endothelial cell density (ecd), complication rates, and graft rejection or failure episodes. all cases were performed under subtenon 's or peribulbar anaesthesia except in staged procedures where phacoemulsification and iol implantation was performed under topical anaesthesia., te, usa) with the ozil torsional handpiece (alcon inc., te, usa) and using a bimanual technique and a 2.2 mm sutureless main incision. the ophthalmic viscoelastic device (ovd) used in combined procedures was healon gv (abbott medical optics, il, usa) while there was no preference regarding the ovd in cases where only cataract was performed. the preloaded one piece aspheric hydrophobic tecnis zcb00 (abbott medical optics, il, usa) was used in all cases. in combined cases, the main incision was enlarged, while in dsaek only procedures a 5.5 mm peripheral corneal incision was created. the stripping of descemet 's membrane was performed before removal of ovd with bimanual automated irrigation / aspiration in combined cases while in dsaek only cases it was performed under air. ovd was thickly coated onto a sheets glide and the endothelial graft placed endothelium down onto the ovd and then was transported to the corneal wound. the graft was delivered into the anterior chamber with a bent insulin syringe using a push technique. the wound was sutured with 10/0 nylon and anterior chamber (ac) reformed with bss plus. following 100% air fill for 10 minutes, air was released to make a bubble just to the size of corneal graft button. at this stage, mydriatics were instilled and the patient was transferred to the ward to rest in a supine position. intraocular pressure was checked at 1 hour postoperatively and air was released if above 30 mm hg. in both groups, the postoperative regime was g. cyclopentolate 1% tds for 2 days, g. chloramphenicol qds for 2 weeks, and g. dexamethasone 0.1% every 2 hours, tapering dose over 6 months. the tissue used was processed by queen victoria eye bank and was precut by the same eye bank technician using the disposable horizon microkeratome after being mounted on the system 's plastic artificial chamber with pressurized air (refractive technologies, cleveland, oh). the microkeratome head was selected after pachymetry was performed with a sonogage horizon system (refractive technologies, cleveland, oh), aiming for a 100 m thickness endothelial graft. all grafts were prepared in a sterile clean room facility licensed by the human tissue authority and transported to the operating room in an optical chamber (independent corneal viewing chamber, bausch & lomb, st. the surgeon trephined the tissue in theatre with the iowa press system and placed the graft in bss plus whilst preparing the recipient eye. the size of the donor corneal disc ranged from 8.5 to 9 mm in diameter. endothelial cell density following surgery was measured using a noncontact specular microscope (em-3000, tomey, usa) at the centre of the cornea. all statistical analyses were performed with spss for windows version 17.0 (pasw, usa). mann - whitney u test, wilcoxon signed rank test, and fisher 's exact test were used. out of the total 202 dsaek operations performed in this period of time in our unit, only 59 cases fulfilled the inclusion and exclusion criteria. out of these 59 cases, 28 eyes had a combined (group 1) and 31 eyes had a staged (group 2) procedure performed. the average age of the overall group of patients (n = 59) was 73.6 9.31 years (range, 5791 years), with 60.3% women and 39.7% men. the average age of the patients with staged procedure (n = 31) was 76.74 8.9 years (range, 6291 years) compared with an average age of 70.15 8.54 years (range, 5790 years) for patients receiving dsaek combined with cataract surgery (n = 28). the donor tissue characteristics and quality used for patients in the two groups were the same and there was no clinical or statistical significant difference between endothelial cell count, donor age and tissue retrieving, storage, and preparation factors. all cases had 6-month follow - up data available while 26 cases from group 1 (92.8%) and 29 cases from group 2 (93.5%) had 12-month follow - up data available. the preoperative logmar bscva was 0.70 0.44 for group 1 and 0.82 0.40 for group 2 (p = 0.26). bscva at 6 and 12 months and ecd at 6 and 12 months are presented in table 2. the percentage of ecd loss at 12 months was 45.3% and 46.2% for group 1 and 2, respectively (p = 0.95). there was no iatrogenic primary graft failure and no rejection episodes occurred 12 months following surgery. fluid interface between recipient cornea and donor disc as well as partial or complete donor disc dislocation within the first week was observed in 6 cases in group 1 (21.42%) and in 1 case in group 2 (3.2%) (p = 0.04). two cases from group 1 had to be taken into theatre for graft repositioning and air injection (7.14%) while all the other cases settled following air injection in the anterior chamber and posturing, performed as an office procedure. despite further manipulations performed, clinical outcome was successful in all 7 cases. there has been lots of discussion within the ophthalmology community regarding whether to do a staged or triple simultaneous procedure for patients with fecd and cataracts. some authors have advocated that a staged procedure should be preferred while others have presented supporting evidence for the combined triple procedure [6, 7 ]. up to date, there is lack of enough published evidence as to which should be the preferred method. there have been no randomized clinical trials and the only study actually comparing the outcomes of the two methods is the early study by terry.. in the specific paper, the authors, following a large prospective case series of 315 eyes that had dsaek, performed a retrospective analysis of patients that underwent a staged or combined procedure and, among other results, presented and compared a cohort of 25 dsaeks without other major comorbidities and 75 triple procedures. their conclusion was that both methods were equally effective with similar endothelial mean cell loss in both groups with 33% in the staged versus 32% in the combined procedure, which compared better to our 46.2% versus 45.3%. although the above authors had a significantly larger group of patients, there seems to be a lot of heterogeneity in the background clinical history as well as the donor tissue used. additionally, the triple procedure group was 3-fold bigger than the dsaek one. in our study, we have performed a direct comparison of the 2 surgical options by making sure that there are no clinical or donor tissue related confounding factors. looking into the donor disc dislocation rates in the literature, it is difficult to make direct comparisons as different studies report dislocation rates differently. dislocations may represent either fluid in the interface of an otherwise well - positioned graft or complete dislocation into the anterior chamber. thus reported dislocation rates in the literature range from 2.5% to 14% while in our 59 cases dislocation rate was 11.8%. although no significant difference as far as graft dislocations was found in the study by terry., in our comparison, there is a statistically and clinically significant difference between the two groups and, by our results, it looks like it is 6 times more likely to develop dislocation or fluid interface with the combined technique. many surgeons believe that the use of ovd during descematorhexis is a risk factor for graft dislocation, something not proven in terry 's study as ovd was used in all their cases. the difference between the ovds used in the 2 studies (healon gv in our cohort versus healon in terry.) could be a contributing factor as the ovd used by us has greater viscosity. although we prefer the use of healon gv for easier visualisation during removal, one could argue that there could still be remnants of ovd that could possibly contribute to that difference in dislocation rate. nevertheless, one point to be taken into consideration by surgeons performing the procedures is the importance of choosing the appropriate ovd or perhaps performing descematorhexis under air or under bss with the use of an ac maintainer. other reasons for this difference in the dislocation rate could be that in the staged procedure the position of the iol is far more stable in the capsular bag making the surgery more straightforward or the fact that when performing the dsaek alone, there is lower vitreous pressure, making it easier to insert, unfold, and manipulate the graft. there are definite limitations in our study as it is a retrospective one where no randomisation has been performed regarding procedure choice and a prospective randomised trial would definitely give more accurate information on the pro et contra of each approach, but due to the lack of efficient evidence in the current literature, we believe that useful conclusions can be drawn from our comparison. the results of our study show that although the complication rate may be higher in the case of combined procedures, the final outcomes are equally good regarding final visual acuity and endothelial cell count. although this is evidence that maybe the staged approach should be the preferred method, one could argue that the combined approach is still more cost effective and convenient as by choosing it the patient will only need a secondary procedure in the operating theatre in only 21.4% of the cases while with the staged approach 2 operations are de facto needed. | purpose. to compare the surgical outcomes of staged and combined phacoemulsification with intraocular lens implant (phaco+iol) and descemet stripping automated endothelial keratoplasty (dsaek) in patients with fuchs ' endothelial dystrophy and cataract. setting. corneoplastic unit and eye bank, queen victoria hospital, east grinstead, uk. methods. retrospective study of patients who had combined phaco+iol and dsaek (group 1) or phaco+iol followed within 2 months by dsaek (group 2). patients who had previous eye surgery or any other ocular comorbidities were excluded. results. there were 28 eyes in group 1 and 31 in group 2. there were no significant differences in the demographics and corneal tissue characteristics of the two groups. the endothelial disc dislocation and rebubbling rate within 1 week in group 1 was 21.42% and in group 2 was 3.2% (p = 0.04), while the endothelial cell density at 12 months was 1510 433 for group 1 and 1535 482 for group 2 (p = 0.89). the mean 12-month logmar visual acuity was 0.28 0.24 for group 1 and 0.33 0.15 for group 2 (p = 0.38). conclusions. although the combined procedure seems to be associated with a higher complication rate the final outcomes seem to be similar to both methods. |
natural products derived from the medicinal plants are used across the globe as pharmaceutical drugs, cosmetics, fertilizers, insecticides, and pesticides. the overexploitation of medicinal plants is a threat to their existence with several taxa becoming extinct. alternate sources of important metabolites have focused on the ability of microbes associated within the living tissues of plants the endophytes since two decades after the discovery of taxol - producing endophytic fungus taxomyces andreanae from taxus brevifolia. all nonvascular and vascular plants examined until now have been found to harbor endophytic microbes with the potential to produce novel secondary metabolites. tropics being the areas of rich biodiversity provide unique biological niches for endophytes with great diversity. tabernaemontana is a genus comprising of 120 species of trees and shrubs of the oleander family, apocynaceae. alkaloids are the predominant phytochemicals found among members of the family with 256 alkaloids characterized from tabernaemontana alone. tabernaemontana heyneana wall. is a taxon endemic to the western ghats of peninsular india. the stem bark, bitter roots, flowers, and latex of fruits are used in folk medicine to treat diseases of skin and toothache and to reduce inflammation. the indole alkaloids and terpenoids isolated from the roots, stem bark, fruits, and leaves possess cytotoxic, anti - implantation, and antioxidant properties [68 ]. l - asparaginase is one such enzyme routinely employed in chemotherapy particularly for cancerous tumors of white blood cells. the enzyme deprives the cancer cells of an essential amino acid asparagine by catalyzing its breakdown ultimately leading to starvation and death. l - asparaginase is specific in its action and does not pose threat to the survival of normal cells. bacterial asparaginases are currently in use under different trade names such as elspar from escherichia coli and erwinase from erwinia chrysanthemi. l - asparaginase derived from eukaryotes may induce relatively least toxicity and feeble immune response [9, 10 ]. the production of asparaginases by fungal endophytes has been reported from plant species with anticancer potentials. since the anticancer activity has been related to the presence of the alkaloids in the plant parts of t. heyneana, the present study deals with the isolation of fungal endophytes and screening them for l - asparaginase activity. bark, twig, leaf, and fruit samples (n = 10) were collected from a healthy tree of t. heyneana wall., growing in the forests of kodagu district, in the talacauvery subcluster of the western ghats (12 17 to 12 27n and 75 26 to 75 33e), karnataka, during the month of january, 2012. bark samples were cut 1.5 m above ground level with a machete, swabbed in alcohol (70%, v / v). the plant samples were placed in separate polyethylene zip lock bags and stored at 4c. the samples were transported to the laboratory and processed within 48 h of collection. a herbarium specimen of the plant is deposited in the herbarium collection of the department. surface sterilization was performed by sequential immersion of samples in 70% ethanol for one min, 3.5% sodium hypochlorite (naocl) for three min, and rinsed three times in sterile distilled water to remove traces of sterilants. the efficacy of surface sterilization was checked by pouring aliquots of the water from final rinse solutions on water agar medium (wa, 2% agar) and incubated for five days. the samples were blotted dry under laminar air flow and cut into small segments of uniform size using sterile scalpel. 200 segments of bark, twig, leaf, fruit, and seeds were placed equidistantly on wa medium supplemented with streptomycin sulphate to inhibit the bacterial growth. the plates were wrapped using clean wrap cling film and incubated at 27c with 12 h light and 12 h dark regimes for six to eight weeks. colonies that emerged from tissue segments were transferred to antibiotic - free potato dextrose agar medium (pda) to enable identification. identification was based on morphological characters and conidial characters using standard identification manuals [1418 ]. the relative frequency of colonization (% cf) of endophytes was calculated as the number of isolates of taxon from each segment divided by the total number of segments plated 100. dominant endophytes expressed as percentage were calculated as percentage colonization frequency divided by sum of percentage colony frequencies of all endophytes 100. the isolation rate (ir) was calculated as the number of isolates obtained from the segments divided by the total number of segments plated and expressed as a fraction. the plate assay method of gulati. was adopted to screen fungal endophytes for l - asparaginase activity on modified czapek dox 's (mcd) agar medium (glucose2.0 g / l, l - asparagine10 g / l, potassium dihydrogen phosphate (kh2po4)1.52 g / l, potassium chloride (kcl)0.52 g / l, magnesium sulphate (mgso47h2o)0.52 phenol red indicator (0.009%) was prepared from a stock solution of 2.5% of the dye in ethanol. the control plates were prepared with mcd medium devoid of asparagine (instead containing kno30.001 g / l as the nitrogenous source) and phenol red indicator to check the ability of test fungi to grow in the medium. the mycelial plugs from four different fungi were inoculated on mcd agar medium marked into four quadrants. the colonies exhibiting pink zones were inoculated on mcd agar medium plates to confirm the activity of enzyme prior to estimation. the positive isolates were cultured in mcd broth medium incubated at 30c in orbital shaker (genei, bangalore) set at 120 rpm for five days. the reaction mixture containing 0.5 ml of 0.04 m l - asparagine, 0.5 ml of 0.5 m tris hcl buffer (ph 8.2), 0.5 ml of enzyme, and 0.5 ml distilled water was incubated at 27c for 30 min. 0.5 ml of 1.5 m trichloroacetic acid (tca) was added to each reaction tube to stop the reaction. 0.1 ml was drawn from the above reaction mixture tube to another tube to which 3.7 ml of distilled water and 0.2 ml of nessler 's reagent were added and incubated for 20 min. the optical density was read at 450 nm using uv - visible spectrophotometer (tpl technology pvt. ltd., one international unit (iu) of l - asparaginase is the amount of enzyme needed to liberate one mol / min of ammonia at 27c. a total of 727 isolates of endophytic fungi belonging to 20 taxa were obtained from the plating of 1000 tissue segments. the relative per cent isolation was highest for isolates of leaf samples (43%) and least for isolates of bark samples (11%). the per cent colonization frequency of endophytes differed for the plant parts used and is represented in table 1. many isolates belonged to the genera fusarium, phomopsis, and colletotrichum which colonized more than one plant part. were isolated from fruits only, while curvularia trifolii and wardomyces sp. were found to occur in seed samples and isolates of nectria sp. were obtained from twig samples only. the isolates of fusarium were recovered from bark, twig, and seeds with three species from bark alone. were found to occur as isolates of bark and twig while colletotrichum spp. were obtained from leaf and twig samples with more isolates being recovered from the former. the dominant genera of bark, twig, leaf, fruit, and seeds were recorded (figure 1). comprised the dominant endophytes of bark and seed samples while phomopsis was the dominant genus in twig and fruit samples. the isolation rate expressed in fraction for endophytes from different plant parts is represented in figure 2. the isolation rate for leaf endophytes was more than one, thus, implying that every leaf was colonized by at least one endophytic fungus. preliminary screening for the enzyme activity by plate assay revealed the enzyme producing ability of nine endophytes. a pink zone was observed around the colonies suggesting that endophytic fungi were able to utilize the substrate asparagine by secreting the enzyme asparaginase which catalyzes the breakdown of the substrate (figure 3). the reaction between the ammonia in the reaction mixture and nessler 's reagent was indicated by the formation of an orange colored solution. the enzyme activities were found to occur in the range of 0.0061.136 unit / ml (table 2). the isolates of f. graminearum from bark and twig exhibited high asparaginase activities of 0.950 iu and 0.836 iu and f. verticillioides showed highest activity among all the endophytic fungi with 1.136 iu of enzyme. the values of optical densities and enzyme activities shown by endophytes differed (table 2). research on endophytes in the past few decades has amounted to our understanding of their nature, interaction with host, and roles played by them in deterring insects, plant pathogens, and other environmental stress. the metabolites derived from endophytes have attracted researchers since their roles in medicine have been addressed. tropical trees have received less attention with regard to endophytic studies in comparison to temperate trees probably due to inaccessible locations. however, in recent years several attempts have been made to bioprospect endophytes of tropical trees and have yielded fruitful outcomes. thus, tropical regions known for the diversity of plant species also have the prospect of housing microbes with great diversity. in the current investigation t. heyneana, a tree endemic to the western ghats of southern india, colletotrichum and phomopsis are reported as endophytes in previous studies from the leaves of t. divaricata [24, 25 ]. these genera are multihost endophytes because they occur consistently in taxonomically diverse tropical tree species of different geographical areas [3, 26 ]. the reason for the frequent occurrence of colletotrichum and phomopsis as endophytes may be attributed to the slimy conidia they produce which are readily dispersed by water in the rain forests. the per cent colonization of endophytes was high in the leaf tissues in comparison to other plant parts. similar observations were made by kharwar. in catharanthus roseus where leaf segments were densely colonized with endophytes when compared to the root and stem segments. tissue specificity observed in this study complies with the results obtained in earlier studies [2729 ]. different tissues of trees rather than the same tissue from different tree species have higher diversity of endophytes. so far, 12 species of the family apocynaceae, namely, allamanda cathartica, alstonia scholaris, alyxia sinensis, catharanthus roseus, cerbera manghas, melodinus suaveolens, nerium oleander, plumeria acutifolia, strophanthus divaricatus, tabernaemontana divaricata, thevetia peruviana, and trachelospermum jasminoides, have been evaluated for fungal endophytes from foliar segments, phloem (c. roseus), and roots. the fungal endophytes of bark have been isolated from few woody plant species from the tropics such as azadirachta indica [30, 31 ], crataeva magna, terminalia arjuna, aegle marmelos, and prosopis cineraria. in the present study, bark, twigs, leaves, fruits, and seeds of t. heyneana were used for the study of endophytic colonization and differences among the isolates in plant parts suggested the occurrence of endophyte taxa in a single tree species. endophytes of t. heyneana examined for enzyme activity demonstrated their ability to metabolize the substrate, l - asparagine. few reports on studies of fungal endophytes for l - asparaginase activity are available [11, 36 ].. isolated from soil and marine algae have been reported to produce asparaginase in earlier studies [10, 37 ]. v. lecanii as endophytes with asparaginase activity has been reported in this study for the first time. though isolates of fusarium and colletotrichum showed pink zones in the agar plate assay, their enzyme activities were found to be low, as estimated by the spectrophotometric method. the reason for the absence of enzyme activity in the quantitative estimation may be attributed to the differences in ability of fungi to produce enzyme in solid and liquid states. the microbes are better sources of l - asparaginase because of the ease with which they can be cultured, extracted, and purified, also facilitating the industrial scale production. e. carotovora and e. coli are currently used as commercial sources of l - asparaginase. their administration induces immune responses ranging from mild to severe in patients suffering from acute lymphoblastic leukemia. t. heyneana, the endemic tree species of the western ghats, has potential medicinal benefits. endophytes of t. heyneana such as f. graminearum have been established as producers of l - asparaginase enzyme. further investigations involving the isolation and purification of enzyme followed by in vitro tests using cancer cell lines can throw light on the usefulness of the endophyte - derived l - asparaginases. | endophytes, the microbes residing within the plant tissues, are important sources of secondary metabolites. tabernaemontana heyneana wall., a medicinal tree, endemic to the western ghats with rich ethnobotanical history and unique chemical diversity, was selected to study fungal endophytes and evaluate them for l - asparaginase activity. healthy plant parts were selected for the isolation of endophytes following standard isolation protocols. a total of 727 isolates belonging to 20 taxa were obtained. the isolates comprised of bark (11%), twig (22%), leaf (43%), fruit (12.0%), and seeds (12%). endophytes such as colletotrichum, curvularia, fusarium, phomopsis, verticillium, and volutella colonized t. heyneana plant parts. fusarium sp., phomopsis spp., isolate thlf01, and fusarium solani were the dominant genera of bark, twig, leaf, fruits, and seed samples, respectively. the endophytes were screened for their ability to utilize l - asparagine by plate assay method. fusarium spp. exhibited a high level of activity among the nine endophytes tested positive for l - asparaginase activity. studies underline the potentials of endophyte - derived fungal l - asparaginases as sources of chemotherapeutic agents. |
railway accidents are one of the major concerns of worldwide traffic safety that impact global public health strategies. recent statistics reports from the european union show a slightly declining trend in train accidents observed since 2004, in all european states. nevertheless, in the eu-27 for 2011, romania was one of the three countries responsible for 48% of rail victims, after poland and germany, with 217 train accidents and 251 persons killed or seriously injured. analysis of the international data revealed that 83.6 % of victims were included in the category of other persons (e.g. : level - crossing users or unauthorised persons on railway premises). this is particularly relevant because psychological studies have documented the traumatic impact that this type of accident may have on railway personnel [210 ]. collisions resulting in death or serious injury of persons who fall or intentionally throw themselves in front of the moving train, are known in the literature as person - under - train (put) incidents. studies have found that accidents causing death or serious injury to other persons are as traumatic for the train driver as collisions between trains where the driver s own life is directly threatened. because of involuntary exposure to put incidents, the likelihood of train drivers to witness the violent death of a person is much higher than that of the general population, and that puts the train driver at risk of psychological trauma. moreover, psychological disability due to involvement in put accidents is likely to affect cognitive and affective functions that are essential for train drivers capacity to work and further ensure traffic safety. the most investigated forms of traumatic reactions are acute stress and posttraumatic stress response, which may develop into disorders. according to the current international standards for mental health (dsm - iv), posttraumatic stress disorder (ptsd) is defined by the following criteria : criterion a : the individual must have experienced a traumatic incident of great severity that caused him to feel intense fear, helplessness and horror. criterion b : the event is persistently re - experienced through intrusive memories, dreams, flashes etc. criterion c : the individual consistently avoids stimuli associated with the trauma and/or has numbed or significantly reduced responsiveness (avoidance). criterion d : the individual shows persistent symptoms of increased arousal, like sleep disturbance, or inability to concentrate, exaggerated startle response (hyperactivation). in exposed train drivers, the ptsd symptom prevalence is not very high, but its presence is constant across studies, stressing the need to manage this occupational hazard. in terms of ptsd comorbidity, depressive disorders are the most common dysfunctions associated with posttraumatic stress in train drivers. in a sample of romanian train drivers we found that exposure to put incidents is high and train drivers with put experiences report significantly more psychological symptoms when compared to train drivers without put incidents. to better understand variations in the prevalence of ptsd symptoms between train drivers exposed to put incidents, most relevant and pervasive vulnerability factors across studies are : a history of psychiatric problems, anterior trauma, current life stressful events, and certain features of the put incidents, for example, their severity or frequency of exposure. train drivers repeated confrontation with put incidents has been interpreted by researchers both as a vulnerability factor and as a factor that can provide inoculation and psychological resilience to such incidents. in our sample we identified that the frequency of exposure to put incidents was the only factor significantly related to ptsd symptoms that train drivers reported. it was interesting to note that train drivers who were involved in repeated put experiences over the years reported less ptsd symptoms, accounting for a habituation effect of repeated traumatic exposure. wanting to further investigate this result, we supplemented the original sample with a new set of data and then explored the differences regarding event particularities, subsequent ptsd and general health symptoms between younger train drivers, with few put experiences, and more experienced train drivers, that reported repeated exposure to put incidents over the years. the overall objective of our study was to assess differences in ptsd, general health symptoms and event particularities between train drivers that were at their first or second put experience, and train drivers that reported repeated put incidents. we obtained the approval for research objectives and procedure from the head of the railway regional department of cluj, traffic division that allowed us access to train drivers involved in traffic safety. we also accessed train drivers in two private companies involved in passenger transport on the railway. of the total 216 train drivers we first approached, 23 refused to participate, and 41 declared they never had a put incident. questionnaires and informed consents were completed individually. the final sample consisted of 193 train drivers (mean age = 38.42, sd = 9.64) currently working for the locomotive depots in cluj, dej, bistria and braov. of them, average professional experience in our sample was of 17.54 years (sd = 9.64). all participants completed a series of questionnaires, as follows : a questionnaire about demographic and circumstantial variables (the frequency of reported put incidents, time passed since the accident, details about the most severe incident, knowledge about standard procedures etc.). train drivers were instructed to think of the most severe incident and describe the symptoms they experienced in the weeks that followed. internal consistency for ies - r in the present sample was adequate (alpha cronbach = 0.75). ies - r is one of the most frequently used measures for traumatic impact of specified events. it has three subscales, corresponding to the three clusters of ptsd symptoms : re - experiencing of the traumatic events, avoidance and hyper arousal. ghq-28 is a measure of mental health screening, with good psychometric qualities (alpha cronbach = 0.70 in the present sample). it has four subscales : severe depression, anxiety and sleep disturbance, somatic symptoms and social dysfunctions. the final sample consisted of 193 train drivers (mean age = 38.42, sd = 9.64) currently working for the locomotive depots in cluj, dej, bistria and braov. of them, 29 (15%) belong to a private railway company. average professional experience in our sample was of 17.54 years (sd = 9.64). all participants completed a series of questionnaires, as follows : a questionnaire about demographic and circumstantial variables (the frequency of reported put incidents, time passed since the accident, details about the most severe incident, knowledge about standard procedures etc.). train drivers were instructed to think of the most severe incident and describe the symptoms they experienced in the weeks that followed. internal consistency for ies - r in the present sample was adequate (alpha cronbach = 0.75). ies - r is one of the most frequently used measures for traumatic impact of specified events. it has three subscales, corresponding to the three clusters of ptsd symptoms : re - experiencing of the traumatic events, avoidance and hyper arousal. ghq-28 is a measure of mental health screening, with good psychometric qualities (alpha cronbach = 0.70 in the present sample). it has four subscales : severe depression, anxiety and sleep disturbance, somatic symptoms and social dysfunctions. of the 193 train drivers, 152 (78.75%) reported at least one put incident. respondents reported as much as 14 put incidents / person, with a mean of 4 (sd = 2.83) incidents / train driver. as for time since the last put incident, the mean number of years reported from the last event was m = 3, sd = 3.67. the average age that the train drivers were when exposed to their first put incident, was 27 years (sd = 6.95). train drivers that did not report being exposed to put incidents were significantly younger (t = 3.5, df = 79, p=0.00) and had significantly less professional experience (t = 5.8, df = 79, p=0.00) then a randomly selected similar size sample of their colleagues with experienced put incidents. for further analysis of data, we divided the sample into four subgroups of train drivers, based on number of reported put incidents, as follows : first category (41 respondents - 21.2%) were non / exposed train drivers (0 put incidents), second category (38 respondents - 19.7%) were train drivers with a low exposure (1 or 2 reported put incidents), third category (65 respondents - 33.7%) consisted of train drivers with medium traumatic exposure (35 reported put incidents) and forth category (49 respondents - 25.4%) consisted of train drivers with a high frequency of reported put incidents (6 incidents or more). most of reported put incidents were accidents (44%) due to lack of attention on part of the victims walking on the railway, or car collisions at crossing levels, 37% were suicides and in 19% of cases, respondents did not know the cause of the accident. most of them involved passengers trains (80%), as opposed to freight trains or other types of railway vehicles. most of them (90 %) involved at least one injured person, and in 40 % of cases at least one person was killed because of the accident. most of the time, the train driver was alone on the locomotive (65%) and had to drive the train to destination immediately after the incident (72%). almost half of the sample saw the victim before and after the collision (45%), but in most cases they could nt offer medical assistance to the victims (86%). some of the train drivers reported sick leave days after the put incident (12%) and relying on more experienced train drivers, for discussing the event and its consequences (13%). to test for significant differences between categories of exposure to put incidents, with regard to contextual particularities, we calculated separate frequencies. we used a contingency chi - square test for finding out if differences were significant. results for each factor are synthesised in table i. we found no significant differences between categories of exposure to put incidents in regard to circumstantial factors. the impact of event scale - revised assesses three categories of symptoms for ptsd : intrusive thoughts (nightmares, flashbacks, the feeling of reliving the event), avoidance (emotional numbness, avoidance of feelings, sensations, ideas and traumatic context) and physiological state of hyper - arousal (irritability, hyper vigilance, difficulty concentrating, exaggerated startle reaction), in correspondence to dsm iv diagnostic criteria for ptsd. for establishing clinical significance of ptsd symptoms, we followed the recommended procedure, using the cut - off value of 33 (on the ies - r scale). of the 152 drivers who reported being involved in put incidents, 142 (93.4%) had scores below the threshold value set for the ies - r. also, 13 (8.6 %) train drivers reported that they did nt experience any specific ptsd symptoms related to experienced put incidents. 6.6% of the participants reported ptsd symptoms over the cut - off score, that ranged up to 44, which is still a low score value when considering a diagnosis of ptsd. to assess the impact of the frequency of put incidents on specific ptsd symptoms, we calculated, using one - way anova test, significance of differences between the three categories of exposed train drivers. the effect of frequency of put incidents on specific ptsd symptoms was significant (f(2,149) = 5.14, p=0.00). the average ptsd symptoms that train drivers in the low frequency category reported was higher than the average number of symptoms reported by train drivers who were repeatedly exposed to put incidents. levene s test for homogeneity of variances was not statistically significant, and we worked with unequal sample sizes, so we used hochberg gt2 correction to assess significance of differences between our three categories of train drivers. the symptom difference between the subgroup of train drivers at their first or second put incident and the third group that reported at least six incidents was significant (hochberg gt2 = 6.57, p=0.00). both differences between first subgroup of train drivers and the two repeatedly exposed categories are significant (subgroup (1, 2) hochberg gt2 = 2.23, p=0.03 ; subgroup (2, 3) hochberg gt2 = 2.91, p=0.00). results stand as evidence that the repeated exposure to these traumatic incidents determines a habituation effect on the train drivers, reducing reports of ptsd symptoms. train drivers also completed the ghq-28 questionnaire, used as an indicator of general health and detection of psychological symptoms. results show that they report low levels of depression, anxiety, somatic discomfort and social dysfunctions, with all of the subscale and total scores below recommended cut - off points for identification of mental health disorders. in terms of the relationship with specific symptoms of posttraumatic stress, anxiety symptoms (r=0.35, p=0.01) and somatic symptoms (r=0.18, p=0.05) significantly correlated with the overall score of ies - r. to highlight the impact of repeated traumatic put incidents on train drivers, we investigated the differences between general symptoms reported by the three categories of train drivers. there were no statistically significant differences between general symptoms reported by the three categories of respondents (f(2,149) = 0.86, p=0.42). to assess differences in general health between train drivers with and without ptsd symptoms, we divided the sample using mean ies - r score as cut - off point. using independent samples t test, we identified a significant difference in general health symptoms between train drivers that reported above average ptsd symptoms (m ghq = 13.48, sd = 3.48) and train drivers with below average ptsd symptoms (m ghq = 11.74, sd = 3.92). thus train drivers that reported higher than average ptsd symptoms also struggled with significantly more symptoms of depression, anxiety, sleep disturbances, social dysfunction and somatic symptoms (t = 2.96, df = 150, p=0.00). of the 193 train drivers, 152 (78.75%) reported at least one put incident. respondents reported as much as 14 put incidents / person, with a mean of 4 (sd = 2.83) incidents / train driver. as for time since the last put incident, the mean number of years reported from the last event was m = 3, sd = 3.67. the average age that the train drivers were when exposed to their first put incident, was 27 years (sd = 6.95). train drivers that did not report being exposed to put incidents were significantly younger (t = 3.5, df = 79, p=0.00) and had significantly less professional experience (t = 5.8, df = 79, p=0.00) then a randomly selected similar size sample of their colleagues with experienced put incidents. for further analysis of data, we divided the sample into four subgroups of train drivers, based on number of reported put incidents, as follows : first category (41 respondents - 21.2%) were non / exposed train drivers (0 put incidents), second category (38 respondents - 19.7%) were train drivers with a low exposure (1 or 2 reported put incidents), third category (65 respondents - 33.7%) consisted of train drivers with medium traumatic exposure (35 reported put incidents) and forth category (49 respondents - 25.4%) consisted of train drivers with a high frequency of reported put incidents (6 incidents or more). most of reported put incidents were accidents (44%) due to lack of attention on part of the victims walking on the railway, or car collisions at crossing levels, 37% were suicides and in 19% of cases, respondents did not know the cause of the accident. most of them involved passengers trains (80%), as opposed to freight trains or other types of railway vehicles. most of them (90 %) involved at least one injured person, and in 40 % of cases at least one person was killed because of the accident. most of the time, the train driver was alone on the locomotive (65%) and had to drive the train to destination immediately after the incident (72%). almost half of the sample saw the victim before and after the collision (45%), but in most cases they could nt offer medical assistance to the victims (86%). some of the train drivers reported sick leave days after the put incident (12%) and relying on more experienced train drivers, for discussing the event and its consequences (13%). to test for significant differences between categories of exposure to put incidents, with regard to contextual particularities, we calculated separate frequencies. we used a contingency chi - square test for finding out if differences were significant. results for each factor are synthesised in table i. we found no significant differences between categories of exposure to put incidents in regard to circumstantial factors. the impact of event scale - revised assesses three categories of symptoms for ptsd : intrusive thoughts (nightmares, flashbacks, the feeling of reliving the event), avoidance (emotional numbness, avoidance of feelings, sensations, ideas and traumatic context) and physiological state of hyper - arousal (irritability, hyper vigilance, difficulty concentrating, exaggerated startle reaction), in correspondence to dsm iv diagnostic criteria for ptsd. for establishing clinical significance of ptsd symptoms, we followed the recommended procedure, using the cut - off value of 33 (on the ies - r scale). of the 152 drivers who reported being involved in put incidents, 142 (93.4%) had scores below the threshold value set for the ies - r. also, 13 (8.6 %) train drivers reported that they did nt experience any specific ptsd symptoms related to experienced put incidents. 6.6% of the participants reported ptsd symptoms over the cut - off score, that ranged up to 44, which is still a low score value when considering a diagnosis of ptsd. to assess the impact of the frequency of put incidents on specific ptsd symptoms, we calculated, using one - way anova test, significance of differences between the three categories of exposed train drivers. the effect of frequency of put incidents on specific ptsd symptoms was significant (f(2,149) = 5.14, p=0.00). the average ptsd symptoms that train drivers in the low frequency category reported was higher than the average number of symptoms reported by train drivers who were repeatedly exposed to put incidents. levene s test for homogeneity of variances was not statistically significant, and we worked with unequal sample sizes, so we used hochberg gt2 correction to assess significance of differences between our three categories of train drivers. the symptom difference between the subgroup of train drivers at their first or second put incident and the third group that reported at least six incidents was significant (hochberg gt2 = 6.57, p=0.00). both differences between first subgroup of train drivers and the two repeatedly exposed categories are significant (subgroup (1, 2) hochberg gt2 = 2.23, p=0.03 ; subgroup (2, 3) hochberg gt2 = 2.91, p=0.00). results stand as evidence that the repeated exposure to these traumatic incidents determines a habituation effect on the train drivers, reducing reports of ptsd symptoms. train drivers also completed the ghq-28 questionnaire, used as an indicator of general health and detection of psychological symptoms. results show that they report low levels of depression, anxiety, somatic discomfort and social dysfunctions, with all of the subscale and total scores below recommended cut - off points for identification of mental health disorders. in terms of the relationship with specific symptoms of posttraumatic stress, anxiety symptoms (r=0.35, p=0.01) and somatic symptoms (r=0.18, p=0.05) significantly correlated with the overall score of ies - r. to highlight the impact of repeated traumatic put incidents on train drivers, we investigated the differences between general symptoms reported by the three categories of train drivers. there were no statistically significant differences between general symptoms reported by the three categories of respondents (f(2,149) = 0.86, p=0.42). to assess differences in general health between train drivers with and without ptsd symptoms, we divided the sample using mean ies - r score as cut - off point. using independent samples t test, we identified a significant difference in general health symptoms between train drivers that reported above average ptsd symptoms (m ghq = 13.48, sd = 3.48) and train drivers with below average ptsd symptoms (m ghq = 11.74, sd = 3.92). thus train drivers that reported higher than average ptsd symptoms also struggled with significantly more symptoms of depression, anxiety, sleep disturbances, social dysfunction and somatic symptoms (t = 2.96, df = 150, p=0.00). one of the main sources of psychological distress faced by rail transport personnel, are put incidents. results of the present study reflect the same tendency as statistical reports of train accidents, showing that romanian train drivers are exposed to a significant number of put incidents. our results are comparable to the findings of studies from other european countries, and tend to display a low, but constant prevalence of ptsd symptoms for train drivers involved in put incidents [26,8 ]. according to circumstantial risk factors identified in studies on train drivers, we previously evaluated the relationship between age, professional experience, frequency and time since the put incident and ptsd symptoms reported. we found that neither age nor professional experience of the train driver, not even time passed since the put incident, were correlated with the intensity of ptsd symptoms. the only significant association we found was between the frequency of put incidents and the intensity of reported ptsd symptoms. thinking that this negative relationship between the two variables may be evidence that over time, train drivers may develop the ability to reduce their reactivity to these incidents we wanted to further investigate differences between train drivers with fewer put experiences and train drivers with average or high level of traumatic job exposure. results confirmed that higher frequency of exposure was associated with lower levels of posttraumatic stress symptoms. authors identified age to be the only factor in a wider range of individual and circumstantial variables that was significantly negatively associated with posttraumatic distress levels following put incidents. because the train drivers mainly reported subclinical ptsd symptoms that were associated with other types of anxiety or somatic symptoms, further research should consider other dimensions of the traumatic impact of such incidents. post - incident reactions can be very different : from the transient state of shock, occurring immediately after the put incident to long - term psychological impairment. new research efforts should be oriented to double the evaluation of ptsd symptoms with the assessment of more subtle changes in neuro - physiological functioning of the train drivers involved in put incidents. results on circumstantial variables as sources of influence, determining the level of psychological distress after the put experience were not all relevant. we suggest that this puzzling finding, which contradicts some of the anterior studies, needs to be more fully assessed. differing from other areas, most accident situations are quite uniform (mostly suicides ; the driver can neither anticipate nor prevent the accident) and the group of train drivers is rather homogenous regarding socio - demographic variables. these circumstances present an opportunity to examine the role of individual disposition with regard to the aetiology of posttraumatic syndromes. it is interesting that no relationship was found between time passed since the accident and specific ptsd symptoms. also, we found interesting results regarding the influence of multiple traumatic experiences, which is associated with reductions in the intensity of reported ptsd symptoms. it would be useful to evaluate how immunization takes place, what are the factors that influence it, and which are the most effective cognitive and emotional coping mechanisms that favour it. our results provide empirical support for the need to address put incidents as occupational hazards for train drivers, putting them at risk for struggling with specific posttraumatic and more general health symptoms. however, we must take into account the limitations of the study, due to the retrospective methods of data collection and exclusively basing our findings on the train drivers subjective reports. empirical evidence of risk and resilience factors relevant for exposed train drivers is highly needed because of the frequency of put incidents and their traumatic potential. identifying the individual and organisational factors that influence the posttraumatic reactions of train drivers will be essential to substantiate effective methods of primary and secondary prevention and intervention procedures when dealing with effects of put incidents. our findings highlight that the frequency of traumatic exposure represents an essential factor that needs to be taken into consideration when identifying particular risk categories of train drivers. first experiences of put incidents seem to have more traumatic impact then repeated ones, establishing newly exposed train drivers as a main category of traumatic risk, and certifying tailored prevention and intervention strategies. | aimsinternational research highlights the occupational risk of train drivers of being exposed to work related traumatic incidents and subsequently developing posttraumatic symptoms or other comorbid dysfunctions.participants and methodsthe article focuses on investigating the effects of repeated traumatic exposure on posttraumatic reactions in a sample of 193 romanian train drivers. we used the impact of event scale - revised (ies - r) to retrospectively evaluate symptoms of posttraumatic stress disorder (ptsd), the general health questionnaire to investigate related mental health symptoms, and a demographic questionnaire to assess contextual factors like frequency or severity of exposure.resultssample reports of exposure to put (person under train) incidents were high. an interesting finding was that train drivers exposed to just one or two put incidents reported significantly more posttraumatic symptoms than train drivers with more put experiences, accounting for a habituation effect of repeated traumatic exposure.conclusionsreported posttraumatic reactions to put incidents and influencing factors provide evidence recommending systematic screening of train drivers for posttraumatic symptoms, general emotional distress and further elaborating prevention and treatment strategies for specific risk categories of these professionals. |
sarcoidosis is a systemic granulomatous disease of unknown origin, characterized by the presence of noncaseating granuloma in affected organs (1). lesions are commonly seen in the lungs, lymphatic system, eyes, skin, liver, spleen, salivary glands, heart, nervous system, muscles, and bones (1, 2). although neurosarcoidosis is a rare manifestation of sarcoidosis, the clinical symptoms can be devastating and occasionally life - threatening. the diagnosis of neurosarcoidosis can be challenging because the disease can present with a lot of symptoms and diverse radiologic findings (1 - 5). among them, spinal sarcoidosis is a very rare entity, occurring in < 1%, and can be manifested as intramedullary, intradural extramedullary, intraspinal epidural spaces and in vertebral bodies (6). in korea, only a few cases of neurosarcoidosis involving brain, spinal nerve root, peripheral nerve and spinal dura, have been reported with or without histological confirmation in the literature. patients with spinal sarcoidosis are considered to be at high risk for severe neurological sequelae (7). the information available about spinal sarcoidosis management and diagnosis comes from a few case reports, small series and expert opinions (8 - 10). however, the documents gave conflicting conclusions regarding the treatment, including corticosteroids and alternative immunosuppressants (6, 7). we recently experienced a patient with isolated spinal cord sarcoidosis, which was confirmed by tissue biopsy and well responsive to high - dose corticosteroid and immunosuppressant. a 54-yr - old woman, with no significant past medical history, presented with progressive 4-extremities weakness and sensory changes, followed by urinary difficulty since 1 month ago and was admitted to our hospital in october 2007. physical examination revealed increased deep tendon reflex, positive babinski sign and decreased motor power with medical research council (mrc) grade 2 strength in the right side and mrc grade 4 strength in the left side. initial magnetic resonance imaging (mri) revealed increased t2 signal from c4 to c6 level, edematous expansion of the cord and intense nodular enhancement (fig. 1a). based on the mri, spinal cord tumor, demyelinating disease involving multiple sclerosis, serological studies for systemic autoimmunity, including rheumatoid factor and antinuclear, anti - dsdna, anti - ssa / ssb, and antiphospholipid antibodies, showed no abnormality. the cerebrospinal fluid (csf) analysis including biochemistry, igg index and oligoclonal band was normal. one month later, the follow - up spinal mri showed the more extended lesion relative to previous mri, and which highly suggested the spinal cord tumor such as intramedullary astrocytoma (fig. based on the suspicion of intramedullary astrocytoma, the laminectomy and tissue biopsy of 2 l, which showed yellowish color, were performed at the central portion of dorsal column in affected cervical cord lesion. postoperatively, patient 's neurologic deficits were not aggravated and, unexpectedly, the histology of biopsy revealed non - caseating granuloma without malignant cell (fig. angiotesin converting enzyme (ace) was mildly elevated to 56 (u / l ; normal,, there was no disease activity on other organs with using brain mri, chest and abdominal computed tomography and nerve conduction study (ncs). the patient was treated with high - dose corticosteroid (60 mg / day) followed by methotrexate (10 mg / week) for over 2 yr. two years later, cervical spinal cord lesion was much improved in the spinal mri (fig. 1d), however clinical symptoms of weakness and sensory change were not definitely improved. recently, the patient had to stop corticosteroid medication because of the cellulitis in the left leg. instead, we tried thalidomide 100 mg daily for 1 month with the goal of titrating up to 400 mg per day, which had been reported to be effective in the refractory neurosarcoidosis (11). however the patient refused high - dose thalidomide due to severe fatigue and high cost, and just treated with low - dose thalidomide (100 mg / day) and treatment did not show distinct effect in our patient. the spinal cord sarcoidosis is very rare and the diagnosis of this entity is difficult, as there are no pathognomic diagnostic study for neurosarcoidosis (3, 12, 13). in general, this disease has been diagnosed clinically using mri, lumbar puncture and attendant laboratory tests, and the diagnosis is possible when systemic sarcoidosis is detected in other organs involving lung, kidney, eye and skin. spinal cord sarcoidosis may present as an idiopathic inflammatory demyelinating disease both clinically and radiologically (5). only a positive biopsy of suspicious lesions in the central nervous system is considered to be definitive confirmation of the diagnosis of isolated neurosarcoidosis. zajicek and colleagues established a diagnostic classification system for neurosarcoidosis that distinguished ' definite ', ' probable ' and ' possible ' neurosarcoidosis based on tissue evidence of non - caseating granuloma and supportive evidence of sarcoid pathology in laboratory and imaging studies (13). however, biopsy should be cautiously considered if possible because of the risk involved in approaching the spinal cord. our patient was initially suspected to have intramedullary astrocytoma or multiple sclerosis based on neurologic symptoms and mri results. of course, the possibility of multiple sclerosis was not high because the clinical symptoms and cervical lesion were gradually progressive for months. the spinal cord biopsy was challenging procedure, nevertheless the cord biopsy and laminectomy were performed for diagnostic confirmation in the consent of patient, and the histology of which was unexpectedly consistent with sarcoidosis. the histology revealed multinucleated giant cells, lymphocytes and aggregated histiocytes within granulomatous inflammation, consistent with non - caseating granuloma seen in sarcoidosis. a few cases with neurosarcoidosis have been reported, however most cases suggested only clinical manifestation without histology, or demonstrated indirect biopsy in the brain, meninges and lymph node (6 - 10). with an initial treatment, we tried high dose corticosteroid and immunosuppressant. systemic corticosteroids and immunosuppressive therapy are the treatment of choice for neurosarcoidosis, however which shows partial response especially in the spinal cord neurosarcoidosis (4, 6 - 10). unfortunately, 25% of neurosarcoidosis still have a refractory course with steroid treatment and, and 20%-40% of those refractory patients will not respond to current conventional immunosuppressant (3). in our case, the clinical symptoms were initially severe and rapidly progressive, and therefore the combined therapy of high - dose corticosteroid and immunosuppressant was necessary. combination with immunosuppressant was useful to improve symptom control and reduce corticosteroid - related side effects in some cases. therefore, making a diagnosis of neurosarcoidosis was therapeutically essential, since corticosteroid and immunosuppressant treatment must be started and continued for years in order to prevent progression and permanent disability. a few reports have documented that the high - dose thalidomide (400 mg / day) was effective in the refractory neurosarcoidosis (11), and so we tried low - dose thalidomide (100 mg / day) for one month in our patient. however there was no clear evidence that thalidomide was effective in our case of neurosarcoidosis, although there was limitation on the dosage and duration of treatment compared with other cases. our case indicates that diagnosis of spinal cord sarcoidosis is not easy and may require histological examination, and high - dose corticosteroid and immunosuppressant will be a good choice in the treatment of spinal cord sarcoidosis, and the thalidomide has to be debated in the treatment of spinal cord sarcoidosis. this is the first korean case, to our knowledge, which shows an isolated spinal cord sarcoidosis confirmed by direct tissue biopsy and good response to corticosteroid and immunosuppressant. | we report a case of 54-yr - old woman who presented with 4-extremities weakness and sensory changes, followed by cervical spinal cord lesion in magnetic resonance imaging. based on the suspicion of spinal tumor, spinal cord biopsy was performed, and the histology revealed multinucleated giant cells, lymphocytes and aggregated histiocytes within granulomatous inflammation, consistent with non - caseating granuloma seen in sarcoidosis. the patient was treated with corticosteroid, immunosuppressant and thalidomide for years. our case indicates that diagnosis of spinal cord sarcoidosis is challenging and may require histological examination, and high - dose corticosteroid and immunosuppressant will be a good choice in the treatment of spinal cord sarcoidosis, and the thalidomide has to be debated in the spinal cord sarcoidosis. |
deep vein thrombosis (dvt) and pulmonary embolism is a serious yet preventable cause of postoperative morbidity and mortality. it is estimated that 20 million cases of lower extremity dvt occur in the usa alone and account for the vast majority of pulmonary embolism each year.1 major orthopedic surgeries of the lower limbs are a high risk for the development of dvt and its incidence has been reported to range from 6 to 75%.24 various measures available for dvt prophylaxis, include early mobilization and physiotherapy, pharmacological means [unfractionated heparin, low molecular weight heparin (lmwh), dextran 40 and 70, warfarin, aspirin ], and mechanical means (mechanical calf muscle stimulation, thigh - high anti - embolic stockings, pneumatic calf muscle compression, electrical stimulation of calf muscle). it has been reported that venous return may be reduced to half after administration of anesthesia, which may even reduce to one - seventh of its preoperative rate.5 this venous stasis, especially of the soleal veins, during the surgery may be an important factor in the development of dvt. the role of peroperative electrostimulation of calf muscles with modern - day handheld electrostimulation device in the prevention of dvt remain sparse in literature.57 we prospectively sought to determine the efficacy of prophylaxis against dvt with peroperative electrostimulation of calf muscles in a consecutive case this was a prospective randomized study, which was carried out between november 2008 and february 2011. the present study included 200 consecutive patients who sustained trauma around the hip joint and underwent surgeries under spinal anesthesia. patients > 25 years requiring surgeries around the hip joint who underwent surgery within 2 weeks of sustaining trauma, and were operated under spinal anesthesia were enrolled. established cases of dvt, or patients taking antithrombotic medication, patients who sustained open fractures, and patients on pacemakers were excluded from the study. patients with other serious life - threatening conditions, pathological fractures, and associated vascular injuries were also excluded from the study. the patients were randomized into two groups of 100 patients each by odd even number, with even numbered patients considered as cases for peroperative calf muscle electrostimulation (group a) and odd numbered ones considered as controls (group b). boston, ma, usa) for all the patients on both the lower limbs using linear probe (5.3812 mhz) a day prior to the surgery to rule out pre - existing dvt, thereby excluding any patient with clinical or subclinical dvt as a result of trauma. first group of patients received thromboprophylaxis using the venioplus (ad rem technology, france) stimulator device for electrostimulation of the calf muscles during surgery was stimulated given to both calf muscles whereas the other group of patients (group b) did not receive any sort of thromboprophylaxis. the stimulator device delivered the low voltage (peak value being usually around 1525 v) and small energy impulses (below 25 c per impulse) to calf muscles. a record was maintained about the involved side, the type of surgery, and the position during surgery for both the groups. all the patients were mobilized in bed as tolerated postoperatively, and static and dynamic exercises were started on the next day after the surgery. it was ensured that none of the patients was immobilized in bed for more than 3 days after the surgery. all the patients were examined daily for clinical signs of dvt like diffuse swelling of the leg and foot, calf tenderness (moses sign) and homan 's sign (pain on passive dorsiflexion of foot). another doppler ultrasound was performed on the 7 postoperative day on the same machine for all the patients on bilateral lower limbs to look for dvt. the doppler assessment included examination of bilateral common femoral, superficial femoral, popliteal, anterior tibial, and posterior tibial veins. a diagnosis of dvt was made when there was visualization of thrombosis, absence of flow, and lack of compressibility or augmentation. the thrombi were classified to be proximal if they were found in popliteal vein or more proximal locations, and distal if they involved tibial or calf muscle veins. the patients having evidence of dvt on doppler examination were treated according to the american college of chest physicians guidelines.8 all the statistical analyses were performed on statistical package for social sciences (spss inc., version 16.0, chicago, il, usa) for windows. fisher 's exact test was used to examine the significance of association (contingency) between the two kinds of interventions. it was referenced for two - tailed p value and 95% confidence interval was constructed around sensitivity proportions using normal approximation method. all the statistical analyses were performed on statistical package for social sciences (spss inc., version 16.0, chicago, il, usa) for windows. fisher 's exact test was used to examine the significance of association (contingency) between the two kinds of interventions. it was referenced for two - tailed p value and 95% confidence interval was constructed around sensitivity proportions using normal approximation method. the mean age of the patients was 54.3 years (19 - 82 years), whereas it was 55.3 (19 - 82 years and 53.3 years (22 - 80 years), respectively, in case and control groups, and the difference was found to be statistically insignificant (p>0.05). the various traumatic conditions for which the patients were operated upon and surgeries are summarized in table 1. majority of the surgeries (66% cases) were conducted in supine position, whereas 34% (n=68) patients were operated in lateral position. various surgical procedures done along with patients diagnoses eight patients (two among group a and six among group b) were diagnosed to have dvt on color doppler ultrasound study on the 7 postoperative day, but the difference was not found to be statistically significant (p=0.279). none of these patients had any clinical signs or symptoms of dvt or pulmonary embolism. among them, four patients were males (two each among group a and b) and another four were females (both in the group b). six patients (four among group b and two among group a) were above 50 years (p<0.05), while two patients (both in group b) were below 50 years of age. four patients were operated in supine position with traction (two each among group a and b), while another four were operated in lateral position (all among group b). four of them (two each among group a and b) were operated with dynamic hip screw ; two underwent hemi - replacement hip arthroplasty, one closed reduction and internal fixation of neck of femur fracture, and another open reduction and internal fixation of acetabulum. all the patients had distal venous thrombus formation involving only the intermuscular (soleal) veins without any proximal migration, which resolved with anticoagulant therapy given in form of low molecular weight heparin (dalteparin) 5000 u subcutaneously twice a day for 7 days along with oral warfarin 5 mg daily for 6 weeks. doppler ultrasonography was done at the end of 6 weeks to confirm resolution of the thrombus. the incidence of dvt in patients undergoing various surgeries around the hip joint are reported to have wide variation, especially in indian scenario.4912 the distribution of lower limb dvt in indian patients is not exactly known and any specific approach to its prevention remains a dilemma. as a consequence most of the thrombi in this population have been reported to be distal, which resolve spontaneously without any long - term consequences.341213 contrast venography has usually been considered as a diagnostic modality of choice for detection of dvt and concerns have been raised on duplex ultrasonography yielding indeterminate studies.14 though nonfilling of contrast in deep veins on venography has been accepted as an indirect sign of dvt, bjrgell. (2000) showed that isolated nonfilling of posterior tibial and deep muscle veins of the calf seen on venography can equally be caused by other pathological conditions like edema, bleeding, ligaments and muscle rupture, baker 's cyst, and superficial thrombophlebitis, or arises without any detectable explanation, thereby leading to an exaggerated number of patients with thrombosis in these studies.15 nowadays, duplex ultrasonography has become the diagnostic standard in most of the hospitals in the united states, making it more representative of real - time medical management.16 the compression ultrasonic technique had an accuracy of 97%, a sensitivity of 100%, and a specificity of 97%. it appears to be an effective technique for diagnosing venous thrombosis, and is safe, well accepted by both patients and staff, can be performed quickly, and carries no inherent risks of the procedure itself owing to its non - invasive nature. it can also be easily repeated, thus making it suitable for monitoring high - risk patients.17 dhillon.3 and piovella.18 reported high incidence of dvt by venography in asian patients undergoing lower limb surgeries without prophylaxis. on the other hand, jain.,10 bagariaetal.,11 mavalankar.,12 atichartakarn.,19 and.20 reported a low incidence of dvt by venography in asian patients undergoing hip surgeries. the results of our study seem to be in tune with the observations of manystudies demonstrating lower incidences of dvtin these patients [tables 2 and 3].10121920 studies valuating dvt during major orthopedic surgeries of lower limbs involving patients of indian subcontinent various studies conducted worldwide on the evaluation of dvt during major orthopedic surgeries of lower limbs we did not find any correlation between the presence of clinical signs of dvt and doppler ultrasound findings in our study as all the eight patients, who demonstrated dvt on ultra sound, did not show any clinical feature of the same. this further confirms the unreliability of physical signs in the diagnosis of dvt, as shown by stulberg.2 all the patients included in our study had surgery performed under spinal anesthesia. it has been suggested that patients undergoing surgery in general anesthesia are 50% more likely to develop dvt than those under spinal anesthesia as vasodilatation that accompanies spinal anesthesia increases the blood flow to lower limbs, thus inhibiting stasis and hypercoagulability.12 effect of limb position and manipulation during surgery causing femoral vein occlusion is well studied in total hip replacement.21 it has been suggested that surgeries in lateral position with hip in flexion, adduction, and internal rotation may lead to femoral vein kinking which may further reduce the venous flow return from the lower limb. however, our study did not show any significant relation between the position during surgery and the occurrence of dvt. lmwh is currently recommended as the preferred agent for thromboprophylaxis,8 but it increases the treatment cost and has risk of bleeding complications and in turn, lead to infection, re - operation1016 to obviate these potential complications, non - pharmacological methods have been considered. since stasis is considered as a major factor in the development of dvt, efforts have been made to increase the venous blood flow in the deep veins during and after surgical interventions. mechanical compression devices have been considered to prevent clot formation by increasing the venous blood flow from the legs and causing the release of endothelial - derived relaxing factors and urokinase,2223 though these devices may have poor patient compliance, incompatibility in patients with lower limb injuries, and ineffectiveness in preventing pelvic thrombi.24 doran. demonstrated that out of the three factors involved in causation of dvt venous stasis, hypercoagulability, and endothelial damage (virchow 's triad) peroperative venous stasis appears to be the most important factor in causing dvt postoperatively, and thus calf muscle stimulation during the operation prevented the reduction in venous flow velocity in legs and reduced the risk of postoperative dvt. also, the biochemical stresses in the form of metabolites produced during the surgery in the operated limb add to the risk of dvt in the limb undergoing the procedure as compared to the opposite nonoperated limb.5 lindstrom. studied the effects of peroperative calf muscle stimulation in patients undergoing major abdominal surgeries with groups of impulses producing a short lasting tetany of calf muscles. it had similar effects on the incidence of postoperative thrombosis as compared with that of dextran 40.6 doran. also demonstrated that during lower limb surgery, the velocity of venous return reduces to a level that dvt can develop in 50% of patients and it can be effectively counteracted by calf muscle electrostimulation whilst the operation is in progress.5 we have used hand held veinoplus stimulator device which delivers electrical impulses into a vicinity of motor points on the muscle via skin patch electrodes in order to squeeze the blood from deep veins of calf. it produces a train of impulses with rectangular voltage waveform when connected to the electrodes. the waveform of current of every impulse is symmetrical and exponential biphasic during the treatment, thus causing powerful and almost symmetrical calf muscle contractions on each side.7 considering the small sample size of our study, it may not be very prudent to draw firm conclusions on the usefulness of calf muscle electrostimulation device (venioplus) in the prevention of dvt following orthopedic surgeries around the hip joint in patients having no additional risk factors. multicentric studies with larger sample size may evaluate therapeutic benefits of this device in lower limb surgeries. | background : the venous stasis of soleal vein during surgery may be an important factor in the development of deep vein thrombosis (dvt). the stimulation of calf muscle during surgery may help in preventing dvt. the present study is conducted to evaluate the role of peroperative calf muscle electrostimulation in prevention of dvt in patients undergoing surgeries around the hip joint.materials and methods : the study comprised 200 patients undergoing surgeries around the hip joint. the patients having risk factors (such as previous myocardial infarction, malignancies, paraplegia or lower limb monoplegia, previous history of dvt or varicose veins, etc.) for the development of dvt were excluded. they were randomized into two groups : 100 cases were given peroperative calf muscle electrostimulation for dvt prophylaxis (group a) and the remaining 100 patients were taken as controls without any prophylaxis (group b). the color doppler ultrasound was performed to exclude pre - existing dvt and on 7th day postoperative to find out the incidence of dvt in both the groups.results:two patients among group a and six patients among group b demonstrated dvt on ultrasonography, but the difference was not found to be statistically significant (p=0.279). none of the patients had any clinical evidence of dvt.conclusion:the role of peroperative calf muscle electrostimulation for dvt prophylaxis remains controversial. the risk of developing dvt in patients undergoing surgeries around the hip joint is very less in patients analysed in our series. |
micrornas (mirnas) are short noncoding regulatory molecules, involved in diverse biological processes. biogenesis of mirnas involve a nuclear phase, where the microprocessor complex, comprising drosha, an rnase iii - like enzyme and its cofactor dgcr8, process primary mirnas (pri - mirnas) into a 70 nt pre - mirna (han., 2004 ; this occurs cotranscriptionally from both independently transcribed and intron - encoded mirnas (ballarino., 2009 ; following drosha - mediated rna cleavage and pre - mirna release from the nascent rna, 5 and 3 nascent rna ends are trimmed by 5-3 xrn2 and 3-5 exosome (morlando., 2008), and the pre - mirna precursor is exported to the cytoplasm (lund., 2004 ; yi., 2003). here, a second rnase iii enzyme, dicer, further processes the pre - mirna into the mature mirna duplex (bernstein., 2001) that targets specific mrnas for degradation or translational inactivation (reviewed in bartel, 2009). mirna levels are tightly regulated at the posttranscriptional level by a number of rna - binding proteins (siomi and siomi, 2010). furthermore, drosha can directly regulate levels of microprocessor complex by cleaving hairpin structures in dgcr8 mrna, thereby decreasing dgcr8 protein levels (han. 2009). along the same lines, drosha knockdown in drosophila leads to upregulation of some mrnas containing conserved rna hairpins, potentially recognized by the microprocessor complex (kadener., 2009). several recent studies demonstrated the ability of microprocessor complex to cleave mrnas, thus regulating their expression. many drosha - dependent mrna cleavage events were identified in mescs, consistent with microprocessor regulation of coding mrnas through direct cleavage (karginov., 2010). drosha can also cleave the tar hairpin of the hiv-1 transcript, resulting in premature termination of rna polymerase ii (pol ii) (wagschal., a recent dgcr8 hits - clip analysis extended these observations and revealed general noncanonical functions of the microprocessor complex (macias., 2012). transcriptome and proteome studies of mice lacking drosha and dicer suggest that both enzymes have nonredundant functions, as their deficiency can induce different phenotypes (chong., 2010). although many rnas were stabilized by drosha depletion, some were downregulated, consistent with drosha possessing independent functions to its role in canonical mirna biogenesis. in human cells drosha exists in two distinct multiprotein complexes (gregory., 2004). the smaller complex, containing just drosha and dgcr8, is necessary and sufficient for mirna processing. the larger complex, displaying only weak pre - mirna processing activity in vitro, contains dead - box rna helicases, double - stranded rna - binding proteins, hnrnp proteins, members of fus / tls family of proteins, and the snip1 protein, implying additional functions in gene expression. thus, dead box helicases p68/p72 increase drosha processing efficiency for a subset of mirnas and at gene - specific promoters interact with transcriptional coactivators and pol ii and regulate alternative splicing (fuller - pace and ali, 2008). nuclear scaffolding protein hnrnpu and members of fus / tls family are also associated with regulation of transcription (wang., 2008). snip1, a component of a large snip1/skip - associated complex, involved in transcriptional regulation and cotranscriptional processing, interacts with drosha and plays a role in mirna biogenesis (fujii., 2006 ; yu., 2008 ars2 is implicated in rna silencing that functions in antiviral defense in flies and cell proliferation in mammals (gruber., 2009 ; it interacts with the nuclear cap - binding complex (cbp20/cbp80) and is involved in mirna biogenesis, suggesting a link between rna silencing and rna - processing pathways. cbp20/cbp80 proteins are also implicated in mirna biogenesis in plants (kim., overall, the existence of this large drosha - complex with only weak mirna - processing activity suggests that drosha may play multiple roles in mirna - independent gene regulation. using genome - wide and gene - specific approaches we now show that drosha binds to the promoter - proximal regions of many human genes in a transcription - dependent manner. similarly, dgcr8 binds promoter - proximal regions of many human genes, suggesting that the whole microprocessor is recruited at promoter regions. we also find that drosha interacts with pol ii and its depletion from human cells causes transcriptional downregulation with a concomitant decrease in nascent and mature mrna levels. this positive function of drosha in gene expression is mediated through its interaction with the rna - binding protein cbp80 and dependent on the n - terminal protein - interaction domain of drosha. thus, results presented in this paper demonstrate an mirna- and cleavage - independent function of drosha. consequently binding of drosha to nascent mirna sequences can be detected using chromatin immunoprecipitation (chip) analysis (morlando., 2008). to investigate genome - wide binding of drosha, we employed a chip - on - chip approach using human 5.1.1 encode array. our results show that, in addition to mirna regions, drosha binds many human genes. this binding is enriched at the transcriptional start sites (tsss), compared to the ends of transcripts (txend), other gene body, or intergenic regions (figure 1a ; full gene list in table s1). in particular, we observed that, out of 160 tss - proximal probes, 55 (34%) had more than 2-fold enrichment of drosha - chip signal over the input signal. this is significantly more than 1,669 (10%) out of 15,998 total probes or 953 (11%) out of 8,333 probes in the gene bodies (p < 0.0001, fisher s exact test). the same level of enrichment was observed for four (40%) out of ten probes overlapping with annotated mirna regions (figure 1aii). by way of illustration, we present selenpb1 and eef1a1 genes, which scored positively in our genomic analysis, where drosha selectively binds to 5-proximal gene regions (figure s1). genes that are not expressed in hela cells such as il4 and tarm1 demonstrated only background drosha signal (figure s1). we also identified genes, such as g6pd, znf687, and fundc2, which are expressed in hela cells but did not demonstrate any significant drosha enrichment (figure s3a). we further confirmed these array data using gene - specific primers in chip experiments. in particular, we observed an enrichment of drosha binding to promoter - proximal region of selenbp1 gene, colocalizing with pol ii peak (figure s2a). we did not detect any significant binding of drosha or pol ii to il4 and tarm1 genes, further confirming chip - on - chip results (figures s2b and s2c). we also did not detect any significant drosha binding over promoter - proximal region, enriched for pol ii, on g6pd gene (figure s3c). to investigate the function of drosha at the beginning of human genes in more detail, we compared pol ii and drosha binding profiles for specific human genes. both pol ii and drosha were enriched over the promoter - proximal regions of human -actin, gapdh, ptb, -actin, and intronless taf7 genes (figures 1b, 1c, and s4a s4c). the enrichment of drosha observed over these genes is higher than over the mir-330 locus within the eml2 gene, used as a positive control for drosha binding (morlando., 2008), we used chip for histone h3, which was found to be depleted from promoter - proximal regions and enriched in the body of these genes (figure 1eiii). to test if drosha binding to these genes is transcription dependent, we treated hela cells with pol ii transcriptional inhibitor actinomycin d. following this treatment, we observed 80% reduction in pre - mrna levels and a substantial reduction in drosha binding to mir-330 locus, -actin, and -actin genes (figures 1e and s4c). in contrast, we saw no effect on histone h3 binding (figure 1eiii). these results indicate that, in addition to mirna sequences, drosha binds 5 end regions of human protein - coding genes in a transcription - dependent manner. furthermore, we also searched for potential correlation between drosha recruitment using our drosha chip - array data and level of pol ii signal determined from available chromatin immunoprecipitation sequencing (chip - seq) experiments in ucsc depository. we have calculated pol ii signal in 1 kb region around the tss for refseq transcripts covered by probes in the drosha array. following this analysis, we identified a weak (pearson r = 0.41) but significant (p = 1e-6) correlation between the drosha probe signal at tss and pol ii chip density (figure s5), indicating that drosha is preferentially recruited to the pol ii - rich regions. overall, these results suggest that drosha binding may be required for enhanced gene expression. drosha is known to bind mirna sequences as part of the microprocessor complex, which also contains dgcr8, a double - stranded rna - binding protein that is deleted in the digeorge syndrome (landthaler., 2004). to investigate whether drosha binds 5 regions of human genes together with dgcr8, we carried out chip experiments in hela cells (figure 2a). dgcr8 was found to be enriched over the mir-330 locus, -actin, gapdh, and selenbp1 promoter - proximal gene regions (figure 2a). these regions overlap drosha binding regions (figures 1, s2, and s4), suggesting that drosha and dgcr8 may form a complex at promoter - proximal regions of human genes. to substantiate these findings in a genome - wide fashion, we analyzed a dgcr8 hits - clip experiment that was performed with endogenous and overexpressed dgcr8 protein in hek293 t cells to identify dgcr8 endogenous rna targets (macias., 2012). in particular, we observed 1,101 dgcr8 clusters, corresponding to 967 genes, in the sense orientation (see supplemental material in macias., 2012) and 196 dgcr8 clusters, corresponding to 174 genes, in antisense orientation (figure 2c ; table s2 for gene identity). interestingly, significant clusters of dgcr8 binding were seen at 1,000/+200 bp from the tsss and just upstream of the transcription termination regions (ttss) (false discovery rate [fdr ] < 0.01) (figure 2b). in particular, we observed significant enrichment of dgcr8 binding in the region within 200 nt downstream of the tss (sense versus antisense ks test p < 2.2e-16 ; figure 2b). dgcr8 binding in this region is most pronounced in genes expressed at medium and high levels when compared to genes expressed at lower levels (ks test p < 2.2e-16 in both cases) (figure s7a). these results clearly indicate that dgcr8, similar to drosha, binds to promoter - proximal regions of human genes. however, it should be noted that when we analyzed dgcr8 binding to spliced mrna transcripts (cdnas) (figure s6), we also see an enrichment over exonic sequences immediately downstream of the tss. again, this is seen in genes with medium and high expression (ks test p < 2.2e-16 in both cases) (figure s7b). this may be related to the reported binding of dgcr8 to multiple exonic regions (macias., 2012), potentially reflecting an additional function. binding of drosha to promoter - proximal regions of human genes may be related to its potential role in processing mirnas from these genomic regions, even though such elements are not annotated in the human genome databases. to test if drosha binds and cleaves mirnas present in promoter - proximal regions, we performed in vitro pri - mirna cleavage assays in hela nuclear extracts (guil and cceres, 2007). using in vitro transcription reactions, we generated radiolabeled rnas from gene regions, identified by drosha chip. we employed mir-17 - 92 cluster within the c13orf25 human genomic region as a positive control for mirna production. following in vitro cleavage assay in hela extracts, mirnas were generated from the control mir-17 - 19a sequences but not from the promoter - proximal region of the -actin gene (figure 2d). we also failed to detect pre - mirnas or rna cleavage products in vitro for promoter - proximal regions of other genes, identified in drosha chip assays (data not shown). these findings suggest that drosha binding over promoter - proximal regions is not associated with mirna synthesis and rna cleavage. to investigate the promoter - associated function of drosha, we performed rnai - induced drosha knockdown in hela cells (figure 3a). following drosha depletion, we observed a small increase in pol ii binding over the promoters of -actin and gapdh genes, using chip analysis (figure 3a). we also observed an 20%40% decrease in pre - mrna levels and 30%60% decrease in poly(a)+ rna levels in hela cells depleted for drosha (figure 3b). in contrast, we did not detect any significant change in the levels of poly(a)+ and pre - mrna corresponding to g6pd, znf687, and fundc2 genes, for which we observed lack of promoter - proximal drosha binding (figures s3b and s3c). if drosha has a direct effect on transcription, we employed nuclear run - on (nro) using br - utp as the labeled nucleotide (core and lis, 2008 ; lin., the advantage of br - utp nro over pol ii chip is the ability of br - utp nro to detect actively transcribing polymerases, as opposed to total pol ii levels detected by chip. as demonstrated in figure 3c, we observed a substantial decrease in the level of nascent transcription for taf7, ptb, and selenbp1 genes in drosha - knocked - down cells. as a negative control for this experiment, we used il4 and tarm1 genes. these genes are not expressed in hela cells ; hence, we did not observe any detectable transcription signal in br - utp nro reactions. these results suggest that drosha has a direct positive effect on transcription, consistent with the genome - wide expression data, where highly expressed genes correlated with increased drosha binding on tsss (figure 1a). furthermore, the antisense transcripts detected over the promoter - proximal region of the -actin gene were also decreased upon drosha depletion (figure s4d). these results further indicate that drosha is not involved in the cleavage of nascent sense or antisense rna transcripts, pointing toward a cleavage - independent function of drosha in human gene expression. we next employed a -actin / egfp reporter plasmid, where transcription of egfp is driven by the -actin promoter, fused to -actin exon 1 and intron 1 regions (qin and gunning, 1997) (figure 3d). as described above, drosha binds the promoter - proximal region of -actin gene (figure s4c). interestingly, in hela cells depleted for drosha we observed 75% reduction in expression of egfp mrna from the plasmid (figure 3d), recapitulating downregulation of endogenous -actin mature and pre - mrnas (figure 3b). these results suggest that drosha plays a positive role in the regulation of -actin / egfp expression. next, we cotransfected -actin / egfp reporter plasmid with rnai - resistant flag - tagged drosha (wt), a catalytic mutant e110aq (aq), unable to process mirnas, and 390 construct into 293 cells, depleted for drosha, using sirna2 (han., 2004). the 390 drosha construct is active in mirna processing but lacks the n - terminal proline - rich and rs - rich domains, proposed to be important for drosha protein - protein interactions (han., 2004) both mrnas and proteins corresponding to wt, aq, and 390 drosha were expressed at a high level in 293 t cells, depleted for endogenous drosha (figure 3ei and 3eii). overexpression of both wt and aq catalytic drosha mutant resulted in the increase of egfp mrna (figure 3eiii). in contrast, overexpression of 390 construct had no significant effect on the expression of egfp mrna. these results suggest that drosha plays a positive role in the regulation of the -actin promoter. importantly, this drosha function is independent of its ability to cleave rna but does require the n - terminal protein - protein interaction domain. we hypothesize that the cleavage - independent stimulatory role of drosha on gene expression may be mediated through its association with protein cofactors. we therefore performed coimmunoprecipitation experiments and found that transiently overexpressed flag - tagged drosha interacts with both unphosphorylated (iia) and phosphorylated (iio) forms of pol ii in 293 t cells, supporting its role in transcriptional regulation. drosha also interacts with cbp80 and ars2 proteins, involved in mirna biogenesis and rna processing pathways, respectively (gruber., 2009 ; sabin., 2009 furthermore, by chip analysis we found that cbp80 protein binds promoter - proximal regions of -actin and gapdh genes, colocalizing with drosha binding (figures 4b and 4c). we next observed that drosha - cbp80 interaction is not affected by the aq mutation in the catalytic domain of drosha but is significantly reduced when the n - terminal domain of drosha is deleted in 390 construct or when both 390 and aq mutations are combined (figure 4d, top panel). these results strongly support the view that drosha interacts with cbp80 through its n - terminal rs domain. we also studied the interaction of drosha with pol ii (figure 4d, middle). similar to cbp80, a catalytic mutation in drosha (aq) does not affect its interaction with pol ii. however, the drosha 390 construct showed reduced interaction with pol ii, and specifically with its phosphorylated form (iio). this suggests that the n - terminal domain of drosha is particularly important for drosha - mediated transcriptional effects in human cells because it mediates interaction of drosha with cbp80 and pol ii. we also observed that interaction of drosha with cbp80 and pol ii is rna independent (figure 4e). consequently, this result provides a molecular explanation for the ability of drosha to regulate gene expression in a mirna- and cleavage - independent manner. we demonstrate the existence of a mirna- and cleavage - independent function of drosha in the regulation of human gene expression. combining whole - genome analysis with gene - specific approaches, we demonstrate that drosha binds to the 5 ends of human genes in a transcription - dependent manner (figure 1). this binding is not associated with mirna biogenesis or rna cleavage but correlates with the level of gene expression (figures 1 and 2). depletion of drosha from hela cells led to direct downregulation of transcription based on nuclear run - on and pol ii accumulation at the promoters of these genes, resulting in reduced levels of pre - mrna and poly(a) mrna (figure 3). chip and hits - clip analysis of dgcr8 also revealed binding sites at promoter - proximal regions, enriched at genes expressed at a higher level, suggesting that drosha and dgcr8 may bind to promoter regions as a complex (figures 2 and s7). a positive function of drosha in the regulation of gene expression was further confirmed using a heterologous reporter construct, where drosha overexpression influences reporter expression (figure 3). this function of drosha is independent of its catalytic activity and mediated through its n - terminal domain, which interacts with the rna - binding protein cbp80 and pol ii, which also bind to the 5 end of human genes (figures 3 and 4). taken together, these results suggest a model whereby drosha promotes gene expression in a cleavage - independent manner by binding to rna hairpins formed at the 5 ends of the nascent rnas and interacting with cbp80 and pol ii, through its n - terminal rs domain (figure 4f). initially, we hypothesized that this positive function of drosha may be associated with generation / regulation of short promoter - associated transcripts (prompts), originally predicted to regulate transcription through changes in chromatin structure, promoter methylation, or pol ii recycling (preker., 2008). prompts may direct drosha to the beginning of the gene through interaction with its binding factors. in our studies, both sense and antisense transcripts, detected over the promoter - proximal regions, were destabilized in drosha - depleted cells (figure s4d), suggesting that drosha does not cleave these transcripts and its function is not mediated through prompts. drosha binding at the beginning of human genes may be also related to pol ii release from promoter - proximal pausing events and be required for the recruitment of transcription / rna processing factors. interestingly, drosha binding coincides with the binding site of the negative elongation factor nelf, which defines the late elongation checkpoint, ensuring conversion to productive elongation mediated by phosphorylation of pol ii ctd, nelf, and dsif by the positive transcription elongation factor b (p - tefb) (egloff., 2009). as both nucleosome positioning and chromatin modifications play an important role in the elongation functions of p - tefb, we also checked whether drosha knockdown causes a change in chromatin modifications. gc - rich sequences are known to be enriched in the promoter regions of human genes (calistri., 2011). transcripts for these sequences are likely to form rna hairpin structures, predicted to be suitable for microprocessor binding mediated by dgcr8 (macias., 2012 ; if drosha were to cleave such rnas, the consequences of drosha cleavage across the whole human genome would be detrimental to cellular survival. we hypothesize that drosha does not cleave promoter - associated rna but is instead utilized as a positive regulator of gene expression. we further analyzed the structures found in promoter transcripts and compared them to mirna, snornas, and protein - coding region structures, found to be bound by dgcr8 in clip analysis. our bioinformatic analysis confirmed that structures in promoter regions have shorter stems, higher minimum free energy, and lower base - pairing probability (figure s8). in addition, promoter transcripts also lack motifs important for the processing of the stem, such as tg dinucleotide at the base of the stem and cnnc motif downstream of the stem, as described in auyeung., this analysis suggests that promoter structures may be less stable, and their recognition and processing by the microprocessor complex may be less efficient compared to mirnas or other sequences cleaved by drosha. evidence that components of the mirna machinery may have additional functions comes from several previous studies. in early - stage thymocytes, drosha recognizes and cleaves mrnas harboring secondary stem - loop structures (chong., 2010). drosha can also regulate viral gene expression of kaposi s sarcoma - associated herpesvirus (kshv) (lin and sullivan, 2011). dgcr8 together with drosha controls the abundance of many cellular rnas, including noncoding rnas, mrnas, and alternatively spliced isoforms (macias., 2012). finally, microprocessor can regulate transcription of some cellular mrna and hiv provirus, causing pol ii premature termination through the cleavage of the tar stem - loop structure (wagschal., 2012). these regulatory activities of drosha each involve recognition and cleavage of target rnas in an analogous manner to its function in mirna biogenesis in contrast to the positive function of drosha on transcription described here. possibly this positive function of drosha is related to the action of rna - binding factors, known to modulate the function of drosha in human cells. such factors are likely to interact with the n - terminal proline - rich and rs domains, proposed to be the main sites of drosha protein - protein interactions. we confirmed the previously described interaction between drosha and cbp80 and ars2 proteins (gruber., 2009 ; sabin., 2009). furthermore, our results also show that drosha interacts with the c - terminal domain (ctd) of pol ii (figure 4). this interaction may be direct or mediated via proteins, containing rs domains and interacting with the ctd, potentially modulating drosha s cleavage - independent function. interestingly, in drosophila the key rnai components dicer 2 and argonaute 2 associate with chromatin and interact with the core transcription machinery, affecting pol ii dynamics (cernilogar., 2011), further supporting the role of rnai machinery in transcriptional regulation. because both drosha and dicer may have additional roles to mirna biogenesis future studies will be necessary to evaluate the full spectrum of molecular functions that these rnase - iii - like enzymes play in human cells. pck - flag wt, aq, and 390 drosha constructs (han., 2004) and -actin / egfp plasmid (qin and gunning, 1997) were described previously. pflag - cmv-2 (flag) was purchased from sigma - aldrich (e7398). total rna was harvested using trizol reagent (invitrogen) followed by dnase i treatment (roche). total rna (2 g) was reverse - transcribed using superscript reverse transcriptase (invitrogen) and random hexamer primers (invitrogen) or gene - specific primers, as described in the supplemental experimental procedures and table s3, followed by qpcr. gene - specific primers were used in all figures, apart from figure 3eiii, where random hexamers were used for quantitative rt - pcr (qrt - pcr), following the manufacturer s description (invitrogen). mir-17 - 19 and -actin gene regions were amplified from genomic dna using t7+-actin (f / r) and t7+mir-17 - 19 (f / r) primers accordingly. in vitro processing the rnai was carried out as described (wollerton., 2004). mrna target sequence for drosha small interfering rna (sirna) duplex was 5-cgaguaggcuucgugacuu-3 (sirna1) and 5-gaguauuuacuugcucag uaa-3 (sirna2). sirna1 was used in all figures apart from figure 3e, where sirna2 was used. pull - downs were carried out in rnase a nontreated extracts, except figure 4e where 50 g / ml rnase a was used to treat cell extracts during the pull - down procedure as described in gruber. western blots were probed with drosha (abcam), actin (sigma - aldrich), gapdh (sigma - aldrich), pol ii (covance), and cbp80 (sigma) antibodies. chip analysis was carried out as previously described (west., 2004). drosha chip - on - chip experiments were carried out as described in de gobbi. hits - clip for dgcr8 was based on a published protocol (wang., 2009) with minor modifications, as described in macias. the br - utp nro was carried out largely as described (lin., 2008 ; skourti - stathaki., nuclear pellets were resuspended in transcription buffer (40 mm tris - hcl [ph 7.9 ], 300 mm kcl, 10 mm mgcl2, 40% glycerol, 2 mm dtt) and 10 mm mix of ratp, rctp, rgtp, and br - utp or rutp (in the control samples). total rna was isolated using trizol reagent (invitrogen) according to manufacturer s instructions and treated with rnase - free dnase i (roche). two microliters of anti - bru antibody (sigma - aldrich) was preincubated with 30 l of protein g dynabeads (upstate) and 10 g trna per sample for 1 hr at 4c. the beads were washed three times with rsb-100 buffer (10 mm tris - hcl [ph 7.4 ], 100 mm nacl, 2.5 mm mgcl2, 0.4% triton x-100) and resuspended in 150 l rsb-100 with 40 u rnase - out (invitrogen) and 5 g of glycogen. total rna was added to the beads and incubated for additional 1 hr at 4c. rna bound to the beads was extracted with trizol reagent followed by dnase i treatment. the rt reaction was performed using superscript iii reverse transcriptase (invitrogen) following the manufacturers instructions. the real - time quantitative pcr was performed using a corbett research rotor - gene gg-3000 machine. the pcr mixture contained quantitest sybr green pcr master mix (qiagen), 2 l of template cdna, and primers from table s3. cycling parameters were 95c for 15 min, followed by 45 cycles of 95c for 15 s, 58c for 20 s, and 72c for 20 s. fluorescence intensities were plotted against the number of cycles by using an algorithm provided by the manufacturer. amount of nascent br - utp rna was calculated by subtracting the background of u - rna produced over a specific gene probe. unless otherwise stated, results are shown as the average values from at least three independent biological experiments sd. 0.05 ; p < 0.05 ; p < 0.05), on the basis of an unpaired, two - tailed distribution determined with a student s t test. analyzed dgcr8 binding at promoter regions by hits - clip and carried analysis in figures s6s8. | summarydrosha is the main rnase iii - like enzyme involved in the process of microrna (mirna) biogenesis in the nucleus. using whole - genome chip - on - chip analysis, we demonstrate that, in addition to mirna sequences, drosha specifically binds promoter - proximal regions of many human genes in a transcription - dependent manner. this binding is not associated with mirna production or rna cleavage. drosha knockdown in hela cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mrna produced from these gene regions. furthermore, we show that this function of drosha is dependent on its n - terminal protein - interaction domain, which associates with the rna - binding protein cbp80 and rna polymerase ii. consequently, we uncover a previously unsuspected rna cleavage - independent function of drosha in the regulation of human gene expression. |
kassebaum reported that the annual rate of change in the maternal mortality ratio was greater than 1% between 2003 and 2013, with the most substantial improvement from 2012 to 2013 at 3.3%. many of the global gains in reducing maternal mortality can be attributed to developments in preventing and treating postpartum hemorrhage (pph). in fact, the biggest absolute reduction was in maternal deaths due to hemorrhage. yet, pph remains the most common cause of maternal death globally2 and has persisted in low - income countries with little change since 1990.1 this is in part due to the prevalence of home deliveries and limited access to life - saving uterotonic drugs in these countries.35 meanwhile, there is also evidence that the rate of retained placenta and pph is increasing in higher - income countries.6,7 pph is often associated with the failure of the uterus to contract after delivery and categorized as blood loss of 500 ml or more following vaginal delivery or 1,000 ml after cesarean delivery.8,9 pph is categorized as primary if it occurs within 24 hours of delivery and secondary if excessive blood loss occurs at 24 hours or more after delivery. the reality is that most cases are primary pph and the time from beginning to death is considerably shorter than other major obstetric complications. first, the initial hemoglobin (hb) level of a woman affects her survival rate from pph.10 the world health organization (who) defines anemia in pregnancy as hb level 1,000 ml of additional blood loss was significantly higher (relative risk [rr ] : 3.6 ; 95% confidence interval [ci ] : 1.0212.88). the women in the misoprostol group were also more likely to undergo intrauterine clinical exploration under anesthesia (rr : 1.66 ; 95% ci : 1.002.76).33 results from a similar trial, but among women who have not been exposed to oxytocin during the third stage of labor, showed that misoprostol is slightly inferior to oxytocin.34 authors conclude that in the absence of oxytocin, misoprostol might be an appropriate first - line treatment for pph. the third rct, published in 2010, assessed whether 600 g of sublingual misoprostol could be used as an adjunct therapy to standard uterotonics. results showed that when compared to a placebo, misoprostol does not offer additional benefits.35 table 2 shows the results from three non - randomized cohort studies with controls. the study by prata,36 the only community - based study with controls for the treatment of pph, used a high dose (1,000 g) of rectal misoprostol and demonstrated the potential use of misoprostol for pph treatment in settings where women were not exposed to conventional uterotonics during the third stage of labor. in fact, winikoff rct findings, although with a lower dose and different route, confirmed the value of misoprostol in settings where oxytocin is not available. the use of high - dose rectal misoprostol was also assessed as an adjunct therapy to oxytocin compared to ergometrine using a retrospective cohort study design.37 results showed no significant differences between the two groups. based on the results from the 2010 rct, we now know that misoprostol has no adjunct therapeutic properties.35 a cohort study in three nigerian hospitals showed that 800 g of sublingual misoprostol was efficacious in stopping bleeding within 20 minutes of use among women diagnosed with pph from uterine atony and not exposed to uterotonics during the third stage of labor, suggesting that the application of results of the 2010 rct in clinical practice produces intended outcomes. misoprostol stopped bleeding in 85% of the pph cases, the remaining needed additional uterotonics.38 the safety profile of misoprostol in obstetrics has long been established and is linked to the pharmacokinetic profile of e2 prostaglandin analog.39 in rcts, misoprostol administered in treatment doses shows increased rr of side effects when compared to placebo. although side effects were reported as transient and occurring in a small group of women, pooled data from a cochrane review show average increases in vomiting (rr : 1.84 ; 95% ci : 1.162.95), shivering (rr : 2.25 ; 95% ci : 1.762.88), pyrexia of 38c (rr : 3.12 ; 95% ci : 2.663.67), and pyrexia of 40c or more (rr : 13.58 ; 95% ci : 4.9337.44).26 the use of misoprostol in intra- and postoperative hemorrhage, as well as in retained placenta, has been investigated. results from a systematic review and meta - analysis evaluating the efficacy and safety of misoprostol for reducing intra - and postoperative hemorrhage showed that misoprostol combined with oxytocin seems to be more efficacious than oxytocin alone.40 the systematic review included 17 studies totaling 3,174 women. seven studies assessed misoprostol vs oxytocin and seven assessed misoprostol plus oxytocin vs oxytocin alone. subsequent rcts found similar results when comparing misoprostol and oxytocin to oxytocin alone during cesarean delivery in women at risk of pph.41,42 a cochrane review of prostaglandins for the management of retained placenta involving 244 women, of whom 194 received a dose of 800 g misoprostol, showed that prostaglandins were not superior compared to placebo. no statistically significant differences were found in the manual removal of placenta, severe pph, need for blood transfusion, or other interventions.43 however, the authors noted that the quality of the evidence was low and called for much larger and sufficiently powered studies to make recommendations. over the past decade, key women s health organizations have promoted the inclusion of misoprostol for the treatment of pph. as early as 2006, the international confederation of midwives and the international federation of gynecology and obstetrics (figo) recommended that misoprostol for the treatment of pph may be appropriate for use in low - resource settings as a stand - alone treatment, in combination with oxytocin, and as a last resort for pph treatment.22 at the same time, the american congress of obstetricians and gynecologists included misoprostol in the list of uterotonics to be used as the first - line treatment for hemorrhage in the event of decreased uterine atony.23 a few years later, the royal college of obstetricians and gynecologists agreed that misoprostol may be an appropriate alternative for pph treatment in settings, where parenteral prostaglandins are not available or where there are contraindications.24 in 2012, figo published its guidelines for the treatment of pph with misoprostol : one dose of misoprostol 800 g sublingually is indicated for the treatment of pph when 40 iu iv oxytocin is not immediately available (irrespective of the prophylactic measures).25 in 2014, the cochrane collaboration published a systematic review on the treatment for primary pph.26 the review assessed the effectiveness and safety of any intervention used for the treatment of pph. of the ten rcts reviewed spanning 4,052 women, seven investigated misoprostol in doses varying from 600 g to 1,000 g, using various routes of administration, including a combination of sublingual and rectal. the finding that seven out of ten studies were assessing the efficacy of misoprostol alone suggests that the role of misoprostol has been one of the most studied interventions in the treatment of pph in recent decades. however, it was not until april 2015 that misoprostol was added to essential medicines who model list (eml) for the treatment of pph. the eml now recommends the use of misoprostol for the prevention and treatment of pph where oxytocin is not available or can not be safely used.27 table 1 presents the results from all the published rcts on the use of misoprostol for the treatment of primary pph after vaginal deliveries. the last systematic review of misoprostol to treat pph was published in 2005 and included three rcts.28 however, given that only seven trials were available, we decided to show them individually in table 1. rcts have used multiple dose regimens and routes, in addition to controlling against placebo or another conventional uterotonic, oxytocin, and/or ergometrine. the first rct was published in 2001 comparing 800 mg of misoprostol rectally to 5 iu oxytocin and 500 g ergometrine intramuscular plus 10 iu oxytocin diluted in 500 ml normal saline iv infusion. results from a relatively small sample size (n=32 in each arm) were promising, indicating that misoprostol is an effective therapy.29 in 2004, hofmeyr published a placebo - controlled trial testing a high - dose misoprostol, but the study was underpowered to show significant differences. in the same year, a study by walraven combined oral (200 g) and sub - lingual (400 g) routes of misoprostol and also showed the therapeutic potential of misoprostol against a placebo. four years later, an rct in pakistan attempted to ascertain whether sublingual misoprostol had additional benefits to a standard oxytocin regimen. although significant reductions in blood loss in the misoprostol group were reported, the study did not reach the intended sample size due to much lower pph rate than expected.32 however, it was not until 2010 that pivotal evidence on the therapeutic potential of misoprostol became available from three rcts. in a double - blind non - inferiority trial, blum demonstrated that 800 g of sublingual misoprostol is clinically equivalent to 40 iu iv oxytocin in women who have received prophylactic oxytocin during the third stage of labor. however, while the time to cessation and additional blood loss of 300 ml and 500 ml was equivalent, the number of women in the misoprostol group with > 1,000 ml of additional blood loss was significantly higher (relative risk [rr ] : 3.6 ; 95% confidence interval [ci ] : 1.0212.88). the women in the misoprostol group were also more likely to undergo intrauterine clinical exploration under anesthesia (rr : 1.66 ; 95% ci : 1.002.76).33 results from a similar trial, but among women who have not been exposed to oxytocin during the third stage of labor, showed that misoprostol is slightly inferior to oxytocin.34 authors conclude that in the absence of oxytocin, misoprostol might be an appropriate first - line treatment for pph. the third rct, published in 2010, assessed whether 600 g of sublingual misoprostol could be used as an adjunct therapy to standard uterotonics. results showed that when compared to a placebo, misoprostol does not offer additional benefits.35 table 2 shows the results from three non - randomized cohort studies with controls. the study by prata,36 the only community - based study with controls for the treatment of pph, used a high dose (1,000 g) of rectal misoprostol and demonstrated the potential use of misoprostol for pph treatment in settings where women were not exposed to conventional uterotonics during the third stage of labor. in fact, winikoff rct findings, although with a lower dose and different route, confirmed the value of misoprostol in settings where oxytocin is not available. the use of high - dose rectal misoprostol was also assessed as an adjunct therapy to oxytocin compared to ergometrine using a retrospective cohort study design.37 results showed no significant differences between the two groups. based on the results from the 2010 rct, we now know that misoprostol has no adjunct therapeutic properties.35 a cohort study in three nigerian hospitals showed that 800 g of sublingual misoprostol was efficacious in stopping bleeding within 20 minutes of use among women diagnosed with pph from uterine atony and not exposed to uterotonics during the third stage of labor, suggesting that the application of results of the 2010 rct in clinical practice produces intended outcomes. misoprostol stopped bleeding in 85% of the pph cases, the remaining needed additional uterotonics.38 the safety profile of misoprostol in obstetrics has long been established and is linked to the pharmacokinetic profile of e2 prostaglandin analog.39 in rcts, misoprostol administered in treatment doses shows increased rr of side effects when compared to placebo. although side effects were reported as transient and occurring in a small group of women, pooled data from a cochrane review show average increases in vomiting (rr : 1.84 ; 95% ci : 1.162.95), shivering (rr : 2.25 ; 95% ci : 1.762.88), pyrexia of 38c (rr : 3.12 ; 95% ci : 2.663.67), and pyrexia of 40c or more (rr : 13.58 ; 95% ci : 4.9337.44).26 the use of misoprostol in intra- and postoperative hemorrhage, as well as in retained placenta, has been investigated. results from a systematic review and meta - analysis evaluating the efficacy and safety of misoprostol for reducing intra - and postoperative hemorrhage showed that misoprostol combined with oxytocin seems to be more efficacious than oxytocin alone.40 the systematic review included 17 studies totaling 3,174 women. seven studies assessed misoprostol vs oxytocin and seven assessed misoprostol plus oxytocin vs oxytocin alone. subsequent rcts found similar results when comparing misoprostol and oxytocin to oxytocin alone during cesarean delivery in women at risk of pph.41,42 a cochrane review of prostaglandins for the management of retained placenta involving 244 women, of whom 194 received a dose of 800 g misoprostol, showed that prostaglandins were not superior compared to placebo. no statistically significant differences were found in the manual removal of placenta, severe pph, need for blood transfusion, or other interventions.43 however, the authors noted that the quality of the evidence was low and called for much larger and sufficiently powered studies to make recommendations. although rcts and non - randomized trials have used different doses and routes of administration, current evidence points to an optimal and effective dose regimen of 800 g of sublingual misoprostol for the treatment of pph, which was endorsed by figo.25 the safety profile of this dose and route is sufficient, but prostaglandin - related side effects such as shivering and vomiting may occur.26 misoprostol s clinical equivalence to 40 iu iv oxytocin, when used for the treatment of pph in women who have received a prophylactic dose of oxytocin during the third stage of labor, provides unique opportunities for low - resource settings. where labor wards are ill - equipped to provide sufficient monitoring and quality iv infusion, service providers can use intramuscular oxytocin during the third stage of labor and sublingual misoprostol if a pph diagnosis is established without hesitation. in addition, given the longer shelf life of misoprostol and relatively better stability in field conditions,39 health service planners, particularly those responsible for drug procurement, can adjust forecasting and purchasing of the drug assuming all estimated primary pph cases will be treated with misoprostol, making it the first - line treatment. current evidence shows that in the absence of oxytocin prophylaxis during the third stage of labor, the drug of choice for the treatment of pph should be 40 iu iv oxytocin. however, in settings where oxytocin is not available, sublingual misoprostol is an appropriate first - line treatment. this finding is of significance for settings where a large number of women deliver at home without a skilled provider or with a minimally trained one, as well as in settings where delivery begins at home and access to health facilities for referral when pph is identified is limited due to the lack of transportation, road security at night, and other issues.3 contrary to expectations based on the evidence prior to 2010, findings from a multicenter rct do not support the use of misoprostol in addition to standard uterotonics (oxytocin or ergometrine) for the treatment of primary pph after vaginal deliveries.35 however, rcts have demonstrated the opposite for cesarean deliveries.4042 thus, the adjunct role of misoprostol to conventional uterotonics is important in reducing intra- and postoperative hemorrhage. in summary, current evidence on misoprostol efficacy is sufficient to move programs forward such that low - income countries, the ones with highest share of pph - attributed burden of disease, can benefit from the ease of administration and storage of the drug. however, it is also in these contexts where the management of pph cases is more challenging, despite knowledge of drug efficacy. for example, the efficacy from clinical trials assumes relatively accurate blood loss measurement, leading to a specific point in time when treatment is administered. thus, standardizing clinical protocols with culturally appropriate ways to measure blood loss after delivery could increase program effectiveness for the treatment of pph. task shifting or sharing with providers at lower - level health centers and health posts, including community health workers, should also be considered, especially when referrals to health facilities are difficult. in many countries, where access to misoprostol for pph is being scaled up,44 providers could benefit from learning not just about prevention but also about the treatment for pph, including clinical officers providing emergency cesarean sections in rural or district hospitals. other ways to potentially increase program effectiveness to treat pph in women who deliver at home would certainly include the use of community health workers and/or traditional birth attendants trained in the recognition of pph and administration of misoprostol. a cochrane review with the objective of determining the safety and effectiveness of a system for advanced distribution of misoprostol for the prevention and treatment of pph found insufficient evidence to support such a system.45 the review identified three studies, and none of them met the inclusion criteria randomized or quasi rcts. thus, the conclusion is based on the lack of evidence rather than results from existing evidence. however, given the current knowledge of pph - attributed mortality and the role of misoprostol to treat pph, a randomized placebo - controlled experiment for advance distribution at community level might pose ethical concerns. the comparison of misoprostol to placebo or to conventional uterotonics would allow programs to make informed decisions about the realm of interventions that are possible to implement in each setting according to provider level and health care system capacity. however, one important question still remains : can misoprostol be given for the treatment of pph after its use for prevention during the third stage of labor ? in addition, even though conventional uterotonics are more commonly used, what is the relative contribution of misoprostol compared to other interventions for pph treatment, such as the non - pneumatic antishock garment, hemostatic drugs, and surgical interventions ? a review of these interventions concluded that more evidence is needed, including the best ways to treat women who do not respond to uterotonics.26 clinical guidelines and treatment protocols should be updated to reflect the current knowledge on the efficacy of 800 g sublingual misoprostol for the treatment of pph. however, improvements in pph treatment, regardless of the uterotonic, should start with a timely and correct diagnosis that can lead to an appropriate case management. women with prior exposure to prophylactic oxytocin, as well as those without exposure to oxytocin and in settings where oxytocin is not available, could all benefit from the therapeutic properties of this drug. women undergoing cesarean section and at risk of pph could also benefit from the efficacy of misoprostol in conjunction with oxytocin. | backgrounda myriad of interventions exist to treat postpartum hemorrhage (pph), ranging from uterotonics and hemostatics to surgical and aortic compression devices. nonetheless, pph remains the leading cause of maternal mortality worldwide. the purpose of this article is to review the available evidence on the efficacy of misoprostol for the treatment of primary pph and discuss implications for health care planning.data and methodsusing pubmed, web of science, and googlescholar, we reviewed the literature on randomized controlled trials of interventions to treat pph with misoprostol and non - randomized field trials with controls. we discuss the current knowledge and implications for health care planning, especially in resource - poor settings.resultsthe treatment of pph with 800 g of misoprostol is equivalent to 40 iu of intravenous oxytocin in women who have received oxytocin for the prevention of pph. the same dose might be an option for the treatment of pph in women who did not receive oxytocin for the prevention of pph and do not have access to oxytocin for treatment. adding misoprostol to standard uterotonics has no additional benefits to women being treated for pph, but the beneficial adjunctive role of misoprostol to conventional uterotonics is important in reducing intra- and postoperative hemorrhage during cesarean section.conclusionmisoprostol is an effective uterotonic agent in the treatment of pph. clinical guidelines and treatment protocols should be updated to reflect the current knowledge on the efficacy of misoprostol for the treatment of pph with 800 g sublingually. |
chromobacterium violaceum is a gram negative, rod shaped, nonsporing, and motile bacteria that can utilize carbohydrate fermentatively and grows readily on the ordinary laboratory culture media at 35c37c.1 it naturally resides in soil and stagnant water of tropical and subtropical regions as saprophyte2 and produces a chemical antioxidant compound called violacein (violet non - diffusible pigment).3 although first identified in 1881, its pathogenic potential was illustrated only in 1905 by woolley from a fatal case of a buffalo in philippines.4 human case of infection caused by this pathogen was first established by lessler from malaysia in 1927.4 this organism is usually considered as nonpathogenic, but it is an opportunistic pathogen of extreme virulence for humans and animals when inoculated into bloodstream via open wound.5 several reports of serious and fatal infections have been reported worldwide, especially from asian and american countries. in majority of these cases, bacteria entered through the breached or abraded skins subsequently after exposure to contaminated soil or water.6 clinical disease caused by this organism ranges from a localized skin lesion to multiple abscesses in the vital organs and fatal septicemia.7,8 here we report a new case of bacteremia caused by c. violaceum at manmohan memorial medical college teaching hospital (mmth), kathmandu, nepal. to the best of our knowledge, this is the first case of culture - proven bacteremia caused by c. violaceum from nepal. written informed consent was taken from the patient for necessary investigations and case publication in scientific journal. a 36-year - old immunocompetent female presented at midnight of 24 august 2016 to the emergency department of mmth, a tertiary care referral hospital in kathmandu, the capital of nepal, with the clinical symptoms of fever, headache, chills, excessive sweating, burning sensation over epigastric region, and shortness of breath for the past 12 hours. she denied any previous illness, but her husband recalled that she had previously been diagnosed with pulmonary tuberculosis and treated with isoniazid and ethambutol as a directly observed therapy 6 years back. she lived in sitapaila, a nearby village, and spent her life as an active housewife until 3 days ago when she plucked her left toe while working in the paddy farmland. thereafter, her left toe became inflamed and swollen with edematous ulceration. on examination, the patient was conscious and alert, but ill - looking. local examination showed indurations above the toe region that was edematous, but not discharging pus or any serous fluid. furthermore, she had blood pressure of 110/70 mmhg, pulse rate of 110 beats per minute, temperature of 39.6c, and respiratory rate of 22 breaths per minute. the patient was provisionally diagnosed as a case of acute febrile illness with a suspicion of wound - related sepsis and immediate management was started. after necessary physical examination, blood and urine samples were collected aseptically for laboratory investigations viz blood culture, urine culture, hematology parameters, widal test, and blood chemistry tests. paracetamol (500 mg) and ibuprofen (400 mg) were administered by parenteral route along with electrolyte fluids. after specimen collection for laboratory investigations, antimicrobial regimen of 400 mg/24-hour of ciprofloxacin and 2 g/24-hour of ceftriaxone were initiated as empiric therapy for gram negative sepsis immediately after admission. the hematological investigations showed a normal (unremarkable) level of total leukocyte count with adequate cellular distribution, but low hemoglobin concentration (table 1). the clinical parameters of blood glucose, urea, electrolytes, and common liver enzymes were found normal (table 2). widal test titer, serological test for enteric fever, was also insignificant in validating that there was no such infectious cause in the disease. ten milliliters of blood sample was aseptically inoculated into brain heart infusion broth and incubated in aerobic atmosphere for 24 hours. urine sample was inoculated into blood agar and macconkey agar plates semi - quantitatively and incubated aerobically for 24 hours at 37c. blood culture broth was inspected next day, and blind subculture was made on blood agar, chocolate agar, and macconkey agar and incubated for next 24 hours. urine culture plates were found with no growth of any bacterial species and reported as sterile. after overnight incubation of subcultured plates from blood culture, the agar plates demonstrated numerous small colonies with a faint violet metallic pigmentation (which later became dark violet on incubation) with no hemolysis (figure 1). for better demonstration of pigment, we transferred the colonies to fresh nutrient agar plate (figure 2) and incubated aerobically for next 24 hours. some colonies on the nutrient agar were initially nonpigmented, but later converted to dark violet on prolonged incubation (72 hours) at room temperature. biochemical characterization of the bacteria was carried out by conventional biochemical tests such as catalase test, oxidase test, triple sugar iron agar test, citrate utilization, urea hydrolysis, sulfide indole motility, oxidation fermentation test, methyl red - voges proskauer test, decarboxylase tests (lysine, arginine, and ornithine). the oxidase test was performed by picking the single isolated colony with the sterile bamboo stick and rubbing over the surface of the filter paper disk impregnated with 1% tetra methyl paraphenylene diamine dihydrochloride reagent. simultaneously, antimicrobial susceptibility test was performed by kirby - bauer disk diffusion method on mueller - hinton agar using commercially available antibiotic disks (figures 4 ; 5) and the result of the susceptibility test was interpreted according to the clinical and laboratory standards institute guidelines for non - fermenting gram negatives.9 the patient was responding better after initial antimicrobial therapy, and fever subsidized after 8 hours. based on the biochemical findings and microscopic observation (gram staining, motility), the organism was confirmed as c. violaceum. antimicrobial susceptibility test indicated that it is resistant to polymixin group of antibiotics, but susceptible to other therapeutic antibiotics (table 4). additional blood culture was performed on day 6 to confirm the prognosis, but was negative for bacterial growth. she was discharged on day 7 on oral ciprofloxacin (1,000 mg/24 hours) and cefixime (500 mg/24 hours) for next 9 days (total 14 days). on followup visit, after completion of the therapy, she made absolute recovery without any complications and the antibiotics were discontinued. the clinical parameters of blood glucose, urea, electrolytes, and common liver enzymes were found normal (table 2). widal test titer, serological test for enteric fever, was also insignificant in validating that there was no such infectious cause in the disease. ten milliliters of blood sample was aseptically inoculated into brain heart infusion broth and incubated in aerobic atmosphere for 24 hours. urine sample was inoculated into blood agar and macconkey agar plates semi - quantitatively and incubated aerobically for 24 hours at 37c. blood culture broth was inspected next day, and blind subculture was made on blood agar, chocolate agar, and macconkey agar and incubated for next 24 hours. urine culture plates were found with no growth of any bacterial species and reported as sterile. after overnight incubation of subcultured plates from blood culture, the agar plates demonstrated numerous small colonies with a faint violet metallic pigmentation (which later became dark violet on incubation) with no hemolysis (figure 1). for better demonstration of pigment, we transferred the colonies to fresh nutrient agar plate (figure 2) and incubated aerobically for next 24 hours. some colonies on the nutrient agar were initially nonpigmented, but later converted to dark violet on prolonged incubation (72 hours) at room temperature. biochemical characterization of the bacteria was carried out by conventional biochemical tests such as catalase test, oxidase test, triple sugar iron agar test, citrate utilization, urea hydrolysis, sulfide indole motility, oxidation fermentation test, methyl red - voges proskauer test, decarboxylase tests (lysine, arginine, and ornithine). the oxidase test was performed by picking the single isolated colony with the sterile bamboo stick and rubbing over the surface of the filter paper disk impregnated with 1% tetra methyl paraphenylene diamine dihydrochloride reagent. simultaneously, antimicrobial susceptibility test was performed by kirby - bauer disk diffusion method on mueller - hinton agar using commercially available antibiotic disks (figures 4 ; 5) and the result of the susceptibility test was interpreted according to the clinical and laboratory standards institute guidelines for non - fermenting gram negatives.9 the patient was responding better after initial antimicrobial therapy, and fever subsidized after 8 hours. based on the biochemical findings and microscopic observation (gram staining, motility), the organism was confirmed as c. violaceum. antimicrobial susceptibility test indicated that it is resistant to polymixin group of antibiotics, but susceptible to other therapeutic antibiotics (table 4). additional blood culture was performed on day 6 to confirm the prognosis, but was negative for bacterial growth. she was discharged on day 7 on oral ciprofloxacin (1,000 mg/24 hours) and cefixime (500 mg/24 hours) for next 9 days (total 14 days). on followup visit, after completion of the therapy, she made absolute recovery without any complications and the antibiotics were discontinued. c. violaceum is the single species of the genus responsible for human infections.10 most clinicians are unaware of this rare bacterium despite its ubiquitous distribution.6 in the published literature, common clinical features in majority of the cases with fatal outcome appear to be sepsis, multiple liver abscesses, and diffuse pustular dermatitis.4 bacteremia associated with c. violaceum has been reported by chen and yang from taiwan,7,11 campbell from vietnam,5 ray from india,4 nanayakkara from sri lanka,12 and others. most of the reported cases had the fatal outcome but successful therapeutic experience with the use of timely therapy has also been reported.5 although c. violaceum is confined to tropical countries, especially in south and southeast asia, there are only two previously reported cases from nepal. of the two cases, the wound sepsis case by ansari has demonstrated the fatality of the case while another asymptomatic bacteriuria by pant was self - limiting.13,14 blood culture confirmed bacteremia associated with this rare pathogen has not been reported from nepal yet. in this case, clinical features of the febrile woman appear to be similar to that of common enteric fever, but skin lesion and isolated bacterial strain helped in timely identification of the rare case of bacteremia. besides, previous studies suggested that the common predisposing factors for c. violaceum associated bacteremia were immunocompromised state, chronic granulomatous disease, steroid therapy, diabetes, and other systemic illnesses,6,10 but in this report there were no such factors to be noted. consumption of contaminated water or food or through exposure of damaged skin to stagnant water or soil is a common route for transmission of this pathogen to healthy individuals. moreover, severe infections after swimming in contaminated water, recreational or stagnant muddy water, and post - surgical cases have also been reported.6 in this case, too, there was sufficient evidence of inoculation of the pathogen and symptoms were also promising although undifferentiated. the ability of the organism to produce a peculiar metabolite violacein giving the colonies their distinctive purple color helped in proper identification of the bacteria. nevertheless, nonpigmented strains of c. violaceum have also been involved in human infections.11,15 chromobacterium is usually susceptible to therapeutic antimicrobials, however genes associated with intrinsic resistance toward penicillins and early cephalosporins have been described.16 isolates have been found susceptible to common therapeutic antimicrobials like chloramphenicol, trimethoprim sulfamethoxazole, tetracyclines, ciprofloxacin, cefepime, and imipenem.17 therefore, broad - spectrum cephalosporins, carbapenems or fluoroquinolones can be used as an appropriate initial choice for c. violaceum infections due to the unavailability of recommended therapeutic guidelines. timely diagnosis and aggressive antimicrobial therapy would be a critical factor for an effective management of c. violaceum and reduce the high mortality associated with these infections.5,7 in our case, timely intervention with administration of broad - spectrum antibiotics to which the organism was sensitive, assisted in proper management of the illness, and the infection did not progress to septicemia and other complications. although human infections with c. violaceum are rare, they are often associated with higher morbidity and mortality. nevertheless, the clinical symptoms and presentation of the infection are not distinctive, it should be included in differential diagnosis of sepsis, especially if exposure to stagnant water and skin breaching is involved. | chromobacterium violaceum is a gram negative saprophytic bacterium, prevalent in tropical and subtropical climates. infections caused by c. violaceum are very uncommon, yet it can cause severe systemic infections with higher mortality when entered into the bloodstream through open wound. a case of symptomatic bacteremia in a woman caused by c. violaceum was identified recently at a tertiary care teaching hospital in nepal. timely diagnosis by microbiological methods and rapid administration of antimicrobials led to a successful treatment of this life - threatening infection in this case. from this experience, we suggest to include this bacterium in the differential diagnosis of sepsis, especially when abraded skin is exposed to soil or stagnant water in tropical areas. the precise antimicrobial selection and timely administration should be considered when this infection is suspected. |
both lipoprotein glomerulopathy (lpg) and fibrillary glomerulonephritis (fgn) are rare causes of end - stage renal disease (esrd). lpg is a rare disease of renal lipoidosis, first described by saito. in 1989. to date, 150 cases of lpg have been reported in the literature, with most of them in japan and east asian countries [2, 3 ]. it has been shown that half of the patients with lpg eventually develop esrd at 127 years after onset of symptoms. however, the literature concerning the outcome of kidney transplantation in patients with lpg was scarce. only five kidney transplants have been reported and lpg recurred early in all the transplanted allografts [48 ]. while usually cases without complications are less likely to be reported, it is noteworthy that cases without lpg recurrence have yet been described. on the other hand, fgn is also a rare deposition disease comprising only 1% of native kidney biopsies [9, 10 ]. it is characterized by the deposition of organized microtubules, measuring 1624 nm in diameter and arranged in a parallel fashion. fgn usually progresses to esrd within months to a few years but the risk of recurrence in kidney transplant is low according to the limited number of cases. herein we report a patient with coexisting lpg and fgn, who underwent deceased kidney transplant > 10 years ago and did not reveal any clinical features of recurrence of diseases after long - term follow - up. a 34-year - old chinese man first presented to our hospital with 1-year history of ankle swelling. laboratory investigations revealed a 24-h urinary protein excretion > 10 g per day, serum albumin 21 g / l, serum creatinine 123 mol / l, total cholesterol 10.1 mmol / l and triglyceride 4.7 mmol / l. light microscopy revealed 13 out of 36 glomeruli showing advanced sclerosis and another six showing segmental sclerosis. electron microscopy showed abundant non - branching fibrillary material ranging from 1619 nm in diameter in the mesangium. electron - lucent areas were noted in the subendothelial space with fragmented filamentous material ranging from 1116 nm in diameter (figure 1a). 1.(a) electron microscopy showing abundant non - branching fibrillary material in the subendothelial space. (b) light microscopy showing marked interstitial inflammation consisting of a large amount of polymorphs in the graft kidney specimen. (c) light microscopy showing some vacuolated and stringy materials inside the dilated capillary lumens in the native kidney specimen. (d) electron microscopy showing the intraluminal materials composed of fine granular material with small lipid vacuoles, consistent with lipoprotein thrombi. (a) electron microscopy showing abundant non - branching fibrillary material in the subendothelial space. (b) light microscopy showing marked interstitial inflammation consisting of a large amount of polymorphs in the graft kidney specimen. (c) light microscopy showing some vacuolated and stringy materials inside the dilated capillary lumens in the native kidney specimen. (d) electron microscopy showing the intraluminal materials composed of fine granular material with small lipid vacuoles, consistent with lipoprotein thrombi. three years later, he presented with uremic symptoms and blood tests showed urea nitrogen 46 mmol / l, creatinine 2011 mol / l, albumin 28 autoimmune markers including antinuclear, antineutrophil cytoplasmic and antiglomerular basement membrane antibodies were all negative. three months later, he received deceased kidney transplant and immunosuppression included prednisolone, tacrolimus and mycophenolate mofetil. his serum creatinine remained stable (at 120 mol / l) without any significant proteinuria. he suffered an episode of acute kidney injury with serum creatinine reaching 201 mol / l during follow - up at 18-months post - transplant. renal graft biopsy was performed and light microscopy revealed marked interstitial inflammation consisting of a large amount of polymorphs which were also noted within the tubular lumens (figure 1b). however, subsequent review of his native kidney biopsy specimen showed that in addition to features of fgn, components of lpg which were characterized by some vacuolated and stringy materials inside the dilated capillary lumens (figure 1c) were also seen. positive and stained pale blue under chromotrope - aniline blue (cab) stain. on electron microscopy, these intraluminal materials contained fine granular material with small lipid vacuoles, consistent with lipoprotein thrombi (figure 1d). these features were not present in the graft biopsy specimen obtained 18 months after transplant. mutational analysis of the apolipoprotein (apoe) gene by polymerase chain reaction and direct dna sequencing revealed an e3/e3 genotype and a heterozygous mutation c.480_488del, which is predicted to result in deletion of leu - arg - lys at codons 162164 (reference sequences nm_000041.2 and np_000032.1). a familial genetic study on his son at 18 years of age showed that he also carries the same mutation, though there was no biochemical evidence of proteinuria or renal disease at the time of testing. his maintenance immunosuppressive regimen remained prednisolone, tacrolimus and mycophenolate mofetil while he was also put on diltiazem and lisinopril for blood pressure control. after > 10 years post - transplant, his latest serum biochemistry and other laboratory examination revealed no significant abnormality. these included serum creatinine 125 mol / l, serum albumin 42 g / l, total cholesterol 4.8 mmol / l, triglyceride 1.2 mmol / l and 24-h urinary protein excretion 10 years. our case shows that the prognosis of patients with coexisting fgn and lpg who have received a kidney transplant can be good. prior studies illustrated that the location of apoe mutations is an important determinants for the development of lpg. however, the relationship between various apoe mutations and lpg recurrence in the kidney graft is not well established because of the limited number of patients. moreover, family studies showed that lpg occurs in some members but not in others even with the same phenotypic and genotypic apoe abnormalities [5, 14 ]. as a result, it has been postulated that some other possibilities such as local mechanisms in the glomeruli or environmental factors in addition to the apoe genotype are required for disease expression. further studies are thus required for clarification of the exact pathogenic mechanism of lpg. in conclusion, our experience shows that kidney transplantation remains a viable therapeutic option for patients with esrd secondary to fgn with lpg. | both lipoprotein glomerulopathy (lpg) and fibrillary glomerulonephritis (fgn) are rare causes of end - stage renal disease (esrd), and the literature concerning the outcome of kidney transplant in patients with lpg or fgn is scarce. we report a patient who suffered from esrd with coexisting fgn and lpg and received deceased kidney transplant > 10 years ago did not reveal any clinical features of disease recurrence during follow - up. our case shows that the prognosis of patients with lpg component who received kidney transplant can be good. kidney transplantation remains a viable therapeutic option for patients with esrd secondary to fgn with lpg. |
malaria is one of the most common parasitic infections in the developing countries and cerebral malaria (cm) is one of the most common causes for non - traumatic encephalopathy in the world. malaria is caused by five species of plasmodium namely plasmodium vivax, plasmodium falciparum, plasmodium malariae, plasmodium ovale and plasmodium knowlesi. however, they are increasing reports of complicated malaria by other species as well. clinical presentation of malaria varies from uncomplicated acute febrile illness to a severe form of malaria when infections are complicated by serious organ failures or abnormalities in the patient 's blood or metabolism. the manifestations of severe malaria include cm, severe anemia, hemoglobinuria due to hemolysis, acute respiratory distress syndrome, abnormalities in blood coagulation, hypotension, acute kidney injury, hyperparasitemia (more than 5% of the red blood cells (rbcs) infected by malarial parasites), metabolic acidosis and hypoglycemia. severe malaria is a medical emergency and should be treated urgently and aggressively. in the indian scenario, for the same reason, these physicians should be aware of the complications, so that earlier and effective treatment can be initiated and patients who need a referral to a tertiary care can also be identified. this was a prospective study done from september 2005 to december 2006 at jiwan jyoti christian hospital in eastern uttar pradesh in india. all the patients above the age of 14 years diagnosed with cm were included in the study. cm was defined as a clinical syndrome of coma (inability to localize a painful stimulus) at least 1 hour after termination of a seizure or correction of hypoglycemia, detection of asexual forms of falciparum malarial parasite on peripheral blood smear and exclusion of other causes of encephalopathy. there were a total of 53 patients with cm of which 38 (71.7%) of them were females. among them 35 (66%) patients were less than 30 years of age [figure 1 ] all of them had presented with fever and altered sensorium with documented malarial parasite on the peripheral blood film. other clinical features [figure 2 ] noted were pallor (35%), icterus (16.98%), hypotension (13.2%), bleeding (3.7%), hepatomegaly (5.66%) and splenomegaly (5.66%). age distribution of patients with cerebral malaria clinical features of patients with malaria majority (84.9%) of the patients had anemia. co - infection with plasmodium vivax was present in 13 (24.53%) of them. treatment received included artesunin compounds (artesunate in 37 and arteether in 3), quinine (9) and quinine doxycycline combination therapy (2). in view of clinical failure 3 of them others included patients who were referred to higher center and those who left the hospital against medical advice. cm is defined as severe p. falciparum malaria with cerebral manifestations, usually coma (glasgow coma scale 30 min after a seizure is also considered to be cm. the 2010 revised criteria for severe malaria are the presence of one or more of the following : prostration, impaired consciousness, failure to feed, respiratory distress (" air hunger "), multiple seizures (more than two episodes in 24 h), circulatory collapse, pulmonary edema (on radiological imaging), abnormal spontaneous bleeding, jaundice, hemoglobinuria, severe anemia, hypoglycemia, acidosis, renal impairment, hyperlactatemia, and hyperparasitemia. according to the latest world health organization (who) estimates, there were about 219 million cases of malaria in 2010 and an estimated 660,000 deaths. africa is the most affected continent : about 90% of all malaria deaths occur there. among the 53 patients with cm, 71.7% were females and 66% were less than 30 years of age. however wasnik. noted a higher incidence in males (75%) most of whom were in the age group of 21 - 30 years of age. seizures are a prominent feature in cm and repeated seizures have been associated with poor outcome. patel noted that 46.8% of the patients with falciparum malaria had anemia whereas wasnik. noted that 65% of the cases had anemia. in our cohort 84.9% had anemia. the causes for anemia in cm patients are obligatory destruction of rbcs containing parasites at merogony, accelerated destruction of non - parasitized rbcs and bone marrow dysfunction. hypoglycemia in cm patients are due to increased peripheral requirement of glucose consequent upon anaerobic glycolysis, increased metabolic demands of febrile illness, obligatory demand of parasites, failure of hepatic gluconeogenesis and glycogenolysis (parasites consume up to 70 times as much glucose as uninfected cells). it is compounded by the stimulation of insulin secretion from pancreatic beta cells by quinine. blood sugars of all the patients in the present study were closely monitored and glucose supplementation was given. renal dysfunction causes morbidity in these patients. biochemical evidence of renal dysfunction was noted in 37.6% patients. out of 526 cases of cm reported from rourkela, in sundargarh district of orissa state, 28.9% had acute renal failure (arf). mortality in this series was particularly high (59%) specifically in those with multiorgan failure. the effect of associated arf on mortality in cm patients indicated, mortality was as high as 39.5% when associated with arf, while it was only 13.9% when unassociated with arf. hyperventilation (kussmaul breathing) with a clear chest on auscultation suggests metabolic acidosis. at our center since there was no facility for estimation of blood gases, we made a clinical diagnosis based on this and was initiated on soda bicarbonate infusion and fluids. clinical improvement as indicated by normal respiratory rate guided us in our management. in our study, 16.9% patients had non cardiogenic pulmonary edema. case fatality rate is very high in these patients and they should be referred to a facility were mechanical ventilation and intensive care is available. the world health organization now recommends using intravenous artesunate in preference to quinine for the treatment of severe p. falciparum malaria in adults. these are derivatives of chinese drug qinghaosou. in a large open label, randomized trial of asian adults with severe malaria, artesunate significantly reduced mortality by 34.7%. majority (75.47%) of our patients. out of these except for 3 patients rest of them had responded to artesunate. noted that the cause of death were arf, metabolic acidosis, aspiration pneumonia and circulatory failure. case fatality rates in the other studies from africa have shown a high mortality rate of 13 - 21%. primary care physicians should be able to diagnose cm from the clinical presentation, which should be supported by detection of p. falciparum or in some cases the other species of malaria on the peripheral blood film. artesunin components as combination therapy are the drug of choice as per who recommendations and can be easily administered with hardly any side effects. non cardiogenic pulmonary edema needing ventilator care and renal failure needing dialysis should be referred to a higher facility. cm once considered a fatal disease has shown remarkable improvement in the outcome with the wide availability of artesunin components. most of the complications of severe falciparum malaria including cm can be managed by a primary care physician. considering the health system in our country if primary care physicians can manage these patients, it will go a long way in reducing the morbidity and mortality of these cases. | introduction : cerebral malaria (cm) is one of the most common causes for non - traumatic encephalopathy in the world. it affects both the urban and rural population. it is a challenge to treat these patients in a resource limited setting ; where majority of these cases present.materials and methods : this was a prospective study carried out from september 2005 to december 2006 at jiwan jyoti christian hospital in eastern uttar pradesh in india. this is a secondary level care with limited resources. we studied the clinical profile, treatment and outcome of all the patients above the age of 14 years diagnosed with cm.results:there were a total of 53 patients with cm of which 38 (71.7%) of them were females. among them, 35 (66%) patients were less than 30 years of age. the clinical features noted were seizure (39.62%), anemia (84.9%), icterus (16.98%), hypotension (13.2%), bleeding (3.7%), hepatomegaly (5.66%), splenomegaly (5.66%), non - cardiogenic pulmonary edema (16.98%) and renal dysfunction (37.36%). co - infection with plasmodium vivax was present in 13 (24.53%) of them. treatment received included artesunin compounds or quinine. median time of defervescence was 2 (interquartile range1 - 3). complete recovery was achieved in 43 (81%) of them. two (3.7%) of them died.conclusion:cm, once considered to be a fatal disease has shown remarkable improvement in the outcome with the wide availability of artesunin and quinine components. to combat the malaria burden, physicians in resource limited setting should be well trained to manage these patients especially in the endemic areas. the key to management is early diagnosis and initiation of treatment based on a high index of suspicion. anticipation and early recognition of the various complications are crucial. |
the term click chemistry describes a collection of organic reactions that proceed rapidly and selectively under mild conditions to covalently link molecular components.(1) among the many click reactions described to date, the huisgen 1,3-dipolar cycloaddition of azides and alkynes(2) has received the most attention. the reaction is highly exergonic (g 61 kcal / mol),(3) the starting materials are easily prepared, the 1,2,3-triazole products are exceptionally stable, and the reaction occurs readily in both organic and aqueous solvents. however, elevated temperatures or pressures are necessary to accelerate the reaction when simple alkynes and azides are employed, since the activation energy for the cycloaddition is high (g + 26 kcal / mol).(3) in the past decade, several strategies have been pursued in order to lower the activation barrier. sharpless and co - workers(4) and meldal and co - workers(5) first reported that cu(i) catalysis dramatically accelerates the reaction of terminal alkynes and azides, regioselectively forming the 1,4-disubstituted triazoles. more recently, ruthenium - based catalysts have been employed to produce the 1,5-disubstituted isomer.(6) these metal - catalyzed variants of the huisgen [3 + 2 ] cycloaddition are now central tools for combinatorial library synthesis,(7) construction of peptidomimetics and glycomimetics,(8) supramolecular synthesis,(9) and labeling of biomolecules in vitro or in fixed samples. however, the reliance of these chemistries on cytotoxic transition metals has largely precluded their use for applications in vivo, despite parallel growth in the use of azides as chemical reporters in biological systems.(15) as an alternative approach to lower the activation barrier for [3 + 2 ] cycloaddition, we have employed the intrinsic ring strain of cyclooctynes, following the precedent of wittig and krebs. these highly strained alkynes (18 kcal / mol of ring strain(18)) react selectively with azides to form regioisomeric mixtures of triazoles at ambient temperatures and pressures without the need for metal catalysis and with no apparent cytotoxicity (eq 1) : we further enhanced the cycloaddition rate by installing propargylic fluorine atoms intended to lower the lumo, thereby increasing its interaction energy with the homo of the azide,(19) an effect that was recently studied using density functional theory calculations.(20) the difluorinated cyclooctyne we termed difo (compound 1 in figure 1) reacted with azides on intact proteins at a rate comparable to that of cu - catalyzed click chemistry and has since been employed for dynamic imaging of cell - surface glycans in live cells(19) and in developing zebrafish embryos.(21) other groups have reported the use of strained oxanorbornadiene(22) and dibenzocyclooctyne(23) reagents as substrates for [3 + 2 ] cycloaddition with azides, suggesting a rich future for cu - free click chemistry. terminal alkynes can be installed in biomolecules using simple building blocks, such as commercial alkynoic acids. in contrast, the synthesis of difo comprised 12 steps and an overall yield of 1%.(19) the final step of the sequence, elimination of a vinyl triflate to form the cyclooctyne, suffered from significant decomposition and low yield. thus, synthetically tractable cyclooctynes with the capabilities of difo are needed in order to expand the use of this cu - free click reagent in biological settings. here we report the design, synthesis, and biological evaluation of the second - generation difo reagents 2 and 3 (figure 1), which retain the difluorinated cyclooctyne core but possess a cc bond to a linker substituent at c4 rather than a co bond at c6 as in 1 (figure 1). this structural change dramatically simplified the synthesis without impact on the kinetics or bioorthogonality of [3 + 2 ] cycloaddition with azides. these synthetically tractable second - generation difo reagents should facilitate further applications of cu - free click chemistry. all of the chemical reagents were analytical grade, obtained from commercial suppliers, and used without further purification, unless otherwise noted. flash chromatography was carried out with merck 60 230400 mesh silica gel according to the procedure described by still.(24) when necessary, deactivated silica gel was prepared by rinsing silica gel thoroughly with a solution of 1% et3n in the starting solvent mixture used for flash chromatography. reactions and chromatography fractions were analyzed with analtech 250 m silica gel g plates and visualized by staining with ceric ammonium molybdate, anisaldehyde, vanillin, or 2,4-dinitrophenylhydrazine or by absorbance of uv light at 245 nm. solvents were removed using a rotary evaporator at reduced pressure (20 torr). unless otherwise noted, h, c{h }, f, and p{h } nmr spectra were obtained with 300, 400, or 500 mhz bruker spectrometers. chemical shifts () are reported in parts per million referenced to the solvent peaks for h and c, to cfcl3 for f, and to h3po4 for p. coupling constants (j) are reported in hertz. low - resolution and high - resolution (hr) fast atom bombardment (fab), electron impact (ei), and electrospray ionization (esi) mass spectra were obtained at the uc berkeley mass spectrometry facility. reversed - phase hplc was performed using a rainin dynamax sd-200 hplc system with 210 nm detection on a microsorb c18 analytical or preparative column. dulbecco s phosphate - buffered saline (pbs) and fetal bovine serum (fbs) were purchased from hyclone laboratory, and rpmi-1640 and f12 media were obtained from invitrogen life technologies, inc. fluorescein isothiocyanate (fitc)-labeled avidin was purchased from sigma - aldrich. flow cytometry analysis was performed on a bd facscalibur flow cytometer using a 488 nm argon laser. cell viability was ascertained on the basis of forward scatter (to sort by size) and side scatter (to sort by granularity). the average fluorescence intensity was calculated from each of three replicate experiments in order to obtain a representative value in arbitrary units. for all of the flow cytometry experiments, data points were collected in triplicate and represent at least two separate experiments. fluorescence microscopy was performed on a zeiss 200 m epifluorescence microscope, and the images were deconvolved using the nearest - neighbor algorithm of slidebook 4.2 (intelligent imaging innovations, inc.). a flame - dried round - bottom flask was charged with 1,3-cyclooctanedione (5.10 g, 36.4 mmol) and mecn (260 ml). cs2co3 (24.3 g, 74.6 mmol) was added, and the reaction mixture was stirred at room temperature (rt) for 30 min. the reaction mixture was cooled to 0 c, and selectfluor (31.0 g, 87.4 mmol) was added, after which the mixture was stirred for an additional 15 min. the system was allowed to warm to rt, stirred for 1.5 h, concentrated under reduced pressure, diluted with 1 m hcl (200 ml), and extracted with diethyl ether (4 200 ml). the combined organic layers were washed with brine (2 100 ml), dried over mgso4, and filtered through a glass frit. the solution was concentrated under reduced pressure and purified by flash chromatography (9:1 hexanes / etoac) to yield a white solid (4.70 g, 73%). h nmr (500 mhz, cdcl3) : 2.67 (m, 4h), 1.81 (m, 4h), 1.63 (m, 2h). c nmr (125 mhz, cdcl3) : 197.9 (t, j = 25.1 hz), 109.5 (t, j = 261.5 hz), 38.7, 26.2, 24.7. ir (thin film, cm) : 3468, 2946, 2867, 1732. hrms (ei) : calcd for c8h10o2f2, 176.0649 ; found, 176.0646. to a flame - dried round - bottom flask were added diketone 4 (1.57 g, 8.92 mmol), phosphonium bromide 5 (4.60 g, 9.37 mmol), and thf (200 ml). the system was cooled to 0 c, and dbu (1.34 ml, 8.92 mmol) was added, after which the reaction mixture was stirred for 20 min at 0 c. after the reaction mixture was warmed to rt, it was stirred for an additional 48 h ; the reaction was quenched with acoh (1.5 ml), and the mixture was diluted with meoh (20 ml), concentrated under reduced pressure, and purified by flash chromatography (05% etoac in a 2:1 mixture of hexanes / toluene) to yield a white solid (2.63 g, 96%). h nmr (400 mhz, cdcl3) : 8.05 (d, 2h, j = 8.4 hz), 7.38 (d, 2h, j = 8.2 hz), 7.23 (s, 1h), 3.92 (s, 3h), 2.70 (t, 2h, j = 6.6 hz), 2.52 (app t, 2h, j = 6.2 hz), 1.86 (m, 2h), 1.53 (m, 4h). c nmr (125 mhz, cdcl3) : 202.1 (t, j = 28.9 hz), 166.8, 140.0, 134.6 (t, j = 19.6 hz), 131.2 (t, j = 10.3 hz), 130.0, 129.7, 129.0, 115.2 (t, j = 253.4 hz), 52.4, 37.5, 27.3, 26.0, 25.7, 25.3 (t, j = 2.5 hz). hrms (fab) : calcd for c17h18o3f2 [m + h ], 309.1302 ; found, 309.1302. to a round - bottom flask were added olefin 6 (2.63 g, 8.53 mmol) and meoh (100 ml). the system was flushed with n2, and a catalytic amount of pd / c was added. the system was again flushed with n2 followed by h2, and the reaction mixture was stirred under an h2 atmosphere (using a balloon) for 24 h. the system was then flushed thoroughly with n2, after which the reaction mixture was diluted with ch2cl2 (100 ml), filtered through celite, and concentrated under reduced pressure. the crude product was purified by flash chromatography (02% etoac in a 2:1 mixture of hexanes / toluene) to yield a white solid (2.47 g, 93%). h nmr (400 mhz, cdcl3) : 7.99 (dm, 2h, j = 8.3 hz), 7.27 (d, 2h, j = 8.14 hz), 3.92 (s, 3h), 3.31 (dd, 1h, j = 13.6, 2.9 hz), 2.822.73 (m, 1h), 2.662.48 (m, 2h), 2.452.28 (m, 1h), 2.172.02 (m, 1h), 1.971.84 (m, 1h), 1.661.42 (m, 4h), 1.411.29 (m, 1h), 1.291.11 (m, 1h). c nmr (125 mhz, cdcl3) : 205.7 (dd, j = 30.4, 25.1 hz), 167.1, 144.7, 130.0, 129.4, 128.6, 119.4 (dd, j = 258.0, 250.7 hz), 52.2, 46.4 (t, j = 21.6), 39.1, 33.8 (t, j = 4.8 hz), 27.2, 24.3 (d, j = 6.9 hz), 24.1 (d, j = 3.4 hz), 22.9. f nmr (376 mhz, cdcl3) : 102.72 (d, 1f, j = 245.5 hz), 122.67 (d, 1f, j = 251.7 hz). hrms (fab) : calcd for c17h20o3f2 [m + h ], 311.1459 ; found, 311.1467. to a flame - dried round - bottom flask was added thf (175 ml) followed by khmds (14.81 ml of a 0.5 m solution in toluene, 7.40 mmol). the reaction mixture was cooled to 78 c with stirring, and ketone 7 (2.19 g, 7.05 mmol) in thf (70 ml) was added dropwise over 20 min. the reaction mixture was stirred for an additional 40 min, and then a solution of tf2nph (2.65 g, 7.40 mmol) in thf (70 ml) was added via syringe. the system was warmed to rt with stirring over 21 h, and the reaction was then quenched with meoh (10 ml). the reaction mixture was concentrated under reduced pressure and purified by flash chromatography (05% etoac in 4:1 hexanes / toluene with 1% et3n) to yield a pale - yellow oil (2.74 g, 88%). h nmr (400 mhz, cd3cn) : 7.93 (d, 2h, j = 8.2 hz), 7.36 (d, 2h, j = 8.2 hz), 6.25 (t, 1h, j = 9.4 hz), 3.85 (s, 3h), 3.24 (dd, 1h, j = 13.5, 3.8 hz), 2.882.70 (m, 1h), 2.61 (dd, 1h, j = 13.5, 10.2 hz), 2.562.42 (m, 1h), 2.412.30 (m, 1h), 1.681.52 (m, 3h), 1.511.44 (m, 2h), 1.431.34 (m, 1h). c nmr (125 mhz, cd3cn) : 167.6, 145.8, 143.6 (t, j = 29.4 hz), 130.5, 130.4, 129.5, 129.1 (t, j = 4.0 hz), 120.5 (dd, j = 246.2, 243.1 hz), 119.5 (q, j = 319.1 hz), 52.7, 46.8 (app t, j = 22.0 hz), 35.2 (app d, j = 5.4 hz), 27.0, 26.2, 23.0, 21.8. f nmr (376 mhz, cd3cn) : 74.45 (s, 3f), 93.93 (d, 1f, j = 272.2 hz), 105.18 (d, 1f, j = 266.9 hz). hrms (fab) : calcd for c18h19o5f5s [m + h ], 443.0952 ; found, 443.0960. to a flame - dried round - bottom flask was added vinyl triflate 8 (2.74 g, 6.20 mmol) in thf (160 ml). the mixture was cooled to 20 c with stirring. in a separate flame - dried round - bottom flask, a 0.199 m solution of lda was made by adding n - butyllithium (8.46 ml of a 2.5 m solution in hexanes, 21.2 mmol) dropwise to a solution of diisopropylamine (3.59 ml, 25.4 mmol) in thf (93.8 ml) while stirring at 78 c. a portion of the lda solution (37.4 ml, 7.44 mmol) was added dropwise to the first mixture over 1 h using a syringe pump at 20 c. the reaction mixture was then brought to rt over 20 min, and the reaction was quenched with meoh (10 ml) ; the mixture was then concentrated under reduced pressure and purified by flash chromatography (03% etoac in 2:1 hexanes / toluene) to yield a white solid (1.58 g, 87%). h nmr (400 mhz, cdcl3) : 7.99 (d, 2h, j = 8.1 hz), 7.27 (d, 2h, j = 7.9 hz), 3.92 (s, 3h), 3.16 (app d, 1h, j = 11.2 hz), 2.602.43 (m, 2h), 2.412.24 (m, 2h), 2.101.88 (m, 2h), 1.871.68 (m, 2h), 1.621.44 (m, 1h), 1.211.08 (m, 1h). c nmr (125 mhz, cdcl3) : 167.2, 145.5, 130.0, 129.4, 128.4, 119.5 (t, j = 238.6 hz), 109.9 (t, j = 11.1 hz), 85.1 (dd, j = 47.2, 41.6 hz), 58.2 (t, j = 24.3 hz), 52.2, 34.5 (d, j = 4.7 hz), 32.6, 30.8 (d, j = 4.4 hz), 28.0, 20.4. f nmr (376 mhz, cdcl3) : 94.32 (d, 1f, j = 260.2 hz), 101.36 (dm, 1f, j = 259.8 hz). hrms (fab) : calcd for c17h18o2f2 [m + h ], 293.1353 ; found, 293.1357. to a round - bottom flask fitted with a reflux condenser were added cyclooctyne methyl ester 9 (1.58 g, 5.42 mmol), lioh (2.59 g, 108 mmol), water (6 ml), and dioxane (24 ml) under an air atmosphere. the reaction mixture was heated to 55 c and stirred for 3 h. the mixture was then cooled to rt and diluted with 1 m hcl until the ph of the solution was < 2. the combined organic layers were then washed (1:1 1 m hcl / brine, 50 ml), dried over mgso4, filtered through a glass frit, and concentrated under reduced pressure. the crude product was then purified by flash chromatography (9:1 to 3:1 hexanes / etoac with 12% acoh) to yield a white solid (1.08 g, 72%). rf = 0.45 (4:1 hexanes / etoac with 1% acoh) ; mp 181.0182.0 (dec). h nmr (400 mhz, cd3cn) : 10.108.80 (br s, 1h), 7.94 (d, 2h, j = 8.2 hz), 7.35 (d, 2h, j = 8.1 hz), 3.10 (d, 1h, j = 10.9 hz), 2.702.50 (m, 2h), 2.432.24 (m, 2h), 2.041.84 (m, 2h) 1.831.67 (m, 2h), 1.551.41 (m, 1h), 1.221.11 (m, 1h). c nmr (125 mhz, acetone - d6) : 167.6, 146.2, 130.8, 130.2, 129.6, 120.2 (dd, j = 239.1, 235.4 hz), 111.6 (app t, j = 11.3 hz), 85.5 (dd, j = 46.7, 41.9 hz), 58.6 (t, j = 24.2 hz), 34.9 (d, j = 4.9 hz), 33.3 (d, j = 2.1 hz), 31.6 (d, j = 4.7 hz), 28.4, 20.5. f nmr (376 mhz, cd3cn) : 93.81 (d, 1f, j = 258.7 hz), 101.32 (ddt, 1f, j = 258.8, 20.3, 7.0 hz). hrms (fab) : calcd for c16h16o2f2 [m + h ], 279.1197 ; found, 279.1190. to a flame - dried round - bottom flask were added dcc (3.66 g, 17.7 mmol), dmap (98.6 mg, 0.807 mmol), 3-methyl-3-oxetanemethanol (1.59 ml, 16.1 mmol), and ch2cl2 (20 ml). the mixture was cooled to 0 c with stirring. a solution of iodoacetic acid (3.00 g, 16.1 mmol) in ch2cl2 (30 ml) the reaction mixture was stirred for 1 h at 0 c and allowed to warm to rt over an additional 1.5 h ; the reaction was quenched with acetic acid (1 ml), and the mixture was stirred for an additional 30 min. the filtrate (300 ml total) was then washed with water (200 ml), saturated nahco3 (2 200 ml), and brine (200 ml). the organic layer was then dried over mgso4, filtered through a glass frit, and concentrated under reduced pressure. the crude product was diluted with ch2cl2 and again filtered through celite to remove any residual dicyclohexylurea that had precipitated. this material was then transferred to a new round - bottom flask and dissolved in thf (100 ml). triphenylphosphine (4.65 g, 17.7 mmol) was added, and the reaction mixture was stirred under n2 at rt for 40 h and then diluted with diethyl ether (100 ml) and filtered through a glass frit to isolate the precipitated product. residual solvent was removed under reduced pressure to yield a pale - yellow solid (7.93 g, 92% over two steps). h nmr (400 mhz, cdcl3) : 7.937.84 (m, 6h), 7.847.78 (m, 3h), 7.737.65 (m, 6h), 5.51 (d, 2h, j = 13.5 hz) 4.26 (s, 4h), 4.15 (s, 2h), 1.22 (s, 3h). c nmr (125 mhz, cdcl3) : 164.3 (d, j = 3.5 hz), 135.4 (d, j = 3.1 hz), 133.9 (d, j = 10.8 hz), 130.4 (d, j = 13.2 hz), 117.4 (d, j = 89.2 hz), 79.1, 71.1, 38.8, 33.6 (d, j = 56.6 hz), 21.0. hrms (esi) : calcd for c25h26o3p, 405.1620 ; found, 405.1631. to a flame - dried round - bottom flask were added phosphonium iodide 10 (5.66 g, 10.6 mmol), ch2cl2 (75 ml), and dbu (1.51 ml, 10.1 mmol), and the reaction mixture was stirred for 20 min. in a separate flame - dried round - bottom flask, difluoroketone 4 (1.78 g, 10.1 mmol) was dissolved in ch2cl2 (125 ml), and this solution was then added to the phosphonium iodide solution. the reaction mixture was allowed to stir for 48 h, concentrated under reduced pressure, and purified by flash chromatography (10:1 to 4:1 hexanes / etoac), yielding a white solid (2.87 g, 94%). h nmr (500 mhz, cdcl3) : 6.49 (s, 1h), 4.51 (d, 2h, j = 6.0 hz), 4.40 (d, 2h, j = 6.0 hz), 4.25 (s, 2h), 2.81 (t, 2h, j = 6.6 hz), 2.66 (t, 2h, j = 6.7 hz), 1.82 (m, 2h), 1.72 (m, 2h), 1.52 (m, 2h), 1.35 (s, 3h). c nmr (125 mhz, cdcl3) : 200.2 (t, j = 28.1 hz), 165.1, 150.4 (t, j = 20.0 hz), 121.1 (t, j = 9.6 hz), 114.2 (t, j = 254.6 hz), 79.7, 69.3, 39.2, 37.5, 26.7, 26.6, 26.0, 25.5, 21.3. f nmr (376 mhz, cd3cn) : 113.03 (s). hrms (fab) : calcd for c15h20o4f2 [m + h ], 303.1408 ; found, 303.1404. to a round - bottom flask were added compound 11 (804 mg, 2.66 mmol) and meoh (30 ml). the system was flushed with n2, and a catalytic amount of pd / c was added. the system was then flushed again with n2 followed by h2, and the reaction mixture was stirred under an h2 atmosphere for 24 h. the system was flushed thoroughly with n2, and the reaction mixture was diluted with ch2cl2 (30 ml) and filtered through celite to remove the catalyst. the crude material was dissolved in ch2cl2 (17 ml) and transferred via syringe to a new flame - dried round - bottom flask, which was under a n2 atmosphere and contained activated 4 molecular sieves. the mixture was then cooled to 0 c with stirring. in a separate flame - dried conical flask, a 0.20 m solution of bf3oet2 (100 l, 0.780 mmol) in ch2cl2 (3.9 ml) was prepared, and a portion of this solution (1.0 ml, 0.20 mmol) was added to the reaction mixture via syringe. the reaction mixture was warmed to rt and stirred for an additional 20 h before the reaction was quenched with et3n (0.5 ml). the reaction mixture was then concentrated under reduced pressure and purified by flash chromatography (20:1 hexanes / etoac with 1% et3n over deactivated silica gel) to yield a white solid (731 mg, 90% over two steps). rf = 0.70 (2:1 hexanes / etoac) ; mp 80.383.3 c. h nmr (500 mhz, cdcl3) : 3.89 (s, 6h), 2.65 (m, 1h), 2.622.46 (m, 2h), 2.21 (d, 1h, j = 14.6 hz), 2.101.94 (m, 2h), 1.90 (br s, 1h), 1.60 (m, 2h), 1.561.42 (m, 2h), 1.36 (m, 2h), 0.80 (s, 3h). c nmr (125 mhz, cdcl3) : 205.8 (dd, j = 29.7, 25.7 hz), 119.3 (dd, j = 257.6, 250.2 hz), 109.0, 72.8, 39.2 (t, j = 21.2 hz), 38.9, 34.6 (t, j = 4.7 hz), 30.5, 27.0, 26.3 (d, j = 7.2 hz), 24.8 (d, j = 2.7 hz), 23.3, 14.7. f nmr (376 mhz, cd3cn) : 103.10 (d, 1f, j = 246.4 hz), 124.20 (dm, 1f, j = 249.0 hz). hrms (fab) : calcd for c15h22o4f2 [m + h ], 305.1564 ; found, 305.1558. to a flame - dried round - bottom flask was added thf (30 ml) followed by khmds (2.66 ml of a 0.5 m solution in toluene, 1.33 mmol). the reaction mixture was cooled to 78 c with stirring, and ketone 12 (354 mg, 1.16 mmol) in thf (15 ml) was added dropwise via syringe over 15 min. the reaction mixture was stirred for an additional 3 h, and then a solution of tf2nph (457 mg, 1.28 mmol) in thf (15 ml) was added via syringe. the system was allowed to slowly warm to rt with stirring over 19 h. the reaction was then quenched with deactivated silica gel, and the mixture was concentrated under reduced pressure and purified by flash chromatography (20:1 to 15:1 hexanes / etoac with 1% et3n over deactivated silica gel) to yield a white solid (407 mg, 80%). h nmr (400 mhz, cdcl3) : 6.05 (t, 1h, j = 9.6 hz), 3.89 (s, 6h), 2.742.56 (m, 1h), 2.512.31 (m, 2h), 2.20 (dd, 1h, j = 14.5, 1.7 hz), 2.001.87 (m, 1h), 1.721.49 (m, 6h), 0.80 (s, 3h). c nmr (125 mhz, cdcl3) 143.2 (t, j = 30.2 hz), 126.9, 119.2 (app t, j = 246.5 hz), 118.6 (q, j = 320.0 hz), 108.9, 72.8, 40.9 (app t, j = 22.3 hz), 35.1, 30.5, 27.2, 26.6, 22.6, 21.8, 14.6. f nmr (376 mhz, cdcl3) : 74.52 (s, 3f), 93.80 (d, 1f, j = 269.8 hz), 104.75 (dm, 1f, j = 278.0 hz). hrms (fab) : calcd for c16h21o6f5s [m + h ], 437.1057 ; found, 437.1050. in a round - bottom flask, vinyl triflate 13 (407 mg, 0.932 mmol) was dissolved in toluene (20 ml) and concentrated under reduced pressure to remove trace moisture. the material was then dissolved in thf (20 ml), and the solution was cooled to 20 c with stirring. in a separate flame - dried round - bottom flask, a 0.20 m solution of lda was made by adding n - butyllithium (1.12 ml of a 2.5 m solution in hexanes, 2.80 mmol) dropwise to a solution of diisopropylamine (475 l, 3.36 mmol) in thf (12.4 ml) at 78 c. a portion of the lda solution (5.6 ml, 1.12 mmol) was added dropwise via syringe pump to the first mixture over 1 h. the reaction mixture was then brought to rt over 30 min, and the reaction was quenched with deactivated silica gel ; the mixture was then concentrated under reduced pressure and purified by flash chromatography (20:1 to 15:1 hexanes / etoac with 1% et3n over deactivated silica gel) to yield a white solid (159 mg, 59%). h nmr (500 mhz, cdcl3) : 3.90 (s, 6h), 2.54 (app dq, 1h, j = 23.5, 9.1 hz), 2.402.25 (m, 2h), 2.252.11 (m, 2h), 2.102.02 (m, 2h), 1.811.71 (m, 1h), 1.65 (dd, 1h, j = 14.6, 10.3 hz), 1.50 (app quint, 1h, j = 7.5 hz), 1.33 (m, 1h), 0.81 (s, 3h). c nmr (125 mhz, cdcl3) : 120.0 (t, j = 238.3 hz), 109.8 (t, j = 11.2 hz), 85.4 (dd, 47.1, 41.8 hz), 72.8, 51.9 (t, j = 23.8 hz), 35.2 (dd, j = 4.5, 1.6 hz), 32.8 (d, j = 4.8 hz), 32.7 (d, j = 2.1 hz), 30.5, 29.9, 28.2, 20.5, 14.8. f nmr (376 mhz, cd3cn) : 94.82 (d, 1f, j = 258.7 hz), 101.81 (ddt, 1f, j = 259.3, 24.1, 7.2 hz). hrms (fab) : calcd for c15h20o3f2 [m + h ], 287.1459 ; found, 287.1461. to a scintillation vial under an air atmosphere were added cyclooctyne orthoester 14 (90.4 mg, 0.316 mmol), meoh (4.5 ml), water (450 l), and ppts (159 mg, 0.632 mmol). the reaction mixture was stirred at rt for 24 h, after which the reaction was quenched with saturated nahco3 (2 ml) and the mixture concentrated under reduced pressure. the crude product was diluted with brine (8 ml) and extracted with etoac (3 10 ml). the combined organic layers were washed with brine (10 ml) and a hcl / brine solution (1:1 1 m hcl / brine, 2 10 ml), dried over mgso4, and filtered through a glass frit. the filtrate was concentrated under reduced pressure to yield a white solid (106.8 mg). a portion of this material (57.8 mg) was transferred to a round - bottom flask, where it was dissolved in dioxane (1 ml) and water (200 l). to this was added lioh (91 mg, 3.8 mmol), and the reaction mixture was stirred at rt for 3 h under an air atmosphere, after which the reaction was quenched with 1 m hcl (5 ml). the reaction mixture was further diluted with brine (3 ml) and extracted with etoac (4 10 ml). the combined organic layers were washed (1:1 1 m hcl / brine, 10 ml), dried over mgso4, and filtered through a glass frit. the filtrate was concentrated and purified by flash chromatography (20:1 hexanes / etoac with 2% acoh) to give a white solid (33 mg, 96% over two steps). rf = 0.66 (1:1 hexanes / etoac with 1% acoh) ; mp 87.488.9 c. h nmr (400 mhz, cdcl3) : 11.9010.60 (br s, 1h), 2.842.69 (m, 2h), 2.462.28 (m, 3h), 2.212.05 (m, 2h), 1.901.74 (m, 2h), 1.67 (m, 1h), 1.41 (m, 1h). c nmr (125 mhz, cdcl3) : 177.8, 119.1 (t, j = 238.6 hz), 110.7 (t, j = 11.2 hz), 84.7 (dd, j = 47.0, 41.6 hz), 52.6 (t, j = 24.4 hz), 33.7 (app d, j = 4.4 hz), 32.9 (d, j = 4.6 hz), 32.7 (d, j = 2.0 hz), 27.9, 20.5. f nmr (376 mhz, cdcl3) 94.64 (d, 1f, j = 260.0 hz), 100.82 (ddt, 1f, j = 260.2, 21.1, 6.8 hz). hrms (esi) : calcd for c10h11o2f2 [m ], 201.0722 ; found, 201.0729. to a flame - dried round - bottom flask were added cyclooctyne 2 (10.5 mg, 0.0377 mmol), ch2cl2 (0.5 ml), and diisopropylethylamine (16.4 l, 0.0943 mmol), and the mixture was cooled to 0 c with stirring. pentafluorophenyl trifluoroacetate (7.1 l, 0.042 mmol) was then added, and after 10 min, the system was warmed to rt. the reaction mixture was stirred an additional 1.5 h, concentrated, filtered through a plug of silica gel eluting with hexanes, and then concentrated to yield a white solid. this material was transferred to a new round - bottom flask and dissolved in anhydrous dmf (0.5 ml). biotinylated amine 16(25) (12.0 mg, 0.0268 mmol) was then added, followed by diisopropylethylamine (7.0 l, 0.040 mmol). the reaction mixture was stirred overnight, concentrated, and purified twice by flash chromatography (1% et3n in ch2cl2, then 5% meoh in ch2cl2) to yield a clear oil (9.7 mg, 36% over two steps). h nmr (400 mhz, cd3od) : 8.41 (m, 1h), 7.93 (m, 1h), 7.77 (d, 2h, j = 8.2 hz), 7.32 (d, 2h, j = 8.2 hz), 4.48 (dd, 1h, j = 7.8, 4.7 hz), 4.29 (dd, 1h, j = 7.9, 4.5 hz), 3.683.53 (m, 10h), 3.48 (q, 4h, j = 6.0 hz), 3.283.16 (m, 4h), 3.10 (d, 1h, j = 10.8 hz), 2.92 (dd, 1h, j = 12.7, 5.0 hz), 2.70 (d, 1h, j = 12.7 hz), 2.632.47 (m, 2h), 2.442.27 (m, 2h), 2.19 (t, 2h, j = 7.4 hz), 2.081.99 (m, 1h), 1.981.79 (m, 4h), 1.791.69 (m, 4h), 1.691.55 (m, 3h), 1.51 (dd, 1h, j = 15.9, 8.1 hz), 1.471.37 (m, 2h), 1.31 (t, 1h, j = 7.3 hz), 1.231.12 (m, 1h). c nmr (125 mhz, cd3od) : 176.1, 170.1, 166.3, 145.2, 134.0, 130.5, 128.7, 121.0 (dd, j = 238.9, 236.0 hz), 111.2 (t, j = 11.2 hz), 86.0 (dd, j = 46.8, 41.8 hz), 71.7, 71.7, 71.4, 71.4, 70.4, 70.1, 63.5, 61.8, 59.5 (t, j = 24.3 hz), 57.2, 41.2, 38.9, 38.0, 37.0, 35.2 (d, j = 4.0 hz), 33.8 (d, j = 1.5 hz), 32.0 (d, j = 4.6 hz), 30.6 (d, j = 3.8 hz), 30.0, 29.7, 29.1, 27.0, 20.8, 9.4. f nmr (376 mhz, cd3od) : 95.40 (d, 1h, j = 259.9 hz), 102.76 (ddt, 1h, j = 259.9, 20.1, 6.8 hz). hrms (fab) : calcd for c36h52n4o6f2s [m + li ], 713.3736 ; found, 713.3736. to a flame - dried round - bottom flask were added cyclooctyne 3 (26.3 mg, 0.130 mmol), ch2cl2 (2.0 ml), and diisopropylethylamine (57.0 l, 0.325 mmol), and the mixture was cooled to 0 c with stirring. pentafluorophenyl trifluoroacetate (25.0 l, 0.143 mmol) was added, and after 10 min, the system was warmed to rt. the reaction mixture was stirred for an additional 1 h, concentrated, and filtered through a plug of silica gel using 1% etoac in hexanes as the eluent to yield a white solid (41.4 mg). a portion of this material (6.5 mg, 0.018 mmol) was then transferred to a flame - dried conical flask and dissolved in anhydrous dmf (0.5 ml). biotinylated amine 16(25) (7.9 mg, 0.018 mmol) was added, followed by diisopropylethylamine (5.0 l, 0.027 mmol). the reaction mixture was stirred for 4 h, concentrated, and purified twice by flash chromatography (010% meoh in ch2cl2) to yield a clear oil. the oil was further purified by reversed - phase hplc (gradient of 570% ch3cn in h2o over 40 min, eluting at 27 min) to yield a clear oil (6.4 mg, 50% over two steps). h nmr (400 mhz, cd3od) : 4.49 (dd, 1h, j = 7.9, 4.3 hz), 4.30 (dd, 1h, j = 7.9, 4.5 hz), 3.683.55 (m, 8h), 3.52 (dt, 4h, j = 6.2, 1.4 hz), 3.27 (m, 4h), 3.21 (m, 1h), 2.93 (dd, 1h, j = 12.8, 5.0 hz), 2.822.66 (m, 2h), 2.49 (dd, 1h, j = 14.7, 3.9 hz), 2.462.28 (m, 2h), 2.20 (t, 2h, j = 7.4 hz), 2.182.00 (m, 3h), 1.851.70 (m, 7h), 1.701.52 (m, 4h), 1.501.33 (m, 3h). c nmr (125 mhz, cd3od) : 176.1, 173.8, 166.3, 120.9 (app t, j = 237.4 hz), 111.8 (app t, j = 11.0 hz), 85.7 (dd, j = 46.6, 42.0 hz), 71.7, 71.7, 71.4, 71.4, 70.1, 70.0, 63.5, 61.8, 57.2, 54.6 (t, j = 24.3 hz), 41.2, 38.1, 38.0, 37.0, 36.3 (d, j = 4.0 hz), 33.9 (d, j = 1.9 hz), 33.5 (d, j = 4.5 hz), 30.6, 30.0, 29.7, 29.0, 27.1, 20.8, 14.6. f nmr (376 mhz, cd3od) : 95.57 (d, 1f, j = 259.0 hz), 102.08 (ddt, 1f, j = 259.4, 20.9, 6.8 hz). hrms (fab) : calcd for c30h48n4o6f2s [m + h ], 631.3341 ; found, 631.3324. cyclooctyne 2 or 3 was mixed at a 1:1 molar ratio (15 mm) with benzyl azide in either (a) cd3cn or (b) a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o (ph 7), and the reaction was monitored by h nmr. the kinetic data were derived by following the change in integration of resonances corresponding to the benzylic protons in benzyl azide (4.4) compared to those of the benzylic protons in the triazole products (5.55.7). the second - order rate constants for the reaction were determined by plotting 1/[azide ] versus time and subsequently using analysis by linear regression. the second - order rate constant k (ms) corresponds to the determined slope. jurkat or chinese hamster ovary (cho) cells were maintained in a 5% co2, water - saturated atmosphere and grown in rpmi-1640 (jurkat) or f12 (cho) media supplemented with 10% fbs, penicillin (100 units / ml), and streptomycin (0.1 mg / ml). cell densities were maintained between 1 10 and 1.6 10 cells / ml. jurkat cells were incubated for 3 days in media containing 25 m ac4mannaz or no sugar. the cells were distributed into a 96-well v - bottom tissue culture plate and washed three times by sequential concentration by centrifugation (2500 g, 3 min, 4 c) and resuspension in 200 l of labeling buffer [pbs (ph 7.4) containing 1% fbs ]. the cells were then incubated at rt with 100 l of 010 m 15ac in labeling buffer for 060 min, with dilutions made from a 2.5 mm stock solution in 7:3 pbs / dmf. after incubation, cells were washed three times and resuspended in 100 l of fitc - labeled avidin (1:200 dilution in labeling buffer of a 1 mg / ml stock solution). after a 15 min incubation in the dark at 4 c, the cells were washed once and then incubated with fitc - labeled avidin for an additional 15 min at 4 c. the cells were washed three times and then diluted to a volume of 400 l for flow cytometry analysis. annexin v - pe staining was performed according to instructions from the manufacturer immediately prior to flow cytometry analysis. cho cells were incubated for 3 days in media containing 25 m ac4mannaz or no sugar in an eight - well labtek ii chambered coverglass (nunc). the cells were washed three times by sequential gentle aspiration of the media and addition of 500 l of media. the cells were then treated with 100 l of a 10 m solution of 15b or 15c (1:250 dilution in media from a 2.5 mm stock solution in 7:3 pbs / dmf) for 60 min at rt. the cells were washed three times, stained with fitc - labeled avidin (100 l of a 1:200 dilution in media from a 1 mg / ml stock solution) for 10 min at rt, washed three times, treated with hoechst 33342 dye (100 l of a 1:1000 dilution in media from a 1 mg / ml dmso stock solution) for 2 min at rt to stain the nuclei, washed twice, and imaged by epifluorescence microscopy. the synthesis of cyclooctyne 2 (scheme 1) began with 1,3-cyclooctanedione, which was prepared in 67% yield as previously described.(26) difluorination of 1,3-dicarbonyl compounds with selectfluor has been reported to occur sluggishly under neutral conditions(27) and more rapidly using enamine intermediates(28) or microwave - assisted strategies.(29) we found that simply treating the diketone with selectfluor and cs2co3 at 0 c produced 2,2-difluoro-1,3-cyclooctanedione (4) in 73% yield. in order to install a linker with a protected carboxylic acid fortuitously, we observed exclusive formation of monosubstituted product 6 even when excess amounts of 5 and base were used, thus enabling efficient desymmetrization of the diketone. the observed chemoselectivity is likely due to the high electrophilicity of the first ketone equivalent compounded with the low nucleophilicity of the stabilized ylide. hydrogenation of the olefin to saturated compound 7 and subsequent conversion of the ketone to a vinyl triflate (8) proceeded in good yield (82% over two steps). cyclooctyne 9 was formed by lda - mediated elimination, and the methyl ester was saponified to yield difo reagent 2 in a total of six steps in 36% overall yield from 1,3-cyclooctanedione. this yield is 25-fold higher than that for the previously reported synthesis of 1(19) and was achieved in half as many steps. a lesson from previous studies is that the hydrophobicity of difo reagents can contribute to nonspecific protein and cell binding. thus, we also synthesized a second - generation difo analogue lacking the nonessential phenyl moiety in the linker. the target, compound 3 (figure 1), possessed a carboxymethyl group to which probes were later attached. a key protecting - group strategy masked the carboxymethyl group as an oxabicycloortho (obo) ester in order to avoid acidic protons that would later interfere with vinyl triflate formation and elimination. as shown in scheme 2, 2,2-difluoro-1,3-cyclooctanedione (4) was reacted under basic conditions with phosphonium salt 10, which bears an oxetane ester, an obo ester precursor.(30) again, we observed selective monosubstitution to yield olefin 11 in 85% yield. hydrogenation of the olefin and conversion of the oxetane ester to the orthoester using bf3oet2(31) yielded ketone 12 in 90% yield. formation of vinyl triflate 13 and elimination to form cyclooctyne 14 proceeded in 80% and 59% yields, respectively. two - step deprotection of the obo ester to carboxylic acid 3 was accomplished by acidic deprotection using ppts to form a simple ester and subsequent saponification using lioh (86% yield).(32) the synthesis of 3 was accomplished with an overall yield of 28% from 1,3-cyclooctanedione. with these reagents in hand, we used h nmr to measure the kinetics of the copper - free click reaction with the model compound benzyl azide. for the reactions with 2 and 3 performed in cd3cn, we obtained second - order rate constants of (4.2 0.1) 10 and (5.2 0.2) 10 m s, respectively ; these values are comparable to that for the reaction of 1 with benzyl azide under identical conditions (7.6 10 ms).(19) to evaluate the kinetics of the [3 + 2 ] cycloaddition in an aqueous environment, we performed reactions of 2 and 3 with benzyl azide in a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o (ph 7) and obtained k values of (9.0 0.3) 10 and (8.6 0.9) 10 m s, respectively. all three reagents are considerably more reactive than cyclooctynes lacking the difluoromethylene group.(33) additionally, we investigated the stabilities of compounds 2 and 3 with respect to biologically relevant nucleophiles. we dissolved each cyclooctyne (20 mm) in a 7:3 mixture of cd3cn and 25 mm potassium phosphate in d2o (ph 7) and then added either 2-mercaptoethanol or 2-aminoethanol (20 mm). we observed no reaction after 24 h, as monitored by h and f nmr, suggesting that compounds 2 and 3 are stable to water, thiols, amines, and alcohols at physiological ph. finally, compounds 2 and 3 were found to be stable for many months when stored at 20 c. an important application of copper - free click chemistry is the nontoxic and rapid detection of azides within living systems, either for molecular imaging or for subsequent affinity capture. difo reagent 1 and its derivatives (e.g., the biotinylated derivative 15a(19)) have proven useful for bioorthogonal labeling of cell - surface glycans bearing azides, and we set out to evaluate the second - generation difo reagents in this context. we first derivatized carboxylic acids 2 and 3 as the biotinylated reagents 15b and 15c, respectively, via formation of the corresponding activated pentafluorophenyl esters and subsequent conjugation to an amine - derivatized biotin reagent (scheme 3). jurkat cells were incubated with 25 m peracetylated n - azidoacetylmannosamine (ac4mannaz) for 3 days, resulting in the metabolic labeling of cell - surface glycans with azido sialic acid (sianaz) residues (figure 2a).(34) the cells were washed and labeled with biotinylated reagents 15b and 15c either at various concentrations (010 m) for 60 min (figure 2b) or for various reaction times (060 min) at 10 m (figure 2c). in all cases, the cells were subsequently stained with fitc - labeled avidin and analyzed by flow cytometry, as described previously.(35) both of the second - generation difo reagents displayed concentration- and time - dependent reaction profiles with cell - surface - associated azides. we were pleased to observe that both reagents displayed very little background fluorescence labeling, though the more hydrophobic 15b resulted in slightly higher background fluorescence than 15c. cells were first incubated with ac4mannaz, which is metabolically converted to cell - surface sianaz residues, and subsequently reacted with difluorinated cyclooctyne probes for visualization. (be) jurkat cells were metabolically labeled with 25 m ac4mannaz (+ az) or no sugar (az) for 3 days. the cells were labeled with 15b or 15c either (b) for 60 min at 0, 1, 2, 5, or 10 m or (c) for 0, 15, 25, 35, or 65 min at 10 m. the cells were then stained with fitc - labeled avidin and analyzed by flow cytometry. (de) the cells were labeled with 15ac or no reagent (no rgt) for 60 min at 10 m and then sequentially stained with fitc - labeled avidin and annexin v - pe, followed by flow cytometry analysis. the error bars indicate the standard deviation of three replicate samples. shown in (d) is the mean fluorescence intensity (mfi) in arbitrary units (au). shown in (e) is the percentage of cells in each sample belonging to the population that stains highly with annexin v - pe. we then compared the efficacy of cell - surface azide labeling of 15b and 15c to the first - generation reagent 15a in a 60 min reaction at 10 m (figure 2d). the observed labeling with 15b and 15c was approximately half of that with 15a, consistent with their relative rate constants. these labeling efficiencies are all significantly higher than those previously reported for mono- or nonfluorinated cyclooctyne reagents or triarylphosphines capable of staudinger ligation.(33) finally, we tested the toxicity of probes 15ac by incubation of cells labeled as above (60 min, 10 m) with phycoerythrin - conjugated annexin v, a marker of apoptosis. as shown in figure 2e, none of the reagents caused a significant change in the percentage of annexin v - positive cells, indicating that difo reagents 15ac are not toxic to jurkat cells. finally, we applied these novel cyclooctynes to image cell - surface glycans in live cho cells. the cells were incubated for 3 days with 25 m ac4mannaz or with no sugar as a negative control and labeled with 10 m 15b or 15c for 60 min. subsequently, the cells were treated with fitc - labeled avidin and hoechst 33342, a live - cell nuclear stain, and imaged by epifluorescence microscopy. we observed clear azide - dependent labeling at the cell surface in the ac4mannaz - treated cells and minimal background fluorescence in the negative control for both 15b and 15c (figure 3), indicating that these second - generation difo reagents can be utilized for live - cell imaging applications. live - cell imaging of cell - surface glycans using second - generation difluorinated cyclooctynes. cho cells were metabolically labeled with (a, b) 25 m ac4mannaz or (c, d) no sugar for 3 days. the cells were labeled for 60 min with 10 m (a, c) 15b or (b, d) 15c, washed, stained with fitc - labeled avidin and hoechst 33342, and imaged by epifluorescence microscopy. images were deconvolved using the nearest - neighbor algorithm of slidebook 4.2 and are shown as maximum intensity z - projection fluorescence images over 7.5 m. we have developed synthetically tractable second - generation difluorinated cyclooctyne reagents for copper - free click chemistry. the synthesis of these reagents involves a novel method for one - step difluorination of 1,3-diketones, a selective wittig reaction to install the linkers, and the use of an orthoester as a cyclooctyne - compatible protecting group. furthermore, these reagents can be prepared efficiently, with overall yields that are more than 20-fold higher than that of the first - generation reagent. critically, we determined that these novel difo reagents are nontoxic to cells and can selectively tag azide - labeled biomolecules in living systems with efficiencies approaching that of the parent compound. we envision that these reagents will enable widespread use of copper - free click chemistry in the context of in vivo imaging and ex vivo labeling of biomolecules and in the generation of novel biocompatible materials.(36) | the 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site - selective labeling of biomolecules in vitro and in vivo. while copper catalysis has been widely employed to activate terminal alkynes for [3 + 2 ] cycloaddition, this method, often termed click chemistry, is currently incompatible with living systems because of the toxicity of the metal. we recently reported a difluorinated cyclooctyne (difo) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. here we report a novel class of difo reagents for copper - free click chemistry that are considerably more synthetically tractable. the new analogues maintained the same elevated rates of [3 + 2 ] cycloaddition as the parent compound and were used for imaging glycans on live cells. these second - generation difo reagents should expand the use of copper - free click chemistry in the hands of biologists. |
mutations in the tyrosine kinase domain of the epidermal growth factor receptor (egfr) gene have been identified in non - small cell lung carcinoma (nsclc), and they were correlated with clinical response to egfr tyrosine kinase inhibitor (tki) treatment. patients with egfr mutations have a higher response rate to egfr - tkis (60%-80%) than those with egfr wild type or unknown mutation status (10%-20%). in addition, egfr - tki has been shown to be superior to carboplatin - paclitaxel as initial treatment. dna - based molecular methods have been used for selection of patients who would benefit from tki therapy ; however, they are tedious, expensive, and not routine in clinical laboratories. recently generated mutation - specific rabbit monoclonal antibodies against the two most common nsclc - associated egfr mutations (e746-a750 deletion [del ] in exon 19 and l858r point mutation [l858r ] in exon 21) for detection of mutant egfr protein by immunohistochemistry (ihc). ihc using these two mutation - specific antibodies has been suggested as a reliable screening method for egfr - tki therapy [4 - 8 ]. however, the immunostaining patterns reported in previous publications were contradictory ; some heterogeneous, while others were described as homogeneous [9 - 11 ]. biopsy samples, however, may be the only tumor materials available for confirming egfr mutation status, particularly in patients whose lung cancer is at an advanced stage and unresectable. in addition, tumor cells in the samples are often low in quantity or of insufficient quality for molecular assays, leading to a second biopsy procedure for obtaining additional tissues. it is therefore important to determine whether biopsy samples are suitable for detection of mutant egfr protein. there has been significant interest in total egfr (tegfr) protein expression, in particular, before the development of mutation - specific antibodies, however, its clinical significance is controversial. some studies reported a better outcome after egfr - tki treatment for tumors overexpressing the tegfr protein, whereas other studies did not [12 - 14 ]. to date, the literature contains limited data regarding the association between tegfr and mutant egfr proteins. in the current study, immunohistochemical analysis of both biopsies and resected tumor tissues from lung adenocarcinoma with known egfr mutation status by direct dna sequencing was performed using mutation - specific antibodies against egfr with e746-a750 deletion in exon 19 and l858r mutation in exon 21. expression of tegfr was also investigated in order to determine possible correlations with mutant egfr proteins detected by mutation - specific antibodies. a total of 154 patients who underwent biopsy (n=78) or surgical resection (n=76) for pulmonary adenocarcinoma at the catholic university st. paraffin - embedded tissues were procured from biopsied or resected specimens in a blinded fashion to the clinical information and egfr mutation status, and ihc was performed using egfr mutation specific antibodies. the study protocol was approved by the institutional review board (irb) of st. tissue corresponding to the precisely identified tumor areas on the hematoxylin and eosin (h&e) slides was scraped for subsequent dna extraction using a qiamp kit (qiagen, valencia, ca). exons 18 to 21 of the tk domain in the egfr gene were amplified by polymerase chain reaction using specific primers, and dna sequencing was performed using the abi 3710 genetic analyzer (applied biosystems, foster city, ca). a single block of paraffin - embedded tissue from biopsy specimens was cut in serial 4-m sections. for construction of tissue microarrays (tmas) from surgically resected specimens, the most representative tumor areas were identified on a selected h&e slide and marked by a pathologist (j.y.). three replicate core samples, each measuring 2.0 mm in diameter, were obtained using a precise instrument, and arrayed on a recipient paraffin block. each case was tested using the following primary antibodies : del - specific monocloncal antibody (pre - diluted, clone sp111, ventana medical systems inc., tucson, az), l858r - specific monoclonal antibody (pre - diluted, clone sp125, ventana medical systems inc.), and tegfr antibody (1:100, clone sp9, spring bioscience, pleasanton, ca). briefly, 4-m sections were deparaffinized in xylene, and rehydrated through a graded series of ethanol. slides were labeled with antibody and protocol - specific bar codes, and loaded into a benchmark xt automated slide stainer (ventana medical systems inc.). the immunoreactions were detected using an ultraview universal dab detection kit (ventana medical systems inc.) and 3,3-diaminobenzidine, followed by counterstaining with hematoxylin and bluing reagent. immunohistochemical assessments were performed by an experienced pathologist (j.y.) without information on molecular - based egfr mutation status. immunoreactivity was scored based on membranous and/or cytoplasmic staining, as follows : 0, no staining or faint staining in 0.05), or in sensitivity, specificity, ppv, and npv were observed between biopsy samples and resected samples. the sensitivity, specificity, ppv, and npv of tegfr antibody were 29%, 77.2%, 46.2%, and 61.7%, respectively (data not shown). no correlation was observed between tegfr expression and egfr mutation (p=0.284), del - specific antibody ihc (p=0.64), or l858r - specific antibody ihc (p=0.125) (table 6). in the current study, we compared the expression status of two egfr mutation specific antibodies between biopsies and resection samples from pulmonary adenocarcinoma, which had been previously genotyped by direct dna sequencing. we also investigated tegfr protein for evaluation of its possible correlation with mutant egfr proteins detected by mutation - specific antibodies. the major findings of our study are as follows : (1) ihc with mutation - specific antibodies correctly identified egfr mutation status in 89% (137/154), showing negative immunostaining in 94.5% (103/109) of patients with egfr wild type or mutations other than e746-a750 deletion and l858r mutation, and positive reaction in 75.6% (34/45) of patients with corresponding mutations ; (2) no significant differences in the egfr mutation frequency were observed between biopsies and resection specimens ; (3) tegfr expression showed no association with immunoreactivity for both mutation - specific antibodies. specific antibodies against egfr with e746-a750 deletion and l858r for identification of mutant egfr proteins by ihc, and reported a sensitivity of 92% and specificity of 99% as compared with direct dna sequencing. although accumulating data have suggested ihc using the two mutation - specific monoclonal antibodies as a simple, rapid, and costeffective screening tool for discriminating lung cancer patients responsive to egfr - based therapies [4 - 10 ], the reported sensitivities and specificities were quite variable. in several investigations on nsclc, the specificities of both antibodies were relatively high (77%-100%), whereas the sensitivities of del - specific and l858r - specific antibodies ranged from 40% to 94% and 24% to 100%, respectively [5,6,9,10,15 - 17 ]. all used the same two antibodies (clone 43b2 specific for e746-a750 deletion and clone 6b6 specific for l858r mutation, cell signaling technology, danvers, ma), so that the wide range of sensitivities and specificities is most likely not due to the antibody used. in addition, we used egfr mutation - specific antibodies purchased from a different manufacturer (clones sp111 and sp125, respectively, ventana medical systems inc.). del - specific ihc showed a sensitivity of 71.4% and specificity of 97.1%, while l858r - specific ihc showed a sensitivity of 77.4% and specificity of 97.6%, compatible with previous reports. similar results were obtained by the most recent investigation using the same antibodies as in the current study. the wide range of sensitivities and specificities may be attributed to scoring systems, which play a critical role in ihc - based analyses. several scoring schemes for interpretation of mutation - specific ihc have been adopted in published papers. immunoreactivity was classified on the basis of staining intensity, sometimes multiplied by the percentage values, both of which are highly dependent on the researcher. some assessed immunostaining in membrane only, others in membrane and/or cytoplasm, or even based on cytoplasmic staining of tumor cells alone. furthermore, when using a four - grading scale (score 0 - 3), 1 + was defined as positive by some investigators, and 2 + was considered positive by others [7 - 10,19 ]. recently, an optimized protocol of an intensity of 2 + or more in membrane and/or cytoplasm of > 10% tumor cells for positivity was shown to be the most appropriate way to interpret the egfr mutation specific ihc. in our study, we performed roc analyses to determine the best cutoff for detection of egfr mutations, and found it to be a score of 1 + or more. consensus of universally accepted criteria in interpreting the results as positive or negative should be reached in order to produce objective and reproducible results before using mutation - specific ihc as a substitute for molecular methods. observed different staining intensity in at least one of three tma cores each from 15/33 positive tumors (45%). on the other hand, xiong. used whole tissue sections and reported homogeneity in staining pattern. this prompted us to investigate whether the type of specimen (biopsy vs. resection) would make a difference in determining mutation status by ihc. in the current study, observed staining patterns were homogeneous in both biopsies and tma cores obtained from each patient. in addition, no significant differences in mutant egfr protein detection rates were found between the biopsy samples and tmas (29.5% vs. 28.9%, p > 0.05). fan. described a heterogeneous expression pattern in some of their cases with no details given, and speculated the causal relationship between heterogeneity and lower sensitivity of del - specific antibody in their biopsy samples. however, they also detected the egfr mutation phenotype in 32.1% of biopsies and 36.3% of resection samples (p > 0.05) with high specificity (97.9%-100%), which is in concordance with our results, suggesting that ihc using two mutation - specific antibodies may be performed as reliably in biopsies as in resection samples. therefore, when biopsied specimens are the only materials available for egfr status in patients at advanced stage, and tumor cells in the samples are insufficient or inadequate for dna - based molecular methods, using ihc with mutation - specific antibodies may preclude another biopsy procedure to obtain ample tissues. the overall sensitivity of ihc with del - specific antibody for all exon 19 mutations was low (44%). this is because del - specific antibody is relatively specific for the frequent e746-a750 deletion and can not detect the alteration. in the current study, 11 of 25 patients with exon 19 mutations had alternative in - frame deletions other than e746-a750 deletion, and thus almost exclusively did not react with del - specific antibody. our observation of the lack of correlation between egfr mutation and tegfr ihc is not surprising. identification of tegfr protein by ihc did not always reflect the detection of egfr mutation, suggesting that mechanisms other than egfr mutation may also play a role in tegfr expression. as in the study herein, all previous publications, to the best of our knowledge, used tegfr antibodies detecting the external domain (ed) of egfr. reported a correlation of tegfr with mutation - specific antibodies, using another ed - specific antibody (clone 2 - 18c9, dako, carpinteria, ca) for tegfr.. a histologically inhomogeneous series of lung cancer may be one possible explanation for this discrepancy. ed - specific antibodies bind to receptors containing ed and do not discriminate between active and inactive receptors, whereas intracellular domain (id)specific antibody would detect active or even truncated forms of the receptor. one study demonstrated that egfr expression evaluated by ed - specific antibody did not predict patient survival, but egfr protein expression using an id - specific antibody was a significant predictor of response to gefitinib in nsclc patients. investigations on whether egfr id - specific antibody ihc is correlated with mutant egfr, and has an impact on diagnostic power in the ihc interpretation of the mutation - specific antibodies, may be of great help in more accurate assessment of egfr mutation status. ihc with egfr mutation specific antibodies exhibited extremely high specificity (94.5%) with good sensitivity (75.6%) in both biopsies and resection specimens specific antibodies may preclude a second biopsy procedure to obtain additional tissues for identification of egfr mutations by molecular biology techniques in biopsies from advanced cancer, especially when tumor cells in the samples are limited. negative cases, however, require further molecular - based analyses for identification of other mutations with low frequency. | purposemutation - specific antibodies have recently been developed for identification of epidermal growth factor receptor (egfr) mutations by immunohistochemistry (ihc). this study was designed to investigate whether the type of specimen (biopsy vs. resection) would make a difference in determining mutation status by ihc, and to evaluate whether biopsies are suitable for detection of mutant egfr protein.materials and methodsihc was performed using mutation - specific antibodies for e746-a750 deletion (del) and l858r point mutation (l858r) in biopsies and tissue microarrays of resected tumors from 154 patients with pulmonary adenocarcinoma. results were then compared with dna sequencing data.resultsmolecular-based assays detected egfr mutations in 62 patients (40.3%), including 14 (9.1%) with del, and 31 (20.1%) with l858r. ihc with two mutation - specific antibodies showed a homogeneous staining pattern, and correctly identified egfr mutation status in 89% (137/154). overall (biopsy / resection) sensitivity, specificity, positive predictive value, and negative predictive value were 75.6% (78.3%/72.7%), 94.5% (90.9%/96.3%), 85% (78.3%/88.9%), and 90.4% (90.9%/89.7%), respectively.conclusionour data showed that ihc using egfr mutation specific antibodies is useful for detection of egfr mutations with high specificity and good sensitivity not only for resection specimens but also for biopsy materials. therefore, ihc using egfr mutation specific antibodies may preclude a second biopsy procedure to obtain additional tissues for identification of egfr mutations by molecular assays in biopsies from advanced cancer, particularly when tumor cells in the samples are limited. |
human chorionic gonadotropin (hcg) is a heterodimeric hormone secreted by the trophoblastic tissue of the placenta as well as certain tumors. small amounts of hcg have been reported to be secreted by the pituitary gland in non - pregnant females and males. various commercial assays are available in the market to detect hcg in both serum and urine. they are also used in the diagnosis, management and follow - up of ectopic pregnancy, trophoblastic diseases and germ cell tumors. in the absence of pregnancy and neoplasia, elevated hcg in females studies have attributed this to false positivity due to the presence of heterophilic antibodies or a physiologic increase in hcg in post - menopausal females. low positive hcg values have led to empirical chemotherapy in some and delay or cancellation of important medical procedures in others. we report a series of non - pregnant female patients with chronic renal failure with positive serum hcg over a period of 1 year from our center. the database of all patients who underwent renal transplant at our center from july 2007 through july 2008 was reviewed. a total of 171 patients with end - stage renal disease were screened for renal transplantation during that year, 109 males and 62 females. the mean age of female patients was 53.7 years (range 23 - 79 years). for pre - transplant screening, all the female patients underwent serum hcg screening to rule out pregnancy, irrespective of their ages. hcg screening was carried out using hcg plus kit from roche pharmaceuticals, usa (0 - 5 iu / five out of 62 (8%) female patients had a positive hcg test (5 the test was repeated in 4 four out of five patients to rule out any discrepancy. a total of 3 patients underwent successful renal transplantation while one is still on the waiting list. two patients were dialysis - dependent while the remaining was being worked up for pre - emptive transplant. the mean value of hcg in post - menopausal patients was 8.4 (range 6 - 10). however, hcg was slightly higher in premenopausal patient. epstein - barr virus serologies were positive in all patients ; two were cytomegalovirus positive. no other common environmental exposures, which could have led to the production of heterophilic antibody, could be identified in them. hcg- is identical to the -subunit of luteinizing hormone (lh), follicle stimulating hormone (fsh) and thyroid stimulating hormone while subunit is exclusive to each hormone. normally produced by syncytiotrophoblast cells of placenta, hcg is also secreted by trophoblastic and gastrointestinal tumors. hcg is produced in very small amounts in men and non - pregnant women, primarily from the anterior pituitary gland. in pregnancy, 30% of hcg the remainder is cleared by other pathways, likely by metabolism in the liver and kidneys. the biological function of hcg is maintaining secretion of progesterone from corpus luteum until the placenta takes over this function. various other degraded and dissociated forms of hcg are present in the serum and urine, with little or no biological activity. besides being used as a marker of pregnancy and certain tumors, hcg screening tests are also carried out before most medical interventions in female patients. most immunoassays use different combinations of antibodies against different sites on hcg or its subunits. some assays detect non - nicked hcg alone, some detect non - nicked hcg and free hcg while others detect both nicked (biologically inactive) and non - nicked hcg. though these tests have high sensitivity and specificity, the heterogeneity in the assays may lead to false positive results. heterophilic antibodies in humans may cross - react with animal immunoglobulin used in the assay and can result in falsely positive tests. these antibodies develop following exposure to serum or tissue antigens of animals and occur in about 3 - 15% of healthy individuals. as a consequence, however, it has been observed that the values in such cases of false positive results range from 100 iu / l to 500 iu / l, a good deal higher than our results. moreover, the assay used in our patients contained blocking antibodies, which bind these heterophilic antibodies and prevent cross reactions. studies have shown that the serum concentration of hcg increases in post - menopausal females. suginami and kawaoi obtained serial sections of pituitary glands at autopsy from women of various chronological ages. they concluded that hcg - like substance was localized in the gonadotrophs of the pituitary glands obtained from post - menopausal females. stenman., in their study, showed that treatment of post - menopausal women with a combination of estrogen and progesterone reduced their serum hcg levels by half. this further points towards the pituitary production of hcg in them. in studies on female cohorts, hcg levels have been found to be proportionate to increases in fsh after menopause. other authors consider pituitary hcg to be produced with increasing menopausal production of lh, owing to the decreased negative feedback by progesterone. case reports have implied that hcg concentrations 5.0 iu / l should not be considered abnormal in post - menopausal women. in a study in females more than 55 years of age, hcg levels > 5 iu / l in 16/240 (6.7%) were observed., in their paper, suggested that the upper limits of normal of serum hcg for post - menopausal women be increased to 14.0 iu / l. thus, this post - menopausal rise in hcg may explain the elevated hcg in four of our patients. the role of chronic kidney disease (ckd) as a cause of elevated hcg levels has not been studied. it is well - known that 30% of hcg being produced is cleared by the kidney and an additional fraction metabolized by it. in our cohort of female ckd patients, hcg levels > 5 iu / l were observed in 5/62 (8.1%). in the subset of patients with age > 55, hcg levels more than 5 iu / ml were noted in 4/30 (13.3%). thus, the presence of ckd in our patients may have served to elevate the hcg levels further in them. however, the factors, which elevate the hcg levels in only some patients with ckd remain poorly understood. a prospective cross - sectional study on pregnant patients showed that mild renal or hepatic impairment has no effect on maternal serum screening, which includes hcg. whether more severe renal disease (such as in our patients) has an effect on hcg excretion is yet to be ascertained. reduced testosterone concentration with the increase in level of gonadotropins, including lh and fsh, in has been attributed to a wide range of factors including aging, chronic inflammation, malnutrition and vascular disease. in these circumstances, a pituitary overproduction of hcg in parallel to fsh / lh, akin to the reports above, also appears plausible. many women experience delays in their management and postponement of surgical procedures because of elevated hcg. instances of needless chemotherapy administered to such women because of the presumption of cancer have also been reported. our patients also had to undergo various other investigations increasing the overall cost and delaying treatment. these included obstetrics / gynecology referrals with follow - up, pelvic ultrasound examinations (one or more) and serial blood testing. the transplant procedure itself was rescheduled in one of the patients on account of the positive hcg. in summary, healthy non - pregnant females can have elevated hcg either due to false positive results or menopausal status. large - scale studies are needed to ascertain the prevalence of a positive hcg test by conventional criteria in post - menopausal females in the general population. correcting hcg levels for age and co - morbidities may be prudent in reducing procedural delays and treatment costs in such women. | women are often subjected to serum human chorionic gonadotropin (hcg) testing prior to diagnostic and therapeutic interventions. a positive result leads to further testing to rule out pregnancy and avoid possible fetal teratogenicity. the impact of chronic kidney disease (ckd) on hcg testing has not been studied. we report a series of 5 women out of 62 with ckd, who had a positive hcg test on routine pre - transplant screening at a single transplant center. we analyzed their case records retrospectively. despite aggressive investigation, their elevated hcg levels remained unexplained. the positive test contributed to delays in transplantation and increased overall cost of treatment. |
mechanical peripheral neuropathies are the consequence of local or extrinsic compression phenomena or impingement by an anatomic neighbor causing a localized entrapment. traumatic neuropathies are the result of either closed injuries or open injuries to peripheral nerves [1, 2 ]. both of these categories are characterized by an important inflammatory component that plays a central role in the pathogenesis of neuropathic pain. inflammatory cells (e.g., macrophages), the production of molecules that mediate inflammation (cytokines), and the production of nervous growth factors are involved [3, 4 ]. in animal models it has been demonstrated that peripheral nerve injuries induce a profound local inflammatory response that involves t cells and macrophages. in particular, in the neuropathic pain model induced by chronic constriction injury (cci) an important macrophage infiltrate has been described in the damaged sciatic nerve [57 ] and in the dorsal root ganglia [8, 9 ]. the inflammatory response paralleled with nervous tissue alterations and pain [7, 10 ]. n - palmitoylethanolamine (pea), the endogenous amide between palmitic acid and ethanolamine, belongs to the family of fatty acid ethanolamides (faes), a class of lipid mediators. pea exerts antinociceptive effects in several animal models [11, 12 ], prevents neurotoxicity and neurodegeneration [13, 14 ], and inhibits peripheral inflammation and mast cell degranulation. anti - inflammatory effects of pea have been associated with peroxisome proliferator - activated receptor-(ppar-) activation, a nuclear receptor fundamental in the control of inflammatory responses, and expressed in various cells of the immune system [19, 20 ]. pea does not elicit anti - inflammatory effects in mutant ppar- null mice (ppar-). indeed, when assessed in either the carrageen hindpaw or phorbol ester ear pinna tests, pea reduced inflammation in wild - type, but not in ppar-, mice. on the other hand, loverme and collegues demonstrated the pivotal role of ppar- in the pea pharmacodynamic mechanism to relieve pain. in a mouse peripheral neuropathy model (cci) we evaluated the effects of repeated pea treatments on the sciatic nerve lesions responsible for neuropathic pain. aimed to highlight the role of ppar- in pea - evoked neurorestoration during neuropathy, a morphological study has been performed in both wild - type and ppar- null mice. all procedures met the european guidelines for the care and use of laboratory animals (86/609/ecc and 2010/63/ue), and those of the italian ministry of health (dl 116/92). male wild - type (wt) and ppar- (ko) (b6.129s4-svjae - pparatm1gonz) mice, previously backcrossed to c57bl6 mice for 10 generations, were bred in our animal facility, where a colony was established and maintained by heterozygous crossing. mice were genotyped as described on the supplier webpage (http://jaxmice.jax.org/), with minor modifications. dna was extracted from tails using the redextract kit (sigma - aldrich, milan, italy). all animals were maintained on a 12 h light/12 h dark cycle with free access to water and standard laboratory chow. pea was from tocris (bristol, uk) ; it was dissolved in peg and tween 80 2 : 1 (sigma - aldrich) and kept overnight under gentle agitation with a microstirring bar. before injection, sterile saline was added so that the final concentrations of peg and tween 80 were 20 and 10% v / v, respectively. drug was injected subcutaneously (s.c.) in a dose of 30 mg kg0.3 ml mouse for consecutively 14 days from the day after surgery. the sciatic nerve of 5 - 6-week - old wt and ko mice were surgically ligated as described. in brief, the animals were anesthetized with ketamine (100 mg kg i.p) and xylazine (10 mg kg i.p.), the left thigh was shaved and scrubbed with betadine, and a small incision (2 cm in length) was made in the middle of the left thigh to expose the sciatic nerve. the nerve was loosely ligated at two distinct sites (spaced at a 2 mm interval) around the entire diameter of the nerve using silk sutures (7 - 0). behavioral tests were performed on the day 14 after surgery. we measured mechanical hyperalgesia using a randall - selitto analgesimeter for mouse (ugo basile, varese, italy). latencies of paw withdrawal to a calibrated pressure were assessed on ipsilateral (ligated) paws on day 14 following ligatures. cut - off force was set at 60 g. to assess for changes in sensation or in the development of mechanical allodynia, sensitivity to tactile stimulation was measured using the dynamic plantar aesthesiometer (dpa, ugo basile, italy). animals were placed in a chamber with a mesh metal floor covered by a plastic dome that enabled the animal to walk freely but not to jump. the mechanical stimulus was then delivered in the mid - plantar skin of the hind paw. the cutoff was fixed at 5 g. each paw was tested twice per session. this test did not require any special pretraining, just an acclimation period to the environment and testing procedure. cutoff force was set at 5 g. after the algesic test, animals were sacrificed and the ipsilateral sciatic nerves, 1 cm proximal and distal to the ligation, were explanted ; the portion containing the ligature was eliminated. spinal cord was removed, and lumbar section was immediately frozen in liquid nitrogen. the tissue samples were osmicated in 1% solution of osmium tetroxide for 2 h under constant agitation. before and after osmication, the tissue was repeatedly rinsed in 0.1 m sodium cacodylate at ph 7.4. after gradual dehydration in ethanol, the osmicated nerve samples were embedded in paraffin (diapath, milan, italy). transverse 5 m sections were cut on a reichert microtome (leica, rijswijk, the netherlands), mounted with canada balsam, and observed under a light microscopy. after the sacrifice, the sciatic nerve was fixed in situ using 4% formalin in phosphate buffer (ph 7.4), nerves were fixed in 4% buffered neutral formalin solution, and then the nerve was embedded in paraffin. finally 5 m sections were stained with azan - mallory for light microscopy studies and were graded for oedema and infiltrate. the sections were semiquantified by an arbitrary scale starting from 1, mild infiltrate and oedema, up to 10, severe infiltrate and widespread oedema. the procedure was carried out by an independent researcher who was masked to the experiment. morphometry was performed on cross sections of osmium - fixed sciatic nerves 10 mm starting from the level of injury or at the corresponding level in uninjured control nerves. the 10 mm cross section corresponded to a level distal or proximal to the injury. counts and measurements were carried out using imagej analysis software. the first step of morphometric analysis of the sciatic nerve consisted of identifying and capturing the entire fascicle image, followed by measurement of the fascicle perimeter and area by contouring its internal epineural (magnification : objective 20x). the next step consisted of capturing sequential inner areas of the fascicle (magnification : objective 100x). the high - magnification micrographs were randomly selected in nonoverlapping areas to cover 5075% of the total cross - sectional area of the nerve. the random selected histological images were converted into binary (black and white) images and cleaned of any blood vessels, degenerated nerve fibers, and artifacts, and the following parameters were then measured : total number and density of nerve fibers, axon diameter, and myelin sheath area. the number of small fibers, defined as fibers 6 m for large and < 6 m for small). cci was able to decrease the myelin thickness of large and small fibers in the distal portion of the ipsilateral nerve in respect to the sham both in wild - type and in knock - out mice (figures 3(a) and 3(b), large fibers ; figures 3(c) and 3(d), small fibers). in regard to axon diameter, a time - dependent decrease was revealed for all the fibers, particularly the small type, both in the distal and in the proximal portions of the ipsilateral nerve ; morphometry revealed a similar profile in ppar- (figures 4(a) and 4(c)) and ppar- (figures 4(b) and 4(d)) animals. in wild - type mice repeated pea administrations, 30 mg kg for 14 days, were able to preserve the nerve morphology. nerve sections of pea - treated mice showed a higher number of fibers in respect to the saline - treated groups (figure 2(a)) ; the myelin thickness in the distal portions of the nerve was decreased to a lesser extent (figures 3(a) and 3(c)) ; the axon diameter was protected in the pea group both in the proximal and in the distal nerve parts, even in the small fibers (figures 4(a) and 4(c)). pea was completely ineffective in ppar- null mice in preventing sciatic nerve alterations evaluated as number of fibers (figure 2(b)), myelin thickness (figures 3(b) and 4(d)) and axon diameters (figures 4(b) and 4(d)). figure 5 shows the infiltrate evaluation 14 days after ligation : inflammatory cells were present in the proximal and, at higher level, in the distal parts of both ppar- (graph (a)) and ppar- (graph (b)) mice. 30 mg kgpea significantly prevented the cellular infiltrate number in wild - type (figure 5(a)) but not in ko animals (figure 5(b)). moreover, both osmium fixed and azan - mallory - stained sections (figure 7) allowed the observation of a massive presence of oedema among the fibers of cci animals. figure 6 show the quantitative oedema evaluation 14 days after operation : the alteration was more evident in the distal portion than in the proximal one without revealable differences due to knock down ppar- gene. pea (30 mg kg s.c. for 14 days) was able to prevent the oedema induction of about 50% in cci wild - type mice (figure 6(a)). no oedema protective effects were observable in pea - treated ppar- animals in respect to vehicle (figure 6(b)). the immune inflammatory cells evaluated were diffusely distributed throughout the nerve tissue in all samples of the cci mice, whereas a mild cd86 positive reaction was detectable in cci mice administered with pea as well as in sham - operated animals. ppar- mice showed a persistence of macrophage infiltrate also in the nerve of pea - treated animals (figures 8 and 9). cci - dependent inflammatory response in the central nervous system was evaluated measuring cox2 levels. as shown in figure 10, in wild - type mice pea pharmacological treatment of peripheral neuropathy is actually restricted to symptomatic drugs that are only partially able to control pain perception. antidepressants, antincovulsants, and opioids can not intervene in nervous tissue alterations that act as a base of neuropathic pain. the present results describe the direct protective effect of repeated pea treatment on lesioned peripheral nerves. cci induces morphometric alterations of the sciatic nerve that dramatically affect the portion distal from the injury and that are also able to induce severe proximal impairment. at the same dose active in pain relief, pea prevents the reduction of myelin sheet thickness and axonal diameter and improves a characteristic degeneration of myelin as highlighted by azan - mallory staining. according to previous results [7, 24, 25 ] cci - mediated nerve architecture derangement is accompanied by a profound local inflammatory reaction which includes oedema, infiltration of hematogenous immune cells, and induction of various soluble factors like cytokines and chemokines. in particular, cd86 is a phenotypic marker of the classically activated m1 macrophages stimulated by proinflammatory cytokines, as ifn, or by lipopolysaccharide and typically recruited after nervous system trauma. m1 macrophages produce high levels of oxidative metabolites (e.g., nitric oxide and superoxide) and proinflammatory cytokines that are essential for host defense and tumor cell killing but that also cause collateral tissue damage. the treatment with pea attenuates the degree of peripheral inflammation, reducing oedema and macrophage infiltration allowing for hypothesizing a synergism between the anti - inflammatory and the neuroprotective mechanisms of pea. on the other hand, an inflammation control mediated by pea induces a cox-2 increase in locations of the central nervous system consistent with the neuroanatomical substrates of spinal nociception. cox-2 produces prostaglandins that contribute to the development and maintenance of spinal hyperexcitability after peripheral nerve injury [28, 29 ]. reducing cox-2 levels, pea seems able to intervene also in these central mechanisms of pain chronicization. to note that a direct inhibition of pro - inflammatory cytokines, using a tnf- antibody, for instance, attenuates pain - related behavior but has no effect on nerve regeneration. pea is a naturally occurring amide between palmitic acid and ethanolamine it is a lipid messenger known to mimic several endocannabinoid - driven actions even though pea does not bind cb1, cb2, and abn - cbd receptors. so far, numerous actions of pea on immune cells such as inhibition of mast cell degranulation, attenuation of leukocyte extravasation, and modulation of cytokine release from macrophages have been described [16, 32 ]. anti - inflammatory effects of pea have been associated with peroxisome proliferator - activated receptor (ppar)-alpha activation. ppar-, well known for its role in lipid metabolism, controls transcriptional programs involved in the development of inflammation through mechanisms that include direct interactions with the proinflammatory transcription factors, nf-b and ap1, and modulation of ikb function. pharmacological studies have demonstrated that ppar- agonists are therapeutically effective in rodent models of inflammatory and autoimmune diseases. our results show that in a neuropathic pain model the ppar- genetic ablation determines a loss of pea effectiveness in reducing oedema prevention and cd-86 positive infiltrating cells. on the other hand, the recruitment of reactive inflammatory cells and subsequent proinflammatory cascades offers a prime target for neuroprotective agents. agonists of ppar- such as fenofibrate and wy-14643 protect against cerebral injury by antioxidant and anti - inflammatory mechanisms and reduce the incidence of stroke in mice [35, 36 ]. using a spinal cord injury model, genovese. demonstrated that dexamethasone utilizes ppar- to reduce inflammation and tissue injury in a rat model of spinal cord trauma. on the contrary, the ppar- agonist gemfibrozil does not promote tissue preservation and behavioral recovery after spinal contusion injury in mice. our study shows the obligatory role of ppar- for the neuroprotective effect of pea in peripheral neuropathy. in the sciatic nerve of cci mice pea exerts a widespread protective effect on both myelin, and axons throughout a ppar--mediated mechanism, since pea treatment fails to rescue nerve tissue in ppar- knock - out animals. the neuroprotective pea properties were suggested by skaper. since dose dependently pea protected cerebellar granule cells from glutamate toxicity in neuronal single cell cultures and prevented histamine - induced cell death in hippocampal cultures. these effects were elicited without involvement of cb receptors. more recently koch. described the protective effect of pea on dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures ; the specific ppar- antagonist gw6471 blocked these effects. pea exerts neuroprotective activities in neurodegenerative diseases. in a mouse model of alzheimer 's and parkinson 's diseases, pea reduced oxidative and apoptotic damages and improve behavioral dysfunctions by a ppar--mediated mechanism [14, 41 ]. in a cellular model, pea was able to blunt -amyloid - induced astrocyte activation and, subsequently, to improve neuronal survival through selective ppar- activation. in neuropathic conditions ppar- seems to join the antihyperalgesic, anti - inflammatory, and neuroprotective effects of pea. on the other hand the inflammatory response to a damage is crucial for both pain sensation and tissue alterations ; the importance of inflammatory mediators is well demonstrated in the pathogenesis of neuropathic pain, where infiltrating macrophages and schwann cells may be involved in the modulation of these mediators in response to nerve injury. starting from the relevance of the ppar- in pea antineuropathic properties, the misunderstood role of peroxisome is intriguing. peroxisomes fulfill multiple tasks in metabolism and adapt contents and functions according to cell type, age, and organism. among the metabolic reactions that take place in peroxisomes, oxygen metabolism, -oxidation of a number of carboxylates that can not be handled by mitochondria, -oxidation of 3-methyl - branched chain and 2-hydroxy fatty acids, ether lipid synthesis, and detoxification of glyoxylate patients with peroxisomal dysfunction present with severe and diverse neurological anomalies, including neuronal migration defects, dysmyelination and inflammatory demyelination, macrophage infiltration, and axon damage, proving that these organelles are indispensible for the normal development and maintenance of the nervous system [4547 ]. thereafter, the peroxisome stimulation could be a broad spectrum approach to prevent nervous tissue damage, and a pea, ppar--mediated, increase in peroxisome number and/or functionality could be also hypothesized. a preclinical study showed that pea - mediated reduction of spinal cord damage was paralleled by an induction of ppar- expression and an up - regulation of potential ppar- target genes, but a clear relationship between ppar- activation and peroxisome boosting is still lacking. the present results demonstrate the neuroprotective properties of pea in a preclinical model of neuropathic pain. antihyperalgesic and neuroprotective properties are related to the anti - inflammatory effect of pea and its ability to prevent macrophage infiltration in the nerve. | neuropathic syndromes which are evoked by lesions to the peripheral or central nervous system are extremely difficult to treat, and available drugs rarely joint an antihyperalgesic with a neurorestorative effect. n - palmitoylethanolamine (pea) exerts antinociceptive effects in several animal models and inhibits peripheral inflammation in rodents. aimed to evaluate the antineuropathic properties of pea, a damage of the sciatic nerve was induced in mice by chronic constriction injury (cci) and a subcutaneous daily treatment with 30 mg kg1 pea was performed. on the day 14, pea prevented pain threshold alterations. histological studies highlighted that cci induced oedema and an important infiltrate of cd86 positive cells in the sciatic nerve. moreover, osmicated preparations revealed a decrease in axon diameter and myelin thickness. repeated treatments with pea reduced the presence of oedema and macrophage infiltrate, and a significant higher myelin sheath, axonal diameter, and a number of fibers were observable. in ppar- null mice pea treatment failed to induce pain relief as well as to rescue the peripheral nerve from inflammation and structural derangement. these results strongly suggest that pea, via a ppar--mediated mechanism, can directly intervene in the nervous tissue alterations responsible for pain, starting to prevent macrophage infiltration. |
the stress - induced cardiomyopathy (scm) is characterized by acute, reversible, and transient left ventricular (lv) systolic dysfunction mimicking acute coronary syndrome without significant stenosis on coronary angiogram.1)2) this syndrome was first described by satoh.3) and was named takotsubo - cardiomyopathy because its shape resembles the tako - tsubo (japanese octopus trap).4) the majority of patients are postmenopausal women who present with acute emotional or physical stress.1)2) clinical features are acute onset chest pain or dyspnea, electrocardiographic st changes with t - wave inversion, elevated cardiac biomarkers, elevated b - type natruretic peptide (bnp), apical or midventricular wall motion abnormality by echocardiography and favorable prognosis.1) the underlying etiology is unclear. there are limited data for clinical manifestations and prognostic factors of scm in korea.5)6) therefore, we sought to clarify the clinical features and prognosis in scm. we retrospectively reviewed and analyzed 39 patients diagnosed with scm from may, 2004, to january, 2009, in wonju christian hospital. scm was diagnosed as : 1) no previous history of cardiac disease, 2) acute onset, 3) a regional wall motion abnormality, typically in the takotsubo or inverted takotsubo shape by echocardiography, and 4) no significant stenosis in the coronary angiogram. we investigated baseline clinical characteristics, laboratory data, hospital course, complications, and clinical outcomes. we defined st - elevation as deviation > 1 mm higher than the baseline in 2 contiguous leads. we defined t - wave inversion as a change from the previous electrocardiogram (ekg) in 2 contiguous leads. echocardiography was performed at admission and rechecked at discharge or improved symptoms. coronary angiography and left ventriculography were performed to evaluate coronary artery disease or lv systolic function. shock was defined as systolic blood pressure < 90 mm hg with signs of end - organ hypoperfusion requiring the use of vasopressor agents. cardiac biomarkers { creatinine kinase mb (ck - mb) fraction and troponin - i }, high sensitive c - reactive protein (hs - crp), and bnp were collected at admission and cardiac biomarkers were rechecked until normalization. after discharge, all patients were followed in an out - patient clinic to reevaluate symptoms, lv systolic function, and adverse events. continuous data were summarized as meansd and were compared by student 's t - test. multivariate logistic regression analysis was performed to determine the independent variables associated with the occurrence of adverse events. past medical history was 10 (26%) of hypertension, 3 (8%) of diabetes mellitus, 5 (13%) of chronic obstructive pulmonary disease, and 5 (13%) current smokers. the initial symptom were dyspnea (n=18, 46%), chest pain (n=10, 26%), mental change (n=8, 21%), collapse (n=1, 3%), fever (n=1, 3%) and seizure (n=1, 3%). the triggering factors of scm were medical illness (n=23, 59%), emotional stress (n=6, 15%), procedure - related (n=3, 8%), trauma (n=3, 8%), and food or drugs (n=2, 5%) (table 2). initial ekg presented t - wave inversion (n=18, 46%), st - elevation (n=11, 28%), st - depression (n=2, 5%), or non - specific findings (n=6, 15%). we observed an increasing trend of scm in spring and winter but there was no statistical significance. the peak ck - mb fraction and troponin i were 15.620.9 ng / ml (reference range : < 5.0 ng / ml) and 6.812.3 ng / ml (reference range : < 0.78 ng / ml), respectively. the bnp at admission was 745.4905.6 pg / ml (reference range : < 100 pg / ml). l (reference range : < 5 mg / l) and 23 patients (59%) had elevated hs - crp at admission. in echocardiography, the mean lv ejection fraction (ef) was 4516% at admission and 6113% at discharge. there were 3 cases of inverted takotsubo shape, 4 cases of transient lv outflow tract obstruction due to a hyperkinetic basal segment of lv, and 1 case of apical thrombus on the site of apical ballooning (fig. 1). the patient who developed apical lv thrombus had a distal embolization in the right common iliac artery, right external iliac artery, and left femoral artery. she had anticoagulation therapy with warfarin and was discharged after 15 days without significant complications. three patients (8%) died due to pneumonia and 13 patients (33%) experienced cardiogenic shock. inotropic agents were administered to 10 patients (26%), and 9 patients (23%) needed mechanical ventilation. eleven patients (28%) received an emergency coronary angiogram to rule out acute st - elevation myocardial infarction. thirty - six patients were discharged after improvement of lv systolic function. during follow - up, there was no scm recurrence or sudden death. the scm patients were divided into two groups according to the presence of cardiogenic shock or death : the adverse clinical event group (n=15) versus the no event group (n=24). these groups showed significantly different age (54.817.5 vs. 65.314.0 years ; p=0.046), hospitalization (22.118.5 vs. 9.46.3 days ; p=0.020), initial ef (34.915.5 vs. 50.613.8% ; p=0.003), initial hs - crp (7680 vs. 2335 mg / l ; p=0.027), initial qtc (477.463.1 vs. 532.969.5 msec ; p=0.018), peak ck - mb fraction (2.81.1 vs. 2.00.9 ng / ml ; p=0.033) and peak troponin i (12.617.5 vs. 2.62.4 ng / ml ; p=0.045) (table 3). multivariate logistic regression analysis (adjusted for age, sex, and risk factors) showed that hs - crp at admission was associated with an odds ratio (or) of 1.41 { 95% confidence interval (ci) 1.02 - 1.97 } for an adverse event. the initial lvef was also significantly associated with an event (or 0.89, 95% ci 0.80 - 0.98, p=0.024). past medical history was 10 (26%) of hypertension, 3 (8%) of diabetes mellitus, 5 (13%) of chronic obstructive pulmonary disease, and 5 (13%) current smokers. the initial symptom were dyspnea (n=18, 46%), chest pain (n=10, 26%), mental change (n=8, 21%), collapse (n=1, 3%), fever (n=1, 3%) and seizure (n=1, 3%). the triggering factors of scm were medical illness (n=23, 59%), emotional stress (n=6, 15%), procedure - related (n=3, 8%), trauma (n=3, 8%), and food or drugs (n=2, 5%) (table 2). initial ekg presented t - wave inversion (n=18, 46%), st - elevation (n=11, 28%), st - depression (n=2, 5%), or non - specific findings (n=6, 15%). we observed an increasing trend of scm in spring and winter but there was no statistical significance. the peak ck - mb fraction and troponin i were 15.620.9 ng / ml (reference range : < 5.0 ng / ml) and 6.812.3 ng / ml (reference range : < 0.78 ng / ml), respectively. the bnp at admission was 745.4905.6 pg / ml (reference range : < 100 pg / ml). l (reference range : < 5 mg / l) and 23 patients (59%) had elevated hs - crp at admission. in echocardiography, the mean lv ejection fraction (ef) was 4516% at admission and 6113% at discharge. there were 3 cases of inverted takotsubo shape, 4 cases of transient lv outflow tract obstruction due to a hyperkinetic basal segment of lv, and 1 case of apical thrombus on the site of apical ballooning (fig. 1). the patient who developed apical lv thrombus had a distal embolization in the right common iliac artery, right external iliac artery, and left femoral artery. she had anticoagulation therapy with warfarin and was discharged after 15 days without significant complications. three patients (8%) died due to pneumonia and 13 patients (33%) experienced cardiogenic shock. inotropic agents were administered to 10 patients (26%), and 9 patients (23%) needed mechanical ventilation. eleven patients (28%) received an emergency coronary angiogram to rule out acute st - elevation myocardial infarction. thirty - six patients were discharged after improvement of lv systolic function. during follow - up the scm patients were divided into two groups according to the presence of cardiogenic shock or death : the adverse clinical event group (n=15) versus the no event group (n=24). these groups showed significantly different age (54.817.5 vs. 65.314.0 years ; p=0.046), hospitalization (22.118.5 vs. 9.46.3 days ; p=0.020), initial ef (34.915.5 vs. 50.613.8% ; p=0.003), initial hs - crp (7680 vs. 2335 mg / l ; p=0.027), initial qtc (477.463.1 vs. 532.969.5 msec ; p=0.018), peak ck - mb fraction (2.81.1 vs. 2.00.9 ng / ml ; p=0.033) and peak troponin i (12.617.5 vs. 2.62.4 ng / ml ; p=0.045) (table 3). multivariate logistic regression analysis (adjusted for age, sex, and risk factors) showed that hs - crp at admission was associated with an odds ratio (or) of 1.41 { 95% confidence interval (ci) 1.02 - 1.97 } for an adverse event. the initial lvef was also significantly associated with an event (or 0.89, 95% ci 0.80 - 0.98, p=0.024). our study shows that elevated hs - crp levels and initial impairment of lv systolic function relate to poor clinical outcome. elevated crp levels are found in scm patients and related to impaired lv systolic function with bnp release.7) enhanced production of acute - phase proteins in the liver by catecholamines may result from direct stimulation or may be mediated by cytokines such as tumor necrosis factor- or interleukin 6.8) pathologic findings of scm revealed the infiltration of inflammatory cells, including activated macrophages.9) collectively, our findings and previous reports suggest that systemic or local inflammatory processes may affect myocardial dysfunction and clinical outcomes in scm. we had different gender - related differences, triggering factors, and initial symptoms than previous studies (table 4). postmenopausal females predominate in scm due to estrogen deficiency after menopause. in animal models, postmenopausal females are more vulnerable to sympathetically mediated myocardial stunning.16) ovariectomised female rats with estradiol supplementation had significantly less stress - induced ventricular dysfunction, suggesting that estrogen possibly downregulates myocardial adrenoreceptors. new animal models propose that estrogen (via immunoreactive estrogen receptors in the central sympathetic neurons) directly attenuates the hypothalamo - sympathoadrenal axis, and also gives rise to cardioprotective substances such as atrial natruretic peptide, and heat shock protein 70.17)18) we found a lower percentage of women (female 69%) with scm, and physical stress was more common than emotional stress. another scm study has mostly male patients (65%) and lv apical ballooning developed in a considerable number of patients with severe physical stress who were admitted to the medical intensive care unit.19) hahn.20) also showed that the triggering factors were physical stress (69%) rather than emotional stress. thus, scm also occurs in men after physical stress, and inflammation (high crp), leading to the development and poor clinical outcome for scm regardless of gender difference. previous studies have reported that chest pain is a more frequent symptom than dyspnea.1)2)20) but, our study showed that dyspnea was more frequent at initial presentation. the patients with dyspnea showed longer hospitalization than patients with chest pain (1818 vs. 108 days). and dyspnea group showed an elevated bnp level (9661,118 vs. 779773 pg / ml), higher hs - crp level (5377 vs. 2229 mg / l) and decreased initial lvef (4217 vs. 5015%), but none significant. the discrepancy between our data and previous reports is unclear, but could relate to the small sample size and wide range of standard deviation. there are 3 cases of inverted takotsubo type, 4 cases of transient lv outflow obstruction type, and 1 case of apical thrombus (fig. variations in regional wall motion in transient lv apical ballooning, transient mid - ventricular ballooning, and other entities involving excess catecholamines relate more to differences in the anatomic location of cardiac adrenergic receptors, the degree of excess sympathetic activity, or individual differences.21) transient severe lv dysfunction can cause serious complication such as cerebrovascular attack, renal infarct, and peripheral arterial obstruction that originates from an lv apical thrombus. our study is restricted to a single center experience and it can not represent general characteristics of the korea population. so, a multicenter prospective study needs to reveal the clinical characteristics and outcomes in korean patients with scm. in our single center experience, the major triggering factor of scm is physical stress due to medical illness or a procedure. elevated hs - crp and decreased lvef at admission were independent risk factors for death or cardiogenic shock in patients with scm. in our single center experience, the major triggering factor of scm is physical stress due to medical illness or a procedure. elevated hs - crp and decreased lvef at admission were independent risk factors for death or cardiogenic shock in patients with scm. | background and objectivesstress - induced cardiomyopathy (scm) is characterized by a transient left ventricular (lv) dysfunction due to emotional and physical stress. there are limited data about the clinical characteristics in korean patients. we sought to clarify the clinical features and prognosis in patients with scm.subjects and methodswe reviewed 39 cases diagnosed with scm in a tertiary hospital. the scm was diagnosed as : 1) no previous history of cardiac disease, 2) acute onset, 3) regional wall motion abnormality, typically in the takotsubo or inverted takotsubo shape by echocardiography, and 4) no significant stenosis in the coronary angiogram. we evaluated clinical characteristics, biomarkers, and prognosis.resultsmean age was 61.316.1 years (female 69%). the triggering factors were physical stress in 32 patients (82%) and emotional stress in 5 patients (13%). the initial symptom was dyspnea (n=18, 46%) rather than chest pain (n=10, 26%). an initial electrocardiogram (ekg) presented t - wave inversion (n=18, 46%), st - elevation (n=11, 28%), and st - depression (n=2, 5%). multivariate logistic regression analysis showed that initial high sensitive c - reactive protein (hs - crp) { odds ratio (or) 1.41, 95% confidence interval (ci) ; 1.02 - 1.97 } and initial left ventricular ejection fraction (lvef) (or 0.89, 95% ci ; 0.80 - 0.98) were significantly associated with death or cardiogenic shock, respectively.conclusionthe major triggering factor of scm is physical stress due to illness or surgical procedures, and the first manifestation is dyspnea rather than chest pain. elevated hs - crp and decreased lvef at admission were independent risk factors for death or cardiogenic shock. |
previous studies indicate that there is a higher prevalence of dental anomalies in children with a cleft condition than in the general population. studies have also demonstrated that both genetics and the surgical repair of the palate influence the occurrence of dental anomalies in the cleft population. anomalies include variations in tooth size, shape, number, structure and formation, and eruption timing. both sets of dentition may be affected and occur more frequently on the cleft - affected side of the maxilla. the most common types of dental anomalies have been reported to be absent or supernumerary teeth, enamel dysplasia and discoloration and delayed root development. in children with cleft lip and palate, the lateral incisor in the alveolar cleft region has the highest prevalence of dental developmental disorders. this may cause functional and esthetic issues for the child and complicating factors for dental and orthodontic treatment. this paper will report the prevalence and type of dental anomalies in the primary and permanent dentition in children with a cleft condition at princess margaret hospital in perth, western australia. the details of current dental patients were selected consecutively from the main dental database via their year of birth for the period 19982001. patients who had not attended for the past 3 years, those with a syndrome and patients without a cleft condition were excluded, leaving a total of 172 subjects for this study. ten subjects did not have any dental anomalies and were excluded, leaving 162 subjects with at least one anomaly. the majority of subjects (92%) were caucasian with 84% residing in the metropolitan area. the mean age of the subjects was 10.8 years (age range of 8.911.9 years) with equal numbers of males and females. the details of current dental patients were selected consecutively from the main dental database via their year of birth for the period 19982001. patients who had not attended for the past 3 years, those with a syndrome and patients without a cleft condition were excluded, leaving a total of 172 subjects for this study. ten subjects did not have any dental anomalies and were excluded, leaving 162 subjects with at least one anomaly. the majority of subjects (92%) were caucasian with 84% residing in the metropolitan area. the mean age of the subjects was 10.8 years (age range of 8.911.9 years) with equal numbers of males and females. dental records and x - rays were examined by one examiner (wn) and verified by a second examiner (rb) to determine dental development. subjects were further divided into cleft type ; unilateral cleft lip and palate (uclp : 25% [left 14% ; right 11% ]), bilateral cleft lip and palate (bclp : 8.6%), ucl : 20%, bcl : 1.2%, and isolated cleft palate (cp : 45%). the following anomalies were reported : agenesis, crowding, delayed development, demineralization, dysplastic, early loss, ectopic, fissural, hypomineralization, hypoplastic, microform, peg lateral, pits and fissures, premaxilla, resorptive, retained, rotated, supernumerary, transposition, miscellaneous dental anomalies, nil and other [appendix 1 for definitions ]. the rates of occurrence of each anomaly were calculated as a percentage of the total sample in each group. microsoft excel 2016 (microsoft corporation, one microsoft way redmond, wa 98052 - 7329, usa) was used for data storage, calculating descriptive statistics, and chart generation. incidence rate difference was analyzed with wilcoxon statistical test using minitab version 17 (minitab inc., quality plaza, 1829 pine hall road, state college, pa 16801 - 3210, usa). chi - square analysis was used to determine if any significant difference existed between genders. the rates of occurrence of each anomaly were calculated as a percentage of the total sample in each group. microsoft excel 2016 (microsoft corporation, one microsoft way redmond, wa 98052 - 7329, usa) was used for data storage, calculating descriptive statistics, and chart generation. incidence rate difference was analyzed with wilcoxon statistical test using minitab version 17 (minitab inc., quality plaza, 1829 pine hall road, state college, pa 16801 - 3210, usa). chi - square analysis was used to determine if any significant difference existed between genders. one hundred sixty - two subjects were grouped into 21 categories of anomaly or abnormality [appendix 1 for definitions ]. overall, 94% of patients were found to have at least one dental anomaly [figure 1 ] with 56 (34%) ; patients having more than one anomaly or abnormality (65% had one reported anomaly ; 22% had two ; 11% had three ; 1.2% had four, and 0.6% had five reported anomalies). dental anomalies and abnormalities were also calculated by gender and cleft type [tables 1 and 2 ]. a significant statistical difference was not found between the frequency of dental anomalies and gender (p < 0.01). occurrence of dental anomalies for all patients dental anomalies by all patients dental anomalies by broad cleft type agenesis was the most prevalent anomaly in this study (15%) [table 1 ]. this affected 14% (22) of patients, predominantly female, and with a cp condition. supernumerary teeth were found to be the third most commonly occurring dental anomaly affecting 10% (16) of patients. the most frequently affected tooth location was 62. with the exception of demineralization and ectopic eruption, all dental anomalies occurred more frequently on the left side. seventy - five percent of agenesis occurred more often on the left side than on the right side. dental anomalies affecting tooth shape occurred exclusively on the cleft - affected side in this study. the most frequently occurring shape anomaly was the occurrence of a peg or conical shaped lateral incisor, and this most commonly (95%) affected tooth 22. fourteen percent (23) of patients had anomalies that occurred as a single condition or very low numbers. these were categorized as miscellaneous dental anomalies and was comprised crib tooth, star lateral, fusion, cavitation, severe enamel staining, and submerging teeth (2), cross - bite, hypomineralization, and palatal eruption (4), transverse migration, deep retentive fissures, necrotic pulp space, and distal flaring. other and was comprised cystic hemangioma, palatal constriction, gingival hyperplasia, limited opening, polyps, abnormal frenum, and glossitis. absent teeth (due to agenesis) were equally represented (50%) by gender [table 1 ]. crowding (82%) resorptive (88%) and retained teeth (85%) were more prevalent in females in this study. anomalies of fissural teeth (58%), supernumerary (63%), and rotated teeth (60%) were more prevalent in males. when dental anomalies were compared by cleft type [table 2 ] agenesis occurred most frequently in patients with a cleft of the lip and palate (50%). other anomalies that were more prevalent in this group were fissural (68%), hypoplastic (60%), and transpositioned (100%) teeth. crowding (73%), demineralization (56%), resorptive (75%), and miscellaneous (61%) anomalies occurred more frequently in patients with a cp. supernumerary teeth (63%) occurred more frequently in patients with a cleft lip only. when compared with further defined cleft types [table 3 ], the most frequent anomaly per cleft type are as follows ; tooth shape disorder of peg lateral (9%) occurred most frequently in bcl ; absent (16%) and premaxilla abnormality (16%) in bclp ; crowding (72%) and other miscellaneous dental anomalies (60%) in cp ; supernumerary (62%) and fissural and rotated teeth (both 31%) in ucl ; absent (25%) and fissural (26%) in ulclp and fissural (26%) in urclp. dental anomalies by refined cleft type the 10 patients whom were found to have no anomalies were represented by equal numbers of male to female and classified within the following cleft types ; six uclp patients, three cp patients, and one ucl patient. a higher prevalence of dental anomalies has been reported in cleft populations compared with the general population and in caucasian subjects. anomalies reported include variance in tooth shape, size, color, structure, position, and the influence of the area of the repaired cleft defect. this study investigated the prevalence of dental anomalies and abnormalities in a predominantly caucasian cleft population. agenesis was the most prevalent anomaly in this study, supporting results reported in other studies of 50% and greater. in this study, the rates of agenesis on the cleft - affected side varied between 75% and 92%, depending on the type of cleft type, with statistically significant differences for cp (p < 0.05), and cleft lip and palate (p < 0.01). previous studies have reported agenesis of the permanent lateral incisor on the cleft - related side to be the most frequently occurring anomaly in children with a cleft condition. this study also found that the permanent maxillary lateral incisor in the area of the cleft was the tooth most frequently absent (64%). loureno ribeiro. suggest that this may be due to the compromised blood supply in the cleft - affected area, either as a congenital condition or the result of surgical repair. supernumerary teeth have been reported to be the second most frequently occurring dental anomaly in the literature. report the prevalence of supernumerary to be from 1.9% to 10.0% in the uclp and cp groups. they further reported a higher rate (22%) of supernumerary teeth present in the permanent dentition in the cleft area in subjects with uclp or cp. it has been suggested that supernumerary teeth in cleft lip and palate result during cleft formation from fragmented lamina of the dentition. in this study, in uclp patients, it has been reported that dental anomalies occur more frequently on the left side (ratio of 2:1). the results from this study supported those findings overall, with the majority of dental anomalies across all cleft types, occurring more frequently on the left side including 75% of agenesis. however, this study also found some differences ; agenesis having a 3:1 ratio, miscellaneous anomalies having a ratio of 4:1, and demineralization occurred exclusively on the right side. dental anomalies affecting tooth shape occurred exclusively on the cleft - affected side in this study. the most frequently occurring tooth shape anomaly was the occurrence of a peg or conical shaped lateral incisor, and this most commonly (95%) affected tooth 22. previous research has shown the prevalence of microdontia to vary in the general population from 1.5% to 2.0%. in this study, when comparing to the general population, there was a only a slightly higher prevalence of microdontia (1.92.4%) on the cleft side for both the uclp groups. tooth anomalies in anterior regions are the most obvious and not only influence appearance but may prove a challenge due to compromised root formation and positioning to achieve the best esthetic outcome. satisfaction with dental esthetic outcome may be very important to a child with a cleft condition that already has a visible difference in appearance from facial asymmetry or scarring from cleft - related surgical repair. it has been reported that in uclp patients, dental anomalies occur with a higher frequency on the cleft - affected side. this study supported the higher occurrence of anomalies on the cleft - affected side with 95% of dental anomalies occurring on the side of the cleft. this occurrence has been suggested to be due to etiological factors involved with a cleft formation, which also influence dental formation. trotman. suggested that the forms of dental anomalies and abnormalities may be related to different cleft types, primarily influenced by the developmental effect of clefting process, and subsequent cleft repair. they further suggest that there may an influential link between the genetic factors, dental anomalies, and cleft type. this study found agenesis to be the most frequent in clefts of the lip and palate, crowding in isolated cp, and the presence of supernumerary teeth in isolated cleft lip. a very high proportion of subjects in this study were found to have at least one dental anomaly, with agenesis being found to be the most frequently reported anomaly occurring equally between genders and most frequently in patients with a cleft of the lip and palate. dental anomalies in patients with a cleft condition may be a complicating factor and must be taken into consideration as part of the treatment planning process and should be undertaken by, or in consultation with, pediatric dental specialists to achieve the best esthetic and functional outcome. | objective : the purpose of this paper was to describe the prevalence and type of dental anomalies in the primary and permanent dentition in children with a cleft condition at princess margaret hospital in perth, western australia.materials and methods : the details of 162 current dental patients extracted from the main dental database through their year of birth for the period 19982001 were selected consecutively. dental records and x - rays were examined by one examiner (wn) and verified by a second examiner (rb) to determine dental development. the mean age of the subjects was 10.8 years with equal numbers of males and females. subjects were further divided into cleft type ; unilateral cleft lip (ucl) and palate, bilateral cleft lip (bcl) and palate, ucl, bcl, and cleft palate.results:one hundred sixty - two subjects were grouped into 21 categories of anomaly or abnormality. prevalence rates for the categories were calculated for the overall group and for gender and cleft type.conclusion:overall, 94% of patients were found to have at least one dental anomaly, with fifty - six (34%) patients having more than one anomaly or abnormality. |
posterior reversible encephalopathy syndrome (pres) is a rare, poorly understood disease with a number of associated factors but no clearly delineated etiology. of described cases, it is estimated that greater than one third of patients will never regain baseline neurologic function. the physiologic causes for pres are difficult to ascertain in light of the myriad of associated diagnoses and presentations. while pres is generally associated with hypertension, more recently it has been linked to drug toxicities (e.g. chemotherapy agents, immunosuppressive medications). however, even with these other instigating factors, hypertension likely still plays a significant role. pres appears to be more common in women though the prevalence data may be skewed by the association of this diagnosis with pregnancy. presentation is highly variable but generally features a depressed level of consciousness among other sequelae of intracranial hypertension. other, less common, presentations range from new - onset seizure activity to focal neurologic deficits. the hallmark vasogenic edema on imaging is similar to that found with eclampsia but is unambiguous enough to be diagnostic [57 ]. significant variation exists, however, such that some authors have observed that this imaging pattern does not necessarily need to be strictly posterior and in some cases it is not we present a patient who developed pres in the immediate post - operative period following complications of a hemicolectomy. anesthesia and sepsis initially confounded her diagnosis but do not appear to have delayed her treatment and eventual complete neurological recovery. the patient is a 67-year - old woman who presented with a near - obstructing adenocarcinoma of the right colon. she underwent an uneventful laparoscopic - assisted extended right hemicolectomy and was discharged in stable condition on post - operative day 5. she presented to our emergency department (ed) on post - operative day 10 complaining of diffuse abdominal pain, hematemesis and subjective fevers. computerized tomography (ct) of the abdomen demonstrated a significant amount of free intraperitoneal fluid and associated pneumoperitoneum ; she was started on broad spectrum antibiotics and taken to the operating room for an exploratory laparotomy. upon entry into the abdomen, she was found to have a significant volume of succus and a small anastomotic leak. the patient 's vital signs were stable and remarkable only for a low grade tachycardia throughout the procedure. however, due to the patient 's persistent obtundation following extubation, she was brought to the intensive care unit (icu). while her oxygen saturation remained above 95% on supplemental oxygen with both oropharyngeal and nasopharyngeal airways in place, she failed to regain consciousness. when she was noted to be apneic, the patient was given naloxone, glycopyrrolate and neostigmine to counteract the opioid and paralytic agents received during her procedure without any clinical improvement in her neurologic status. while bedside blood glucose testing was within normal limits, an arterial blood gas demonstrated a profound respiratory acidosis with a ph of 7.03, a pco2 of 107 and a pao2 of 375. she was re - intubated for hypercapnic respiratory failure and all sedating medications were held. this included hypertonic saline, head of bed elevation to 30 degrees, maintenance of normotension and normoglycemia. she was also started on levetiracetam as seizure prophylaxis. a magnetic resonance imaging (mri) non - contrast head ct showing hydrocephalus with tonsillar herniation and vasogenic edema of the parietal and occipital lobes. vasogenic edema involving the bilateral cerebral hemispheres, with relative sparing of the frontal lobes suggestive of pres. pneumocephalus is present secondary to interval placement of an external ventricular drain (seen in the right lateral ventricle). non - contrast head ct showing hydrocephalus with tonsillar herniation and vasogenic edema of the parietal and occipital lobes. vasogenic edema involving the bilateral cerebral hemispheres, with relative sparing of the frontal lobes suggestive of pres. pneumocephalus is present secondary to interval placement of an external ventricular drain (seen in the right lateral ventricle). with the initiation of treatment described above, the patient slowly began to recover neurologic function. by post - operative day 33 five months later, she underwent a successful ileostomy reversal and was back to her pre - operative quality of life. she experienced complete resolution of her neurological symptoms and normalization of her neuroimaging (fig. 3). figure 3:mri brain 3 months post - diagnosis, axial t2 sequence. pres was identified as a clinical entity in 1996 by hinchey. in their seminal paper, they described patients with renal insufficiency and hypertension that had extensive bilateral white matter changes within their cerebrum, brainstem, and cerebellum. of note, seven of the patients they described were on immunosuppressant therapy and were treated in part by decreased or stopped immunosuppression. prior to hinchey 's formal codification of the diagnosis, this specific set of clinical findings had been attributed to eclampsia given the relative predominance of these findings in pregnant patients with associated hypertension, neurologic sequelae and characteristic vasogenic edema. it is hypothesized that pres is a direct complication of endothelial injury related to a loss of cerebral autoregulation in the setting of poorly controlled hypertension. recovery generally occurs within 56 days with supportive care including maintenance of normotension, treatment of sepsis and the cessation of any offending medications. while pres has been associated with sepsis, colorectal cancer and a history of chemotherapy administration ; fulminant presentation in the immediate post - operative period in the setting of normotension has not been described. while subtle manifestations were seen in two of the four total perioperative cases identified, these patients exhibited mild to moderate new onset hypertension in the immediate post - operative period. in the case we describe, the patient was obtunded from the immediate postoperative period until diagnosis and subsequent management aimed at reducing intracranial hypertension. we theorize that the systemic vasodilation and capillary leak associated with this patient 's abdominal sepsis was exacerbated by the induction of anesthesia and precipitated pres. despite her resultant vasogenic edema and intracranial hypertension progressing to tonsillar herniation, timely surgical intervention and optimal medical management facilitated a complete neurologic recovery. the views expressed in this manuscript are those of the authors and do not reflect the official policy or position of the department of the army, department of defense, or the us government. | posterior reversible encephalopathy syndrome (pres) is an unusual disease of unknown incidence and cause. there are a wide range of associated, predisposing medical causes to include pregnancy, renal failure, immunosuppressive medication administration and hypertension. the diagnosis is made following the radiographic identification of characteristic vasogenic edema in the setting of neurologic impairment. a significant portion of patients will have long - term, if not permanent, sequelae of the disease. we present a patient who developed pres following a hemicolectomy that was complicated by an anastomotic leak. she went on to a complete recovery following surgical treatment of the leak and supportive care. |
leptin as a neuroendocrine hormone has effects on the glucose metabolism, sexual maturation, reproduction, pituitary - adrenal axis, immune system, thyroid, and growth hormones level [36 ]. probably this hormone is an important risk factor in carcinogenesis, because obesity itself can promote tumorgenesis and is a risk factor for cancer over time [7, 8 ]. on the other hand, leptin plays an important role in the oxidation reactions such as fatty acid oxidation and angiogenesis. there are many reports concerning the effect of leptin on stimulation of cell mitosis and its involvement in carcinogenic stages of breast, clone, prostate, lung, kidney, and ovary cells [1116 ]. studies have shown that leptin by increase of cell proliferation and inhibition of apoptosis is involved in creating certain types of tumors [1719 ]. leptin acts through its receptor on the cell surface and its receptor expression also increases following the activity of pi3k / akt pathway and increases the activity of antiapoptosis molecules such as bcl - xl and xiap. in some cancer cells, expression of leptin receptor levels and stimulation by leptin will lead to increase of cell proliferation. leptin stimulates expression of some molecules such as cyclind1, cdk2 and c - myc that result in cell cycle progression and cell proliferation [21, 22 ]. the important molecular pathways, such as jak / stat3, pi3k / akt, erk / mapk, in many cancer cells can be activated by leptin / leptin receptors [2124 ]. furthermore, leptin with the induction of vegf and vegf - r2 molecules expression plays an important role in the tumorigenesis. these molecules are involved in many malignancies such as colon, stomach, endometrial, ovarian, and breast cancer [2630 ]. additionally, increased serum levels of leptin and its receptor have been associated with distant metastases, disease recurrence, and lower survival in patients with breast cancer. this hormone probably through its receptor and activation of the pi3k / akt pathway plays an important role in papillary thyroid cancer pathogenesis. it also seems that the oncogenic effects of leptin on papillary thyroid carcinoma cells are related to the stimulating cell proliferation and apoptosis inhibition. involvement of thyroid hormones on basal metabolism and regulating appetite and weight control in many scientific reports is given explicitly [10, 3234 ]. the most common endocrine malignancy is thyroid cancer, and papillary form of thyroid cancer is the most common type of thyroid cancer (8090%). the aim of this study was determining the serum leptin levels in patients with papillary thyroid cancer and its comparison with healthy subjects. patientsthe case population consisted of 83 individuals, including 35 males and 48 females, 14 to 62 years (mean age 38.6 years) with papillary thyroid cancer (ptc). they were referred to research institute for endocrine sciences, shahid beheshti university of medical science. also, 90 persons were selected as control group (40 male and 50 females) from referred to the laboratory with normal thyroid function tests (tsh : 0.3 = 3.5 miu / l, t4 : 4.5 = 12.5 g / dl, t = up : 25 = 35% and t3 : 75 = 210 ng / ml) with age, sex, bmi matched with case group. this study has been approved by institutional review board and ethics committee of obesity research center, research institute for endocrine sciences, shahid beheshti university of medical sciences. the case population consisted of 83 individuals, including 35 males and 48 females, 14 to 62 years (mean age 38.6 years) with papillary thyroid cancer (ptc). they were referred to research institute for endocrine sciences, shahid beheshti university of medical science. also, 90 persons were selected as control group (40 male and 50 females) from referred to the laboratory with normal thyroid function tests (tsh : 0.3 = 3.5 miu / l, t4 : 4.5 = 12.5 g / dl, t = up : 25 = 35% and t3 : 75 = 210 ng / ml) with age, sex, bmi matched with case group. this study has been approved by institutional review board and ethics committee of obesity research center, research institute for endocrine sciences, shahid beheshti university of medical sciences. blood sampling was performed in both studied groups. for preparation of serum, 3 ml of whole blood was collected from antecubital vein in sitting position and was incubated 10 min in rt for coagulation. then sera separated by 10 min centrifugation at 3000 rpm and the obtained sera were aliquoted in three 0.5 ml microtubes. the isolated serum samples from each individual were stored in 1 ml eppendorf microtubes at 80c (japan 's sanyo c company). anthropometric characteristics, including height and weight of patients and control group, were measured by height measuring scaled balance (seca, german company) ; height with 0.5 cm and weight with 250 g sensitivity were reported. these data were used to calculate the body mass index (kg / m). those individuals, who were using drugs affecting thyroid function and obesity drugs, were excluded. in groups the used kits were prepared from the canadian company (dbc company, ontario, canada). human thyrotropin and leptin hormones were determined based on sandwich elisa method, whereas a thyroxin hormone was measured according to the competitive eia method. the sensitivity of thyroxine, thyrotropin, and leptin kits was 0.6 g / dl, 0.1 mu / l, and 0.4 ng / ml, respectively. additionally, the coefficients of variation for these assays were 6.2%, 7.1%, and 6.5%, respectively. according to the normal distribution of data obtained by testing kolmogorov - smirnov (ks) (p = 0.68 for case group and p = 0.52 for control group), the frequency, mean and standard deviation were used to describe characteristics. the independent t - test was used to compare mean (except leptin with geometric mean and ci 95%) of variables between two groups further, data was analyzed using statistical software (spss 15), and significant level was considered at 0.05. the results of thyroid hormones test, including thyroxine and thyrotropin in both control and patients groups, are given in table 2. since the leptin hormone secreted from adipose tissue is different in male and female, therefore the different levels of measured leptin hormone in two groups are shown in table 3 (gender based). height, weight, and body mass index between males and females of both groups were significant (p < 0.05). in addition, a significant difference (p < 0.05) was observed between the leptin hormone levels in males and females in both healthy and cancer groups. the amount of leptin hormone in cancer patients was higher than that in normal individuals, significantly (p < 0.05). our data showed that the serum leptin levels of iranian patients with papillary thyroid carcinoma were significantly higher than those in control group subjects. this increased level was observed in both males and females with papillary thyroid carcinoma. as this increased level was observed in both gender and different ages, so it could be related to thyroid carcinoma and it is independent of sex and age. even though in this study the leptin level was higher in females than males in both groups, this is probably related to more adipose tissue mass in women. both leptin and thyroid hormones cause thermogenesis and reduce body weight therefore maybe it is considered as a first association between the two hormones. but the most studies have not shown significant changes in leptin levels in hypothyroidism and hyperthyroidism disorders [36, 37 ]. but in their study only 34 cases were investigated, the status of thyroid function in patients and healthy group was not evaluated, and age - matching was not considered. in our study, 83 persons were matched for age, sex, and bmi. assessing thyroid function in patients and healthy individuals was performed, and no significant difference was observed in both groups. in both above studies, bmi in women was higher than in men, which was quite predictable. in both studies leptin levels in women were higher than those in men that is because of increased fat mass in women. in one study cheng. showed that expression of leptin and/or leptin receptor in papillary thyroid cancer was associated with neoplasm aggressiveness, including tumor size and lymph node metastasis. interestingly, in another study, uddin. demonstrated that leptin plays an important role in papillary thyroid cancer pathogenesis through pi3k / akt pathway via its receptor (ob - r) and is a potential prognostic marker associated with an aggressive phenotype and poor survival. one of the limitations of our study was inability to followup the patients after surgery. therefore, reduction or normalization of high leptin levels in thyroid cancer patients was not assessed. however, a significant increase of serum leptin levels in iranian patients with papillary thyroid carcinoma maybe used as a reliable marker to diagnose or confirm papillary thyroid cancer. in addition if the leptin levels in cancer patients decrease after thyroidectomy, it will be used for the followup treatment, possibly. so a before - after study is recommended for future investigations instead of case control study. thus, leptin level measurement can be used to followup the treatment of patients. strongly high leptin level in papillary thyroid cancer patients in comparison with health subject it means that adipose tissue secreted hormones, proteins, and peptides potentially may have application in diagnosis, confirmation, and/or treatment followup. | introduction. leptin as an adipose - tissue - related peptide hormone contributes to the control of food intake, energy expenditure, and other activities such as cell proliferation. therefore, association of leptin level with thyroid cancer has been suggested recently. considering that thyroid cancer is the most common endocrine cancer, the aim of this study was evaluation of leptin levels in thyroid cancer. materials and methods. 83 patients with papillary thyroid cancer (35 males and 48 females) with 90 healthy persons as control group (40 male and 50 females) were selected. serum thyroxine, thyrotropin, and leptin levels were determined in both groups. as a body fat tissue affects leptin level, so height and weight were measured and body mass index was calculated too. results. there was no statistically significant difference in age, serum thyroxine, and thyrotropin levels. bmi in women was more than in men in both groups. serum leptin levels in thyroid cancer group were significantly higher than control group (p < 0.05). conclusion. the results of this study showed an acceptable association between the hormone leptin levels with papillary thyroid cancer, so it may be considerad as a correlated peptide which may help in the diagnosis or confirmation of thyroid cancer beside in other specific tumor markers. |
the uterus has been considered an organ adjusting and controlling the important physiological functions, pregnancy, childbirth, a sexual organ, a source of energy, and an organ maintaining the attractiveness and beauty of women. moreover, it comprises an important part of women s self - image, and loss of uterus means the loss of sense of femininity (1 - 2). about 600.000 women undergo hysterectomy every year in the united states, and this number has remained rather constant (3, 4). although many advantages of hysterectomy have been known, it is still unknown how it affects the vaginal length and sexual functioning (5). moreover, women s sexual desires are an essential human right and contribute to women s comfort and welfare (6). candidates for hysterectomy are always worried about its potential negative effects on their sexual functioning and the relationship with their sexual partner (7). the effects of hysterectomy on the quality of life and sexual functioning differ from one woman to another. the complaints after hysterectomy include the loss of libido, decreased frequency of intercourse, decreased sexual responsiveness, difficulty with reaching orgasm, diminished sensation of the vagina, dyspareunia (painful intercourse), vaginal shortening, loss of penile penetration, and loss of vaginal elasticity and lubrication (8). a study (2009) stated that more than half of patients were suffering from feelings of premature aging and loss of libido after hysterectomy (9). in another study, the sexual pleasure (frequency of sexual activity, increased more than half of patients were suffering from feelings of premature aging and loss of libido, and decreased sexual disorders) improved considerably in most patients (10). regarding the contradictions about positive and negative effects of hysterectomy on women s sexual functioning, this study was conducted to review the studies on the effect of hysterectomy on postoperative women s sexual function. this study was a narrative review and performed in 5 steps : a) determining the research questions, b) search methods for identification of relevant studies, c) choosing the studies, d) classifying, sorting out, and summarizing the data, and e) reporting the results. determining the research questions : how does hysterectomy affect women s sexual functioning ? search methods for identification of relevant studies : the studies were identified using academic research articles and relevant keywords and through advanced searching in electronic publications, including cochrane library, magiran, pro quest, springer, science direct ; and databases of iran medex, pubmed, and sid of 1999 - 2015. the keywords hysterectomy, desire, arousal, orgasm, pain, and dyspareunia were searched. moreover, reference lists of published articles were reviewed in order to increase the sensitivity and choose more studies. choosing the studies : the full text or abstract of all articles, documentations, and reports obtained through the advanced search was retrieved. once the repeated materials were eliminated, the irrelevant articles were eliminated through reviewing the title, abstract, and full text of the articles, and thus relevant articles were chosen. having searched the keywords, we selected 150 articles, of which 25 articles were eliminated because they were repeated. then, the title and abstract of the other 125 articles were reviewed, and 54 irrelevant articles were eliminated. of the remaining 71 articles, 41 articles were excluded once their full text was reviewed, and 4 articles were added upon reviewing the references of those articles. eventually, 34 articles were used to write this review study (figure 1 shows the procedures through which the studies were chosen). literature search and review flowchart for selection of primary studies articles used in writing this review study included english and persian case - control, cross - sectional, and prospective and retrospective cohort studies. inclusion criteria : the studies examining sexual functioning in women undergoing hysterectomy were included in the study. exclusion criteria : the studies examining sexual functioning in women not undergoing hysterectomy were excluded from the study. classifying, sorting out, and summarizing the data : the obtained data were classified as shown in table 2. reporting the results : the reported data comprised 5 categories. the review of the studies yielded 5 main categories of results as follows : the effect of hysterectomy on sexual desire, the effect of hysterectomy on sexual arousal, the effect of hysterectomy on orgasm, the effect of hysterectomy on dyspareunia, and the effect of hysterectomy on sexual satisfaction (table 1). two studies reported that most of the patients did not experience any changes in their sexual desire (11 - 12). in a study, women who had been sexually active before the surgery maintained their sexual activity after the surgery and reported the same frequency of sexual activity after 6 months (13). meston s study did not show any significant difference between women undergoing hysterectomy and women with fibroids but not undergoing hysterectomy in terms of desire (14). another study reported that neither the body image nor the libido decreased after hysterectomy, and no important changes occurred in this regard (15). however, the women undergoing hysterectomy in some studies experienced considerable improvement of sexual desire (6, 10, 16). furthermore in gutl.s study, sexual dysfunctions, such as the loss of sexual desire, significantly decreased after the abdominal and vaginal hysterectomy, and women reported the improvement in their sexual desire and sexual satisfaction 3 months and 2 years after the surgery (17). however, some other studies mentioned the decreased sexual desire after hysterectomy. in this regard, jensen. found there was a long - term lack of sexual interest in patients 12 months after radical hysterectomy as compared with that before the diagnosis of the cancer and however, many patients who were sexually active before the diagnosis of cancer became active again 12 months after the surgery although they reported fewer sexual intercourses (18). a study revealed that the poor libido was one of the sexual concerns reported after hysterectomy (19). some other studies also showed that sexual desire and frequency of intercourse decreased significantly (20 - 21). furthermore, problems related to sexual desire and feeling of women undergoing hysterectomy were significantly higher than those before the surgery (22). bayram and sahin revealed that sexual activity significantly decreased 3 months after hysterectomy and proved obvious symptoms of depression that had affected the sexual functioning negatively (23). study on women undergoing radical hysterectomy for treatment of initial stages of the cervical cancer reported dysfunction of all sexual aspects, including a decrease in sexual desire that was lower than the decrease in other aspects (24). study also showed that tah and tah+ bso mainly decreased the sexual desire (25). some studies showed positive effects of hysterectomy on sexual arousal, and some other studies showed negative effects in this regard. most women in goetsch s study experienced higher sexual arousal after abdominal and vaginal hysterectomy, and only 25% of the women reported decreased sexual arousal (26). anonymous also reported that the problems related to sexual arousal generally decreased after hysterectomy (13). in some studies, vaginal dryness decreased after hysterectomy, and women significantly improved in terms of sexual arousal and activity 3 months and 2 years after hysterectomy (6, 16 - 17). however, some other studies reported that hysterectomy increased vaginal dryness and abnormal vaginal contractions. one of the sexual problems after hysterectomy was inadequate vaginal lubrication that was more constant outcome and eventually decreased sexual satisfaction (9, 12, 19, 27). in meston s study, women undergoing hysterectomy reported low normal level of vaginal lubrication that implied the potential sexual - mental arousal following the hysterectomy although no significant difference was found between groups undergoing hysterectomy and groups with fibroids but not undergoing hysterectomy in terms of sexual arousal (14). study reported significant deterioration of the sensation of cold and warm stimuli in anterior and posterior vaginal wall after hysterectomy (28). tangjitgamol s study reported disorders in all aspects of sexual functioning, including decreased sexual arousal and vaginal lubrication, as the most obvious changes were the decreased sexual frequency and vaginal lubrication, and sexual arousal decreased to a less extent (24). in pieterse study, the patients who had undergone radical hysterectomy for treatment of the initial stages of their cervical cancer reported a negatively significant effect of the surgery, in comparison to their condition before the surgery and to the patients in the control group, on their sexual functioning, such as the less lubrication, narrowness and shortening of the vagina, and numb areas around the labia, during 24 months of follow - up (18, 29). moreover, maas. found that women with a history of radical hysterectomy showed a significant decrease in maximum vaginal pulse amplitude during sexual arousal, and the variation in vaginal pulse amplitude during sexual arousal occurred with regard to the fact that all the patients experienced equally strong sexual arousal (30). in some studies, most of the patients did not experience any changes in the frequency and intensity of orgasm (11, 14). goetsch also found that the intensity of orgasm and nipple stimulation after vaginal and abdominal hysterectomy were similar to those before the surgery or increased, and 13% of the women reported a decrease in the intensity of orgasm after the surgery (26). study showed that hysterectomy had caused sexual disorders, such as the decreased pleasure after the intercourse and reaching orgasm (12). some studies reported the failure to have orgasm as one of the sexual problems after hysterectomy, which was significantly higher than that before the surgery (19, 22). reported disorders in all sexual functions, including a decrease in frequency of orgasm after radical hysterectomy (24). study, the patients had experienced severe problems with orgasm and unpleasant sexual intercourses regarding their shortened vagina during 6 months after radical hysterectomy as compared with the patients in the control group (31). regard, two other studies reported that the problems with orgasm decreased after the surgery, and there were very few exacerbated problems (13, 17). in some similar studies, women experienced significant improvement of orgasm after hysterectomy, and sexual pleasure considerably improved in most of the patients regardless of the type of surgery (6, 10, 32). in rhodes study, the frequency of orgasm increased after hysterectomy, and the failure to have orgasm significantly decreased 12 months and 24 months after the surgery (16). although libido and frequency of intercourses after abdominal hysterectomy was significantly lower than those before the surgery in kuscu. study, no difference was observed in terms of dyspareunia and sexual satisfaction (25). study, hysterectomy affected the unusual vaginal contractions and the fear and avoidance of a sexual intercourse but not the pain during intercourse (12). badakhsh. also reported a decrease in dyspareunia, an increase in vaginal dryness, and a decrease in sexual satisfaction following hysterectomy (9). found that deep dyspareunia decreased 6 - 12 after hysterectomy, while superficial dyspareunia decreased 6 months after the surgery but increased after 12 months (31). according to dragisic., it seems that hysterectomy causes pain during sexual intercourses (11). the sexual dysfunctions in pieters s study during 24 months of follow - up after hysterectomy included the narrowness and shortening of vagina and dyspareunia (29). study included dyspareunia associated with the shortening of the vagina and decreased vaginal lubrication (19). jensen. reported that the patients who had undergone radical hysterectomy for treatment of the initial stages of the cervical cancer experienced severe dyspareunia during the first 3 months after the surgery, and radical hysterectomy had adverse long - term and short - term effects on sexual functioning, including dyspareunia and pain and anxiety due to the shortening of vagina during the intercourse as short - term adverse effects (18). some studies also mentioned the dyspareunia caused by a decrease in vaginal lubrication and vaginal narrowness and shortening as influential sexual problems after radical hysterectomy (24, 33 - 34). however, some other studies showed a decrease in bleeding disorders and dyspareunia after hysterectomy, which resulted in improvement of sexual functioning, satisfaction and quality of life (13, 16, 21). in gult. study, sexual dysfunctions, such as dyspareunia and vaginismus, significantly decreased after abdominal and vaginal hysterectomy (17). based on the review of the relevant studies, the effect of hysterectomy on sexual satisfaction differed from one study to another. in this regard, two studies did not find any difference in the sexual satisfaction before and after the surgery (14, 25). however, women who had undergone hysterectomy for their benign gynecological conditions in two other studies experienced high degrees of sexual satisfaction (10, 27). anonymous also reported that the favorable sexual satisfaction after the surgery was similar to that before the surgery or even increased, which was not unexpected due to the decreased problems related to the pain, arousal, and orgasm (13). for instance, badakhsh. found a significant increase in number of people without sexual satisfaction or with poor sexual satisfaction after hysterectomy and a decrease in number of people with favorable and optimum sexual satisfaction, which occurred by the psychological changes following the surgery and increased vaginal dryness (9). two other studies also reported that the patients sexual satisfaction decreased after the surgery (22, 24). study, as the decrease was independent from the type of surgery and oophorectomy (20). the present review study focused on the effect of hysterectomy on women s sexual functioning and did not review the studies on the effect of couples sexual performance and relationship. furthermore, the studies on the effect of hysterectomy on women s sexual functioning were reviewed regardless of the method of surgery and emotional, mental, and social consequences of hysterectomy. the present study reviewed the effect of hysterectomy on women s general sexual functions, including desire, sexual arousal, orgasm, pain, and sexual satisfaction through reviewing a broad range of studies performed in several years. two studies reported that most of the patients did not experience any changes in their sexual desire (11 - 12). in a study, women who had been sexually active before the surgery maintained their sexual activity after the surgery and reported the same frequency of sexual activity after 6 months (13). meston s study did not show any significant difference between women undergoing hysterectomy and women with fibroids but not undergoing hysterectomy in terms of desire (14). another study reported that neither the body image nor the libido decreased after hysterectomy, and no important changes occurred in this regard (15). however, the women undergoing hysterectomy in some studies experienced considerable improvement of sexual desire (6, 10, 16). furthermore in gutl.s study, sexual dysfunctions, such as the loss of sexual desire, significantly decreased after the abdominal and vaginal hysterectomy, and women reported the improvement in their sexual desire and sexual satisfaction 3 months and 2 years after the surgery (17). however, some other studies mentioned the decreased sexual desire after hysterectomy. in this regard, jensen. found there was a long - term lack of sexual interest in patients 12 months after radical hysterectomy as compared with that before the diagnosis of the cancer and however, many patients who were sexually active before the diagnosis of cancer became active again 12 months after the surgery although they reported fewer sexual intercourses (18). a study revealed that the poor libido was one of the sexual concerns reported after hysterectomy (19). some other studies also showed that sexual desire and frequency of intercourse decreased significantly (20 - 21). furthermore, problems related to sexual desire and feeling of women undergoing hysterectomy were significantly higher than those before the surgery (22). bayram and sahin revealed that sexual activity significantly decreased 3 months after hysterectomy and proved obvious symptoms of depression that had affected the sexual functioning negatively (23). study on women undergoing radical hysterectomy for treatment of initial stages of the cervical cancer reported dysfunction of all sexual aspects, including a decrease in sexual desire that was lower than the decrease in other aspects (24). study also showed that tah and tah+ bso mainly decreased the sexual desire (25). some studies showed positive effects of hysterectomy on sexual arousal, and some other studies showed negative effects in this regard. most women in goetsch s study experienced higher sexual arousal after abdominal and vaginal hysterectomy, and only 25% of the women reported decreased sexual arousal (26). anonymous also reported that the problems related to sexual arousal generally decreased after hysterectomy (13). in some studies, vaginal dryness decreased after hysterectomy, and women significantly improved in terms of sexual arousal and activity 3 months and 2 years after hysterectomy (6, 16 - 17). however, some other studies reported that hysterectomy increased vaginal dryness and abnormal vaginal contractions. one of the sexual problems after hysterectomy was inadequate vaginal lubrication that was more constant outcome and eventually decreased sexual satisfaction (9, 12, 19, 27). in meston s study, women undergoing hysterectomy reported low normal level of vaginal lubrication that implied the potential sexual - mental arousal following the hysterectomy although no significant difference was found between groups undergoing hysterectomy and groups with fibroids but not undergoing hysterectomy in terms of sexual arousal (14). study reported significant deterioration of the sensation of cold and warm stimuli in anterior and posterior vaginal wall after hysterectomy (28). tangjitgamol s study reported disorders in all aspects of sexual functioning, including decreased sexual arousal and vaginal lubrication, as the most obvious changes were the decreased sexual frequency and vaginal lubrication, and sexual arousal decreased to a less extent (24). in pieterse study, the patients who had undergone radical hysterectomy for treatment of the initial stages of their cervical cancer reported a negatively significant effect of the surgery, in comparison to their condition before the surgery and to the patients in the control group, on their sexual functioning, such as the less lubrication, narrowness and shortening of the vagina, and numb areas around the labia, during 24 months of follow - up (18, 29). moreover, maas. found that women with a history of radical hysterectomy showed a significant decrease in maximum vaginal pulse amplitude during sexual arousal, and the variation in vaginal pulse amplitude during sexual arousal occurred with regard to the fact that all the patients experienced equally strong sexual arousal (30). in some studies, most of the patients did not experience any changes in the frequency and intensity of orgasm (11, 14). goetsch also found that the intensity of orgasm and nipple stimulation after vaginal and abdominal hysterectomy were similar to those before the surgery or increased, and 13% of the women reported a decrease in the intensity of orgasm after the surgery (26). study showed that hysterectomy had caused sexual disorders, such as the decreased pleasure after the intercourse and reaching orgasm (12). some studies reported the failure to have orgasm as one of the sexual problems after hysterectomy, which was significantly higher than that before the surgery (19, 22). reported disorders in all sexual functions, including a decrease in frequency of orgasm after radical hysterectomy (24). study, the patients had experienced severe problems with orgasm and unpleasant sexual intercourses regarding their shortened vagina during 6 months after radical hysterectomy as compared with the patients in the control group (31). regard, two other studies reported that the problems with orgasm decreased after the surgery, and there were very few exacerbated problems (13, 17). in some similar studies, women experienced significant improvement of orgasm after hysterectomy, and sexual pleasure considerably improved in most of the patients regardless of the type of surgery (6, 10, 32). in rhodes study, the frequency of orgasm increased after hysterectomy, and the failure to have orgasm significantly decreased 12 months and 24 months after the surgery (16). although libido and frequency of intercourses after abdominal hysterectomy was significantly lower than those before the surgery in kuscu. study, no difference was observed in terms of dyspareunia and sexual satisfaction (25). study, hysterectomy affected the unusual vaginal contractions and the fear and avoidance of a sexual intercourse but not the pain during intercourse (12). badakhsh. also reported a decrease in dyspareunia, an increase in vaginal dryness, and a decrease in sexual satisfaction following hysterectomy (9). thakar. found that deep dyspareunia decreased 6 - 12 after hysterectomy, while superficial dyspareunia decreased 6 months after the surgery but increased after 12 months (31). according to dragisic. the sexual dysfunctions in pieters s study during 24 months of follow - up after hysterectomy included the narrowness and shortening of vagina and dyspareunia (29). similarly, sexual problems reported in bayram. study included dyspareunia associated with the shortening of the vagina and decreased vaginal lubrication (19). jensen. reported that the patients who had undergone radical hysterectomy for treatment of the initial stages of the cervical cancer experienced severe dyspareunia during the first 3 months after the surgery, and radical hysterectomy had adverse long - term and short - term effects on sexual functioning, including dyspareunia and pain and anxiety due to the shortening of vagina during the intercourse as short - term adverse effects (18). some studies also mentioned the dyspareunia caused by a decrease in vaginal lubrication and vaginal narrowness and shortening as influential sexual problems after radical hysterectomy (24, 33 - 34). however, some other studies showed a decrease in bleeding disorders and dyspareunia after hysterectomy, which resulted in improvement of sexual functioning, satisfaction and quality of life (13, 16, 21). in gult. study, sexual dysfunctions, such as dyspareunia and vaginismus, significantly decreased after abdominal and vaginal hysterectomy (17). based on the review of the relevant studies, the effect of hysterectomy on sexual satisfaction differed from one study to another. in this regard, two studies did not find any difference in the sexual satisfaction before and after the surgery (14, 25). however, women who had undergone hysterectomy for their benign gynecological conditions in two other studies experienced high degrees of sexual satisfaction (10, 27). anonymous also reported that the favorable sexual satisfaction after the surgery was similar to that before the surgery or even increased, which was not unexpected due to the decreased problems related to the pain, arousal, and orgasm (13). for instance, badakhsh. found a significant increase in number of people without sexual satisfaction or with poor sexual satisfaction after hysterectomy and a decrease in number of people with favorable and optimum sexual satisfaction, which occurred by the psychological changes following the surgery and increased vaginal dryness (9). two other studies also reported that the patients sexual satisfaction decreased after the surgery (22, 24). study, as the decrease was independent from the type of surgery and oophorectomy (20). the present review study focused on the effect of hysterectomy on women s sexual functioning and did not review the studies on the effect of couples sexual performance and relationship. furthermore, the studies on the effect of hysterectomy on women s sexual functioning were reviewed regardless of the method of surgery and emotional, mental, and social consequences of hysterectomy. the present study reviewed the effect of hysterectomy on women s general sexual functions, including desire, sexual arousal, orgasm, pain, and sexual satisfaction through reviewing a broad range of studies performed in several years. the examination of the sexual functioning after hysterectomy is a complicated multifactorial process that depends on various factors, such as the body image ; the sexual partner s performance ; communicative matters ; the reason for undergoing hysterectomy ; the patient s sexual functioning before the surgery ; type of hysterectomy ; mental, social, and emotional factors ; and total quality of life in patients. according to the studies reviewed in this study, most of the sexual disorders improve after hysterectomy for uterine benign diseases, and most of the patients who were sexually active before the surgery experienced the same or better sexual functioning after the surgery. however, radical hysterectomy for gynecological cancers causes more negative effects on sexual functioning due to the elimination of a large part of pelvic ligaments and pelvic autonomic nerves. one of the important points about these patients before and after the surgery is the psychological supports for them, adaptation with postoperative problems, and especially the sexual partner s support for them. an important solution for making these women ready to face with postoperative sexual complications is to train them on the basis of needs assessment in order that the patients undergoing hysterectomy be ready and capable of coping with the complications, and their sexual functioning improves after the surgery. application in education in general, patients do not receive adequate information about their sexual health and consequences before hysterectomy. the existing barriers in this regard include cultural restrictions, personal and shame from both patients and physicians, and inadequate education and low educational levels. furthermore, the definitions and consequences of the surgery for women s sexual matters should be integrated and clear, and it is necessary to precisely explain sexual and communicative problems with the sexual partner to patients, preferably by a physician of the same sex. application in research this study reviewed the effect of hysterectomy on women s sexual functioning and revealed the need to perform studies on sexual training requirements of women undergoing hysterectomy. moreover, this study laid the ground for further studies on the effect of hysterectomy on sexual functioning in terms of the method of surgery and the effect of hysterectomy on women s physical and emotional issues. | background : regarding the contradictions about positive and negative effects of hysterectomy on women s sexual functioning, this study was conducted to review the studies on the effect of hysterectomy on postoperative women s sexual function.method:this study was a narrative review and performed in 5 steps : a) determining the research questions, b) search methods for identification of relevant studies, c) choosing the studies, d) classifying, sorting out, and summarizing the data, and e) reporting the results.findings:the review of the studies yielded 5 main categories of results as follows : the effect of hysterectomy on sexual desire, the effect of hysterectomy on sexual arousal, the effect of hysterectomy on orgasm, the effect of hysterectomy on dyspareunia, and the effect of hysterectomy on sexual satisfaction.conclusion:according to the studies reviewed in this study, most of the sexual disorders improve after hysterectomy for uterine benign diseases, and most of the patients who were sexually active before the surgery experienced the same or better sexual functioning after the surgery. an important solution for making these women ready to face with postoperative sexual complications is to train them on the basis of needs assessment in order that the patients undergoing hysterectomy be ready and capable of coping with the complications, and their sexual functioning improves after the surgery. |
almost 263,020 oral cavity cancers, and 127,654 oral cancer deaths occur worldwide each year. on january 1, 2010, in the us there were around 275,193 women and men alive who had a history of oral and oro - pharyngeal cancer (181,084 men and 94,109 women). the 5-year survival rate of patients with oral cancer remains almost unchanged regardless of various treatment improvements in the last thirty years. individuals at high risk of developing oral cancer (oc) are mainly older, males, heavy tobacco smokers and alcohol users, and have a poor diet and low socioeconomic status. recent studies have implicated hpv infection as an independent risk factor for oro - pharyngeal cancers. oral squamous cell carcinoma initiates in a multi - step process in which normal cells are transformed into preneoplastic cells and then to cancer. during this process within the oral cavity, lesions such as leukoplakia, erythroplakia, lichen planus and submucous fibrosis have a propensity for malignancy. oral potentially malignant disorders (opmd) transform to oral cancers through various histopathological stages from hyperkeratosis / hyperplasia, to various degrees of dysplasia (categorized by mild, moderate, or severe according to the presence and severity of cell atypia and other structural aspects of the epithelium), to carcinoma in situ (cis), and finally to invasive cancer. histopathological evaluation for the grade of epithelial dysplasia is the most common method used to ascertain malignant potential of individuals with oral pre - cancerous lesions. early detection for oral cancer has the potential to decrease the morbidity and mortality of the disease, especially in high - risk individuals. to date only one randomized clinical trial evaluated the effect of oral cancer screening and demonstrated that periodic oral examination has the potential to reduce mortality from oral cancer in high - risk individuals. visual and tactile examination remains the most common tool available to detect any mucosal changes and requires a 90-s exam, yet few oral health care providers are conducting a thorough oral mucosal exam. the aim of the present study was to 1) estimate the prevalence of opmds and 2) identify the associated risk factors in a large dental population. all new patients aged 18 or older, attending the oral diagnosis clinic in the department of general dentistry at boston university henry m. goldman school of dental medicine from july 8, 2013, through march 8, 2014 were included in this study. each patient was asked questions on : socio - demographic information including age and gender ; self - reported medical history ; family history of cancer ; tobacco smoking and alcohol consumption ; height and weight to determine body mass index (bmi). a thorough visual oral soft tissue examination was performed on each patient by a dental student first and then by an attending dentist to identify any opmd or any other mucosal lesion using mouth mirrors and a sterile piece of gauze to retract the tongue. the diagnostic criteria for the recognition of opmd (erythroplakia, leukoplakia, oral lichen planus and submucous fibrosis) were based on the who recommendations. although oral lichen planus is classified as an opmd there is still controversy on its pre - malignant nature. patients with leukoplastic and/or erythroplastic lesions and without a definitive clinical diagnosis were reevaluated and considered for biopsy for diagnostic purposes (figure 1). individuals with opmd were considered as cases and those without any opmd as controls. figure 1oral mucosal lesions and oral potentially malignant disorders (opmds) in a dental population we described the distribution of patient characteristics, including demographics, tobacco smoking, and daily alcohol consumption. to explore the association between opmds and risk factors that might be expected to be associated with opmds, we used logistic regression models to estimate the odds ratios (ors) and 95% confidence intervals (cis). statistical analyses were performed using stata, version 9.2 (stata corp lp, college station, tx, usa). for all analyses, a p value of < 0.05 (2-tailed) was considered statistically significant. all new patients aged 18 or older, attending the oral diagnosis clinic in the department of general dentistry at boston university henry m. goldman school of dental medicine from july 8, 2013, through march 8, 2014 were included in this study. each patient was asked questions on : socio - demographic information including age and gender ; self - reported medical history ; family history of cancer ; tobacco smoking and alcohol consumption ; height and weight to determine body mass index (bmi). a thorough visual oral soft tissue examination was performed on each patient by a dental student first and then by an attending dentist to identify any opmd or any other mucosal lesion using mouth mirrors and a sterile piece of gauze to retract the tongue. the diagnostic criteria for the recognition of opmd (erythroplakia, leukoplakia, oral lichen planus and submucous fibrosis) were based on the who recommendations. although oral lichen planus is classified as an opmd there is still controversy on its pre - malignant nature. patients with leukoplastic and/or erythroplastic lesions and without a definitive clinical diagnosis were reevaluated and considered for biopsy for diagnostic purposes (figure 1). individuals with opmd were considered as cases and those without any opmd as controls. figure 1oral mucosal lesions and oral potentially malignant disorders (opmds) in a dental population we described the distribution of patient characteristics, including demographics, tobacco smoking, and daily alcohol consumption. to explore the association between opmds and risk factors that might be expected to be associated with opmds, we used logistic regression models to estimate the odds ratios (ors) and 95% confidence intervals (cis). statistical analyses were performed using stata, version 9.2 (stata corp lp, college station, tx, usa). for all analyses, a p value of < 0.05 (2-tailed) was considered statistically significant. a total of 3,142 patients (54.3%, females) received a comprehensive examination of the oral cavity (table 1). at the time of the oral exam, patients ranged in age from 18 to 97 years, with a median age of 43 years. tobacco smoking and alcohol consumption was reported in 75.8% and 63.9% of the individuals, respectively. a total of 142 patients (4.5%) were identified as having a mucosal lesion (78 white lesions, 30 ulcerative lesions, 34 mixed lesions ; figure 1) among these, 37 patients (1.2%) had a suspicious opmd and received an oral biopsy for definitive diagnosis. one patient was affected by submucous fibrosis, three were diagnosed with leukoplakia and nine with oral lichen planus. table 1patients characteristics totalopmd (n=3,142)(n=27) n (%) n (%) age 18 - 30906 (28.8)2 (7.5)31 - 501,134 (36.1)12 (44.4)50 + 1,102 (35.1)13 (48.1)median (range)43.0 (18 - 97)49 (23 - 88) gender female1,706 (54.3)11 (40.7)male1,436 (45.7)16 (59.3) daily tobacco use never2,259 (75.8)17 (63.0)ever722 (24.2)10 (37.0) daily alcohol consumption never1,906 (63.9)19 (70.4)ever1,078 (36.1)8 (29.6)opmd : oral potentially malignant disorders opmd : oral potentially malignant disorders male patients were associated with higher odds of having opmd (or 1.7, 95% ci 0.83.8 ; p=0.16 ; table 2). individuals who were current tobacco smokers were twice as likely to have an opmd (or 1.9, 95% ci 0.84.1 ; p=0.12) compared to never smokers. increasing age was associated with having opmds (p<0.05). daily alcohol consumption did not increase the risk of having an opmd (or 0.7. we observed no significant associations for systemic diseases, bmi and opmds (data not shown). table 2multivariate analysis for oral potentially malignant disorders opmd no (n=3,115)yes (n=27)odds ratio (95% ci)p for trend age 18 - 30904 (99.8)2 (0.2)1.0<0.0131 - 501,122 (98.9)12 (1.1)4.8 (1.1 - 21.7) 50 + 1,089 (98.8)13 (1.2)5.4 (1.2 - 24.0) gender female1,695 (99.4)11 (0.6)1.00.16male1,420 (98.9)16 (1.1)1.7 (0.8 - 3.8) daily tobacco use never2,242 (99.3)17 (0.7)1.00.12ever712 (98.6)10 (1.4)1.9 (0.8 - 4.1) daily alcohol consumption no1,887 (99.0)19 (1.0)1.00.48yes1,070 (99.3)9 (0.7)0.7 (0.3 - 1.7) opmd : oral potentially malignant disorders opmd : oral potentially malignant disorders we conducted a large study in a population of dental patients and found that around 1% had an opmd upon oral examination (subsequently confirmed by histopathological examination). the oral visual and tactile examination remains a non - invasive tool that can result in earlier diagnosis of opmds, but also a large number of other oral mucosal diseases. (2011) showed that smoking was associated with a more than two - fold increase in the odds of having an opmd (or 2.5, 95% ci : 1.34.8). (2005) reported that individuals who were current smokers had a 4.7-fold (95% ci : 3.26.8) increased risk of having an opmd. however, when alcohol consumption was considered, we did not find any statistical significant association with opmds whereas chung,. (2005) found that opmds among individuals reporting alcohol drinking were significantly higher than in non - drinkers (or 3.6, 95% ci : 2.45.3). our findings support that oral cancer screenings should not be a separate procedure rather they should be part of the complete dental examination for all patients. primary prevention of oscc should focus on the prevention of cancer by avoiding known carcinogens (e.g., heavy tobacco consumption). secondary cancer prevention includes early detection of cancer through screening programs in a population at risk and asymptomatic, as well as prevention of the transformation of opmds. dentists and all members of the oral health team have the unique opportunity to prevent smoking uptake and promote smoking cessation among their patients and may therefore reduce the prevalence of opmds. in addition, patients at high risk may be referred to specialists for behavioral counseling interventions to reduce tobacco use or heavy alcohol consumption. oral mucosal lesions are easily detected through direct visualization, and so oral health providers, otolaryngologists, primary care physicians and nurse practitioners should be effectively trained to perform a comprehensive oral mucosal examination and identify abnormal lesions. as in all studies, larger studies are necessary to further explore the association between smoking tobacco, alcohol consumption and opmds. second, our results may not be generalizable to the population at large, as only dental patients were included. randomized - controlled trials may be useful to further investigate the sensibility and specificity of the oral visual examination in the context of opmds. the commission on dental accreditation (coda) in the united states has recently approved a new dental curriculum to help dental students become proficient in oral cancer screening (comprehensive general dentistry, coda 2 - 23 b). optimal oral visual screening for oc remains a simple and essential tool to identify any suspicious lesions and potentially increase survival. a thorough visual and tactile examination in dental patients, particularly those with a history of smoking and elderly is warranted. dentists and dental students should stay alert for signs of oral potential malignancy and counsel their patients about risk factors for oral cancer. although opmds were rare, our results confirm the importance of a thorough chairside screening by dentists to detect any mucosal changes. | objectives oral cancer (oc) may be preceded by clinically evident oral potentially malignant disorders (opmds). oral carcinogenesis is a multistep process that begins as epithelial hyperplasia and progresses to oral epithelial dysplasia and finally to fully malignant phenotypes. the aim of our study was to estimate the prevalence of opmds in a large population of dental patients.methods patients were seen in the oral diagnosis and oral medicine clinics at boston university henry m. goldman school of dental medicine between july 2013 and february 2014 and received a comprehensive oral examination to identify any possible mucosal lesions. patients with a suspected opmd (submucous fibrosis, oral lichen planus, leukoplakia and erythroplakia) that did not resolve in 23 weeks received a biopsy for definitive diagnosis. logistic regression models were used to explore the relationship between opmds and associated risk factors.results a total of 3,142 patients received a comprehensive oral examination [median age : 43 (range : 1897) ; 54.3% females ]. among these, 4.5% had an oral mucosal lesion with 0.9% being an opmd (one submucous fibrosis, three epithelial dysplasias, fourteen with hyperkeratosis / epithelial hyperplasia and nine with oral lichen planus). males and current smokers were associated with higher odds of having opmd (or 1.7, 95% ci 0.83.8 ; or 1.9, 95%ci 0.84.1). increasing age was associated with having opmds (p<0.01).conclusion optimal oral visual screening for oc remains a simple and essential tool to identify any suspicious lesions and potentially increase survival. although opmds were rare, our results confirm the importance of a thorough chairside screening by dentists and dental students to detect any mucosal changes. |
it has been recognized since at least as early as the mid-1500s that inhaled minerals (i.e., inorganic particles) can pose a risk. extensive research has focused on the biological mechanisms responsible for asbestos- and silica - induced diseases, but much less attention has been paid to the mineralogical properties and geochemical mechanisms that might influence a mineral 's biological activity. several important mineralogical characteristics control a mineral 's reactivity in geochemical reactions and are likely to determine its biological reactivity. in addition to the traditionally considered variables of particle size and shape, mineralogical characteristics such as dissolution behavior, ion exchange, sorptive properties, and the nature of the mineral surface (e.g., surface reactivity) play important roles in determining the toxicity and carcinogenicity of a particle. ultimately, a mineral 's species (which provides direct information on a mineral 's structure and composition) is probably one of the most significant yet most neglected factors that must be considered in studies of toxicity and carcinogenicity.imagesfigure 4. |
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induced parturition in cows may have several technological and medical advantages over spontaneous delivery, such as avoiding unattended night - time calving, providing colostrum to the newborn in time, preventing dystocia caused by relative fetal oversize, dealing with fetal deformities, and hydroallantois. calving can be successfully induced by an intramuscular injection of prostaglandin f2 (pgf2). it acts on the corpus luteum to cause luteolysis and stops the production of progesterone. time required from treatment to calf delivery ranges from 24 to 72 hours, on average 48 hours. however, the major problem arising from this procedure is a dramatic rise in the incidence of retained placenta (rp), up to 80% in some cases, although no other serious harmful effects on cows and calves have been reported. retained placenta (rp) is a reproductive disorder seen in cows and water buffalos. it is characterized by the inability of the animal to expel fetal membranes within 12 hours after parturition. its incidence varies from 1.3 to 39.2% of overall parturitions, resulting in serious economic losses in the dairy industry throughout the world. the etiology of rp is complex and not yet fully understood. in the majority of cases, it is directly caused by a disturbance of the prepartal loosening mechanism in the placentomes. furthermore, cows with rp are at high risk of developing metritis and silent heat, resulting in prolonged service period and low fertility rate [6, 7 ]. various factors, such as age, breed, heredity, environment, hormonal status, nutrition, and immunity had been implied to contribute to the development of rp. however, no single factor was sufficient to provide a satisfactory explanation for the mechanism underlying the development of this disorder. it is well established that innate and acquired immune defense mechanisms are lowest in the peripartum period due to metabolic strain during pregnancy, calving, and early lactation. two immune mechanisms had been proposed to explain the detachment of the fetal membranes during parturition. both were based on the hypothesis that upon parturition, when blood supply to the placenta begins to cease, the placenta becomes a foreign body that the maternal immune system must recognize, attack, and expel. gunnink demonstrated that leukocytes were less able to recognize cotyledon tissue harvested from cows with rp compared non - rp. however, kimura. argued that 38 hours needed to expel fetal membranes was a too short time for lymphocytes to mount an effective rejection process ; hence, they focused on the role of innate immunity mediated by neutrophils through their oxygen - dependent killing capabilities. pregnancy in dairy cows is considered to induce oxidative stress, which in turn can be a significant underlying factor to dysfunctional host immune and inflammatory responses that can increase the incidence of perinatal disorders during the transition period [11, 12 ]. proposed a pathway of rp development starting with an imbalance of the antioxidant capacity, followed by a decrease in estrogen production, resulting in decreased pgf2 and accumulation of arachidonic and linoleic acids in the placental tissue. therefore, it is necessary that the animal organism should be able to maintain its antioxidative / oxidative processes in balance during peripartal period. methods for quantifying oxidative stress mostly include direct or indirect measures of antioxidants and oxidants, or their metabolic products present in animal tissues. malondialdehyde (mda) is one of the several low - molecular weight end - products that arise from oxidative decomposition of polyunsaturated fatty acids. mda readily reacts with thiobarbituric acid producing a red pigment that can be measured spectrophotometrically. selenium is well established as one of the key elements in the antioxidative defense of a living organism. in the active form, it is incorporated in glutathione peroxidases (gpx), a family of enzymes that use the reductive potential of glutathione (gsh) to convert hydroperoxides to corresponding alcohols. according to bernabucci. [16, 17 ], plasma gpx activity in cows raises several days prior to parturition and gradually declines afterwards, while in the erythrocytes, the activity remains constant. however, in its reaction with the peroxyl radicals vitamin e produces hydroperoxides that are still toxic and must be removed by gpx, hence the functional interdependence of selenium and vitamin e. the first report on the relationship between se and vitamin e status of cows and retention of placenta was published by trinder. drawing substantial attention to this subject. experiments conducted by jaskowski and wentink. established a minimal selenium concentration in cow 's blood plasma of 30 ng / ml, below which the incidence of rp would significantly rise, namely, from 5 - 6% to 2022%. preventive effects of different doses of se and/or vitamin e against rp prepartally supplemented to cows, have been reported over the years by a number of authors [2025 ]. all three known types of iodothyronine deiodinases (i d), enzymes which activate and/or inactivate thyronines through deiodination, contain se in the form of selenocysteine in their catalytic sites. however, i d activities are not as tightly linked to se status as the activity of gpx. therefore, serum thyroxine (t4) and triiodothyronine (t3) levels as well as t4/t3 ratio may only be slightly affected by marginal se deficiency. according to nixon the stage of lactation and season of the year have a strong influence on changes in serum thyronines.. reported that serums t4 and t3 concentrations in periparturient cows start to decrease 14 days prior to delivery and recover slowly within the first few weeks after parturition. high incidence of retained placenta, together with a wide variability in the intervals between prostaglandin f2 treatment and calf delivery, remains the primary limiting factor for the practical use of pgf2 in the synchronization of parturition on dairy farms. the aim of this trial was to determine to which extent a preparturient supplementation with different doses of se and vitamin e would influence the incidence of rp, as well as selenium, mda, and thyronines status in cows treated with pgf2 for the induction of parturition. thirty - three (33) holstein - frisian cows included in this investigation were randomly distributed to 3 groups and supplemented with sodium selenite (ss) and tocopherol acetate (tac) as follows : control group (n = 9) was not supplemented, and it served as a negative control;group a (n = 11) was supplemented 10 mg ss and 400 mg tac;group b (n = 13) was supplemented 20 mg ss and 800 mg tac. control group (n = 9) was not supplemented, and it served as a negative control ; group a (n = 11) was supplemented 10 mg ss and 400 mg tac ; group b (n = 13) was supplemented 20 mg ss and 800 mg tac. the supplement was administered by a single intramuscular injection between days 250 and 255 of gestation ; parturition was induced using a single intramuscular injection of pgf2 (2 ml, 500 g of cloprostenol) not before day 275 of gestation ; venous blood samples for analysis were taken 12 hours postpartum. all animals were clinically healthy, multiparous, and single calve, with no previous record of retained placenta or other disorders. determination of selenium in whole blood samples of cows was carried out using atomic absorption spectrometry - hydride technique. samples of whole blood (0.5 g) were accurately weighted using analytical balance denver instrument, model tb-215d (denver instruments, usa), transferred into teflon microwave vessels, and digested with 8 ml of 69% nitric acid (sigma - aldrich, usa) and 2 ml of 30% hydrogen peroxide (fluka analytical, usa). microwave oven (milestone, germany, model touch control) was set to the following program : temperature ramp from ambient temperature at 180c followed by 15 min of holding time and 20 min of cooling time. digested samples were transferred to volumetric flasks and diluted using 5 m hydrochloric acid (sigma - aldrich, usa) to the final volume of 25 ml. determination of selenium concentration was carried out using solaar, series 4 spectrometer equipped with vp70 hydride module and ec90 electrical furnace for precise temperature control of the analytical cuvette (thermo electron, uk). measuring absorption at 196 nm was performed after seh4 formation in the hydride system with 5% nabh4 (j. t. baker, the netherlands) and 0.6% naoh (merck, germany). stabilization and baseline delay times were 40 and 60 seconds, respectively, and reading time was 7 seconds in three replicates. quantification of se content was performed using five - point calibration curve (1040 g / kg, including zero) of reference standard solutions (merck, germany). quality control was achieved using blank samples fortified in 20 g / kg of se and certified reference material (bcr 189). good linearity was obtained from the calibration curve (r = 0.998), and the measured concentration of the reference material was in the range of the reference value. briefly, a 3 ml of 0.1% orthophosphoric acid, 1 ml of 0.6% thiobarbituric acid, and 0.1 ml of 0.28% hydrated ferrous sulfate solution were added to 0.3 ml of serum. the produced chromogen was extracted with n - butyl alcohol (4 ml). after centrifugation (2200 g, 10 minutes), the butanol layer was separated for spectrophotometric measurement at 535 nm. glutathione peroxidase activity was measured in whole blood samples by the coupled test described by gnzler.. blood samples were hemolyzed using drabkin 's reagent (1.6 mm kcn, 1.2 mm k2fe (cn)6, and 0.023 m nahco3). glutathione (gsh) as the donor of hydrogen becomes oxidized to gs - sg. in the second phase of this coupled reaction, gs - sg is reduced to gsh by nadph and glutathione reductase (gr). final concentrations of used reagents were 100 mm phosphate buffer (ph 7.4), 4 mm edta, 6 mm gsh, 0.375 iu / ml gr, 0.3 mm nadph and 1.575 mm tbh. low concentration of tbh (under 2.32 mm) as used in this method determines only the activity of se - dependent gpx. the reduction of nadph was followed for 3 min at 366 nm using a cecil ce2021 spectrophotometer (uk) with a peltier thermostat unit. absorbance (a) values were taken at 30 seconds intervals, and the results were expressed in microkatals per liter (kat / l). concentrations of t3 and t4 were measured in heparinized plasma samples using commercial standard ria kits (inep, zemun). the assay is based on the competition between unlabelled t3 and t4 and a fixed quantity of l labeled t3 and t4 for a limited number of binding sites on t3 and t4 specific antibodies (bound to the tubes). allowing a fixed amount of tracer and antibody to react with different amounts of unlabelled ligand, the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. antigen - antibody complex is bound to the tubes, and the supernatant is then separated by decantation. counting the radioactivity of the bound phase obtained results were arranged and analyzed from two aspects : table 1 displays experimental results according to se and vitamin e treatments as previously described, and table 2 displays the results according to the absence / presence of retained placenta in cows, diagnosed 12 hours postpartum, independently from se and vitamin e treatment. analysis was performed using ms excel 2007 and graph pad prism 5 statistical software packages. the treatment of cows with 10 mg sodium selenite (ss) and 400 mg tocopherol acetate (tac) reduced the incidence of retained placenta from 66.7% in the control group to 38.2% in group a, while treatment with 20 mg ss and 800 mg tac further reduced rp to 30.8% in group b (table 1). blood selenium concentrations and gpx activities in both treated groups (a or b) were significantly higher compared to the control ; however, there were no significant differences between groups a and b. blood plasma mda content was lowest in group b (3.95 0.88 m), significantly lower than both control (5.70 0.94 m) and group a (4.59 1.20 m). total plasma t4 levels were highest in group a (59.55 13.00 nm) and lowest in group b (43.2 16.2 nm), while mean t3 concentrations ranged from 1.1 0.4 nm in group b to 1.7 0.6 nm in control group. comparison between cows with and without the diagnosed rp (independently from ss and tac treatment), revealed the following (table 2) : cows with rp had significantly lower blood se content and gpx activity and higher plasma mda concentrations compared to those free of rp. plasma t3 did not differ significantly, while plasma t4 concentration was significantly higher in cows with rp. the primary aim of this study was to test the assumption that prepartum supplementation of cows with se and vitamin e should exhibit the protective effect against the onset of retained placenta in cows with prostaglandin f2 induced parturition in a similar fashion as in animals with spontaneous parturition. to our best knowledge, this is the first such attempt ; therefore, the only available frame of reference for our data is the research done on animals with spontaneous labour. in cows supplemented with se and vitamin e, 20 days prior to parturition induced by pgf2, the incidence of retained placenta was effectively halved, from 66.7% in the control group to 38.2% and 30.8% in groups a and b, respectively (table 1). similarly, julien. demonstrated that a single 20-day prepartum injection of 50 mg of sodium selenite and 680 iu of -tocopherol acetate effectively reduced the incidence of rp from 51.2% in the control group of cows to 8.8% in the treated group. selenium alone was at least as effective as a combination of selenium and vitamin e. the higher the dose of selenium administered, the lower the incidence of rp was registered. in all these experiments, the percentage of rp in herds was reduced to values close to zero, while in our trial the lowest value remained at 30.8%. therefore, we estimate that further increase in se dosage would not produce a linear but rather asymptotic decrease in rp incidence the supplement dosage should be considered with great care, balancing between sufficient supply and avoiding toxicity. marked discrepancies in dose / effect ratios described in the literature may be the result of different basal se status of animals, which is highly dependable on naturally occurring se levels in locally produced feedstuffs. according to investigations conducted by jovanovi. in the region surrounding the experimental farm, se content in cereals and hay ranged from 40 to 62 gse / kg. furthermore, as the retention of placenta is known to be a multifactorial disorder, our results demonstrate that certain effects of pgf2 may not fall into the domain of oxidant - antioxidant balance in the body and can not be overcome solely by the antioxidative actions of se and vitamin e. blood selenium concentration is considered to be a good indicator of the long - term se status in most animal species. selenium concentration was significantly elevated (table 1) in groups a (163 28 ng / ml) and b (188 30 ng / ml) supplemented with 10 mg and 20 mg sodium selenite, respectively, compared to unsupplemented control animals (129 18 ng / ml). cows suffering from retained placenta (table 2) had significantly lower (p < 0.05) blood se content (138 40 ng / ml) compared to non - rp animals (176 33 ng / ml). collected blood samples from 254 se unsupplemented dairy herds in norway and found that their blood se concentrations ranged from 60 to 120 ng / g and proposed the content of 100150 ngse / g as a delimiting range below which a higher incidence of rp could be expected. blood selenium concentrations measured in their study were slightly below the range of the values found in unsupplemented cows in our experiment. this reflects the fact that norwegian crops are slightly more selenium deficient than those in the northern part of serbia. however, both sets of data corroborate that the blood concentration of 150 ngse / ml could represent the boundary between higher and lower probability for the onset of rp. the activity of selenium - dependent glutathione peroxidase (gpx) in the blood of dairy cows from groups a and b (182 32 and 186 33 kat / l, resp.) was twice as high as in the control (91 15 kat / l) ; however, activities did not significantly differ between groups a and b (table 1). blood gpx activity was significantly lower in cows with rp than in non - rp cows (table 2). comparable results were presented in studies conducted by wischral. and kankofer.. plasma mda concentrations were 5.70 0.94 m in control, 4.59 1.20 m in group a, and 3.95 0.88 m in group b. mda was significantly lower in treated animals (p < 0.05 in group a and p < 0.01 in group b) compared to control (table 1). however, all of these values are generally substantially higher than those published by wischral. we assume that high levels of mda may be due to elevated oxidative stress caused by premature parturition induced by pgf2. animals with rp had significantly higher (p < 0.01) plasma mda than non - rp cows (table 2), which is in accordance with the findings of the above - mentioned authors. concentrations of plasma thyroid hormones, t4 and t3, in our experiment (table 1) were below those published by jovanovi. for 12 months old heifers fed se - adequate feed but corresponded to the levels published by pethes. and djokovi however, t4 and t3 did not apparently depend upon blood selenium supplementation of the animals. on the other hand, plasma t4 was significantly higher in cows with diagnosed rp, compared to non - rp (table 2). our study demonstrated that in cows treated with single intramuscular injection of 20 mg sodium selenite and 800 mg tocopherol acetate three weeks prepartum there was a significant increase in plasma gpx activity and decrease in mda concentration. consequently, incidence of retained placenta in supplemented compared to nonsupplemented animals was reduced in half. this indicates that oxidative stress may be the principal, but not the only, cause of increased rp in animals with induced parturition. further investigation is needed, using reasonably higher supplement doses of antioxidants, to show whether the incidence of rp can be brought to values closer to zero in a similar fashion as in cows with spontaneous calving. | the incidence of retained placenta (rp) in cows increases in cases of parturition induced by prostaglandin f2. we analyzed the effects of different doses of supplemental selenium and vitamin e on the incidence of rp, blood selenium, plasma thyronines, and malondialdehyde concentration. thirty - three clinically healthy, multiparous holstein - frisian cows were assigned to 3 groups and supplemented with a single intramuscular injection of sodium selenite (ss) and tocopherol acetate (tac) between days 250 to 255 of gestation : control unsupplemented ; group a10 mg ss + 400 mg tac ; group b20 mg ss + 800 mg tac. parturition was induced using pgf2 not before day 275 of gestation. the rp incidence was reduced from 66.7% in the control to 38.2 and 30.8% in groups a and b, respectively. blood selenium and glutathione peroxidase activity in treated groups were significantly higher compared to control, with no significant difference between groups a and b. plasma malondialdehyde in group b was significantly lower than that in control and group a, while thyronines levels were not affected. comparison of rp and non - rp cows, independently of supplement treatment, revealed higher blood selenium and glutathione peroxidase activity and lower mda and thyroxine in non - rp animals, while triiodothyronine level did not differ. |
management of ao type c3 distal femur fractures with multifragmentary articular involvement is challenging, with most series reporting average to poor results and frequent complications such as malunion, knee stiffness, and secondary osteoarthritis.1234567891011 only a few studies123456 have exclusively reported the outcome of complex c3 type fractures of the distal femur, with only three such studies125 published in english literature. various surgical approaches have been suggested to achieve an adequate exposure of the distal femoral articular surface, including medial / lateral parapatellar approaches,712 swashbuckler approach,13 tibial tubercle osteotomy,514 and combined medial and lateral approaches.3 external ring fixators have been used with mixed results by several previous authors12 to address the comminution in these complex injuries. some have used dual medial and lateral plates in these.345 most previous authors have employed primary bone grafting to enhance union in these complex fractures.1234 the use of swashbuckler approach for c3 fractures has not been reported yet in the literature. we have reported the results in a consecutive series of patients with c3 type distal femur fractures, operated by a single surgeon in a tertiary level trauma center, with a single lateral locked plate (distal femur locking compression plate [df - lcp ]), using a modified swashbuckler approach, without primary bone grafting. 12 consecutive adult patients operated with open reduction and internal fixation of distal femur fractures with multifragmentary articular involvement (ao / ota type 33c3), between september 2012 and march 2014 were included in this prospective study. extraarticular fractures (type a), partial articular fractures (type b) and simple articular fractures (type c1 and c2) of the distal femur were not included in the study. patients with ipsilateral fractures in the same lower extremity and pathological fractures were excluded from the study to avoid confounding of results. all patients were initially managed according to advanced trauma life support guidelines.15 the fractured limb was splinted in a bohler - braun frame, with the application of skin traction. all the patients were operated within 10 days of the initial trauma. radiological evaluation included anteroposterior and lateral x - rays of the femur with knee, along with a pelvic x - ray to rule out proximal fractures. computed tomography (ct) scans with three - dimensional reconstruction were done, whenever feasible, to better delineate the fracture anatomy and to allow detailed subgroup classification. four of the 12 patients could not afford a ct scan and were operated based on plain films. the ao / ota classification16 was used to further classify the c3 fractures up to the subgroup level, based on the severity of extra - articular involvement [figure 1 ]. the extra - articular component was also described according to the ao / ota subgroup classification of 33a fractures [figure 2 ], which is more detailed and useful in the management of the extra - articular component. ao / ota classification of distal femur type c fractures ao / ota classification of distal femur type a fractures all the patients were operated in the supine position on a radiolucent table by the senior author (aa), who also has a considerable experience in knee arthroplasties [figures 3 and 4 ]. if eversion of the patella was tight, the lateral attachment of the patellar tendon was released to ease eversion, similar to our practice in total knee arthroplasties. we used a leg - holding tray from our total knee arthroplasty set [figure 3 ] to facilitate limb positioning in varying degrees of flexion. in full flexion, an excellent exposure of the distal articular surface of the femur (including the posterior part of the medial femoral condyle) is obtained [figures 3 and 4 ]. (a - c) preoperative x - rays anteroposterior and lateral views of knee joint and computed tomography scans (case no. (d) clinical photograph showing patient positioning and draping with leg - holding tray. (f) immediate postoperative anteroposterior and lateral x - rays of knee joint showing implant in situ. (g) three months postoperative x - rays anteroposterior and lateral views showing fracture consolidation. (h and i) clinical photograph showing range of motion of the knee at 3 months (a - c) preoperative x - rays anteroposterior and lateral views of knee joint and computed tomography scans (case no. (f) immediate postoperative anteroposterior and lateral x - rays showing implant in situ. (i and j) clinical photographs of patient showing range of motion of the knee at 3 months the articular block was reconstructed first, which was subsequently reattached to the metadiaphysis. the distal femur articular surface was carefully inspected for all fracture lines, and the fracture classification was confirmed. the provisional reduction was done with pointed clamps and k - wires, followed by insertion of lag screws. the usual sequence of reduction was first, any sagittal split of posterior condyles, followed by the coronal splits (hoffa / trochlear fragments), addressing the intercondylar component last. osteochondral fragments were fixed with headless or countersunk screws (for large fragments > 1 cm) or k - wires (for intermediate fragments 510 mm). our patient population can not afford biodegradable pins, otherwise we would have preferred them over k - wires. k - wires were put in a divergent fashion, preferably purchasing the far cortex (bicortical) and were cut flush with the articular surface (lost k - wire technique). after anatomical reconstruction and rigid fixation of the articular block, attention was diverted to the extra - articular fracture, with repeat assessment of the fracture pattern. our philosophy of management of the extra - articular component in distal femur fractures is as follows. we prefer open reduction and compression plating (hybrid fixation) for simple metaphyseal fractures (ao types a1.2, a1.3) or those with an intact wedge (ao type a2.1). fractures with wedge comminution (ao type a2.2, a2.3) or segmental comminution (ao type a3.1, a3.2, a3.3) were managed by indirect reduction and bridge plating using transarticular approach and retrograde plate osteosynthesis (tarpo) technique.7 the length, alignment, and rotation were carefully restored similar to the normal side. medial dissection was avoided in all cases to preserve the biology, and primary bone grafting was not done in any case. the anterolateral incision can be easily extended proximally, as in a standard lateral approach, if open compression plating of the fracture is to be done. the implant used was a distal femur lcp made of stainless steel (ssepl, vadodara, india). it provides provision of inserting up to three 6.5 mm locking cancellous screws, and up to four additional 5 mm locking screws in the articular block. we aimed to put four to five locked screws in the articular block, and four bicortical screws in the metadiaphysis (in case of compression plating) or three to five locking screws (in bridge plating). the working length of a bridge plate was kept at least 2.5 times the working length of the fracture, with a screw density ratio of 0.40.5. the knee was mobilized on the 1 or 2 postoperative day, depending on the degree of postoperative pain. touchdown or partial weight - bearing was begun, depending on the fracture stability, and progressed to full weight bearing upon fracture union at 612 weeks. the patients were discharged only after 90 flexion of the knee was achieved, usually by the 4 or 5 postoperative day. the clinical and radiological evaluation was done by the junior author (vk), who was not a part of the operating team. the patients were clinically examined at the time of discharge from hospital for any malrotation or limb length discrepancy. a limb malrotation 1 cm) or k - wires (for intermediate fragments 510 mm). our patient population can not afford biodegradable pins, otherwise we would have preferred them over k - wires. k - wires were put in a divergent fashion, preferably purchasing the far cortex (bicortical) and were cut flush with the articular surface (lost k - wire technique). after anatomical reconstruction and rigid fixation of the articular block, attention was diverted to the extra - articular fracture, with repeat assessment of the fracture pattern. our philosophy of management of the extra - articular component in distal femur fractures is as follows. we prefer open reduction and compression plating (hybrid fixation) for simple metaphyseal fractures (ao types a1.2, a1.3) or those with an intact wedge (ao type a2.1). fractures with wedge comminution (ao type a2.2, a2.3) or segmental comminution (ao type a3.1, a3.2, a3.3) were managed by indirect reduction and bridge plating using transarticular approach and retrograde plate osteosynthesis (tarpo) technique.7 the length, alignment, and rotation were carefully restored similar to the normal side. medial dissection was avoided in all cases to preserve the biology, and primary bone grafting was not done in any case. the anterolateral incision can be easily extended proximally, as in a standard lateral approach, if open compression plating of the fracture is to be done. the implant used was a distal femur lcp made of stainless steel (ssepl, vadodara, india). it provides provision of inserting up to three 6.5 mm locking cancellous screws, and up to four additional 5 mm locking screws in the articular block. we aimed to put four to five locked screws in the articular block, and four bicortical screws in the metadiaphysis (in case of compression plating) or three to five locking screws (in bridge plating). the working length of a bridge plate was kept at least 2.5 times the working length of the fracture, with a screw density ratio of 0.40.5. the knee was mobilized on the 1 or 2 postoperative day, depending on the degree of postoperative pain. touchdown or partial weight - bearing was begun, depending on the fracture stability, and progressed to full weight bearing upon fracture union at 612 weeks. the patients were discharged only after 90 flexion of the knee was achieved, usually by the 4 or 5 postoperative day. the clinical and radiological evaluation was done by the junior author (vk), who was not a part of the operating team. the patients were clinically examined at the time of discharge from hospital for any malrotation or limb length discrepancy. a limb malrotation 90 flexion after surgery. 1, 5, and 7) returned at 1214 weeks with 130) on table. two of the patients showed back out of one of the locking screws at 6 and 12 weeks followup, but their fractures were healing well and no screw exchange was needed. one patient developed a superficial infection, which settled with regular wound care and oral antibiotics. table 2 shows the immediate postoperative alignment of the operated leg in the coronal, sagittal and axial planes. the primary fixation in this case was in 510 varus and had to be accepted during the course of the fixation due to extensive medial comminution involving the epiphysis [case no. the alignment was maintained at 6 weeks, but a further varus collapse to around 15 varus was noted at final fracture healing at 14 weeks. the alignment was maintained between the immediate postoperative and final assessments in all other patients. coronal, sagittal, and rotational alignment of the operated limbs immediately after surgery the outcome was rated as excellent in seven patients, good in three patients and fair in two patients according to the ksks [table 3 ] at 1 year. three patients showed radiological changes of osteoarthritis, two in the tibiofemoral compartments [table 3, case no. 3 and 5 ] and one in the patellofemoral compartment [table 3, case no. only one of these patients experienced pain during routine day - to - day activities, while the other two were asymptomatic. one other patient [table 3, case no. 7 ] had asymptomatic osteoarthritis in the knee at the time of injury which showed no progression during followup. intraarticular fractures of the distal femur are challenging injuries. these require an extensive surgical approach to visualize and reduce the articular fragments, particularly in complex fractures. the insult to the periarticular soft tissues caused both by the initial trauma and subsequent surgical approach, causes difficulty in early postoperative rehabilitation predisposing to knee stiffness. hence, the outcome of intraarticular fractures of the distal femur remains unpredictable, and these are fraught with complications such as malunion, nonunion, stiffness, and secondary osteoarthritis. multiple surgical approaches have been described previously to obtain a good exposure of distal femoral articular surface, including medial parapatellar approach,12 lateral parapatellar (anterolateral) approach,7 tibial tubercle osteotomy,514 and combined medial and lateral approaches.3 the latter two approaches are too extensive and frequently lead to complications, such as delayed wound healing, flap necrosis and delayed healing of osteotomy [table 4 ]. the parapatellar approaches provide a sufficient articular exposure but involve splitting of the quadriceps mechanism, which may lead to scarring or adhesions. moreover, they are difficult to extend proximally, if open compression plating of the extra - articular fracture is planned. the proximal extension of a medial parapatellar approach, as described by henry,19 involves cutting through the rectus tendon, which may not be desirable. results of c3 type fractures of distal femur fixed using different techniques starr.13 described a modified anterior approach to the distal femur, which facilitated complete exposure of distal femur articular surface and quicker rehabilitation. we use an anterolateral skin incision for all distal femur fractures as a universal it can be limited to the standard lateral approach for simple fractures, extended distally as a swashbuckler approach in complex fractures, and extended proximally for open compression plating as in standard lateral approach. starr., in their original report, had avoided the use of tourniquet, citing that it can prevent medial retraction of quadriceps muscle.13 however, with our modified technique, we did not face any difficulty in retraction of the quadriceps, even in cases in which a tourniquet was applied. the outcome of distal femur fractures has improved in recent times, with the use of biological and indirect reduction techniques and percutaneous osteosynthesis with bridge plates, usually locked ones. zlowodzki.20 in a systematic review of different fixation techniques in the operative management of acute nonperiprosthetic distal femur fractures found that the use of locked plates is associated with a decreased relative risk of nonunions and infections as compared to compression plates. however, they found an increased relative risk of fixation failures and secondary surgical procedure with the use of locked plates.20 several other studies have expressed concerns that locking plates may cause callus inhibition, with reported nonunion rates up to 20% in distal femur fractures.2122 furthermore, the precontoured locked plates may not conform to the bony contours in all patients and may lead to malalignment.23 bridge plating by tarpo technique requires a demanding surgical technique and extensive use of intraoperative imaging.7 in our experience, fractures of the distal femur (both types a and c) without extra - articular comminution (a1.2, a1.3, and a2.1) have a good outcome with open compression plating (hybrid technique), which remains our procedure of choice for these, for reasons of technical ease, accurate pretraumatic alignment and more reliable healing. we reserve the use of bridge plating by tarpo technique for fractures with extra - articular comminution. we have analyzed the reported outcomes of complex c3 type fractures in previously published studies [table 4].123456 most of them reported a varying proportion of patients (7.7%16.7%) with poor results due to complications such as superficial infection, flap necrosis and stiffness. srbu.6 in a study of c3 fractures fixed by a lateral locked plate using tarpo approach reported 100% good to excellent results, though with a lower mean flexion of the knee (108) as compared to our series (120). this may be explained by the different outcome scores employed in the two studies. as previously reported by other authors, patients knee function assessed by the kss is noted as less optimal as compared to the neer score.24 a standardized and validated outcome scoring system needs to be developed for distal femur fractures to allow comparability across studies. we attribute the good knee flexion obtained in our series to anatomical articular reduction, rigid fixation of articular fracture lines and early mobilization. concomitant addressal of ligament injuries, as was done in our series, is important for early rehabilitation as patients are apprehensive to move unstable joints. most previous studies1234 on c3 fractures of the distal femur have employed the use of primary bone grafting to enhance union. with the advent of minimally invasive reduction techniques and bridge plating, we used the tarpo technique for comminuted extra - articular fractures and did not employ primary bone grafting in our series. only one patient required secondary bone grafting at 3 months, which was due to technical reasons (slight gap left at fracture site). in our experience, a single lateral locked plate gives sufficient stability for most distal femur fractures, even with extensive comminution. before the advent of locking plates, there was a propensity for varus collapse with a single lateral plate in comminuted fractures, and double plating with bone grafting was recommended.12 however, a single locked lateral plate gives sufficient axial and torsional rigidity,25 even in cases with medial comminution, and the use of dual plates is no longer required. the strength of our study is the inclusion of only the most complex fractures of the distal femur (ao type c3). the study was a prospective one, with all cases operated by a single surgeon, using a single surgical approach, and the same type of implant, eliminating any confounding of results due to variation in surgical technique, reduction skills, or implant properties. since we have only included the complex c3 type distal femur fractures, the total number of patients was limited in our study, similar to other such studies.123456 another limitation in our study is the short followup period. the true incidence of posttraumatic osteoarthritis can be estimated only by a long term study though early trends are reflected in our study. our study shows a good to excellent functional outcome of complex c3 type distal femur fractures, fixed with a single lateral locked plate, using a modified swashbuckler approach. | background : complex ao type c3 fractures of the distal femur are challenging injuries, fraught with complications such as malunion and stiffness. we prospectively evaluated a consecutive series of patients with complex ao type c3 distal femur fractures to determine the clinicoradiological outcome after fixation with a single locked plate using modified swashbuckler approach.materials and methods:12 patients with c3 type distal femur fractures treated with a lateral locked plate, using a modified swashbuckler approach, were included in the study. the extraarticular component was managed either by compression plating or bridge plating (transarticular approach and retrograde plate osteosynthesis) depending on the fracture pattern. primary bone grafting was not done in any case. the clinical outcome at 1 year was determined using the knee society score (kss). the presence of any secondary osteoarthritis in the knee joint was noted at final followup.results:all fractures united at a mean of 14.3 4.7 weeks (range 626 weeks). there were no significant complications such as nonunion, deep infection, and implant failure. one of the patients underwent secondary bone grafting at 3 months. the mean range of motion of the knee was 120 14.8 (range 105150). seven patients had excellent, three patients had good and two patients had a fair outcome according to the kss at 1 year. at a mean followup of 17.6 months, three patients showed radiological evidence of secondary osteoarthritis of the knee joint. however, only one of these patients was symptomatic.conclusion:the results of complex c3 type distal femur fractures, fixed with a single lateral locked plate using a modified swashbuckler approach, are encouraging, with a majority of patients achieving good to excellent outcome at 1 year. |
the oxidation of alkanes to alcohols or ketones and the dehydrogenation of alkanes to alkenes are both widely studied targets for c h bond functionalization. for example, the oxidation of cyclohexane to a mixture of cyclohexanol and cyclohexanone is a large - scale commercial process for the production of adipic acid. the oxidation of propene at the allylic c h bond to form acrolein also is a well - known large - scale c h bond oxidation process, and the oxidation of allylic c h bonds to allylic esters is being studied actively for applications in target - oriented synthesis. the dehydrogenation of light alkanes is being studied as a route to ethylene, propene, butene, butadiene, isobutene, and isoprene, with hydrogen as the single side product or with an oxidant to consume the hydrogen and make the reaction, called oxidative dehydrogenation (odh), favorable thermodynamically. although alkane dehydrogenation and allylic oxidation are both known reactions, the combination of these two reactions in a single process is rare. one can envision that such a process could occur by initial dehydrogenation of an alkane to an alkene, followed by oxidation of allylic c h bonds in the alkene product. indeed, one form of such a reaction is the well - established synthesis of maleic anhydride from butane. however, the combination of dehydrogenation and selective oxidation of the alkene to an allylic alcohol derivative directly from an alkane is poorly developed. recently, two examples of copper - catalyzed oxidative dehydrogenative cross - coupling reactions of an aldehyde and toluene with cyclohexane to generate allylic esters have been reported. however, the yields of these reactions were generally low and occurred with limited substrate scope. copper - catalyzed combinations of alkane dehydrogenation and aziridination or epoxidation also have been reported, but the epoxide and aziridine are just one component of a mixture of products, and they formed with a maximum of 34 turnovers. thus, a high - yield combination of dehydrogenation and c h bond oxidation of an alkane to form an allylic alcohol derivative that occurs with tolerance for a wide range of functional groups is not known. herein, we report the copper - catalyzed oxidative dehydrogenative carboxylation (odc) of unactivated alkanes in the presence of carboxylic acid derivatives to form the corresponding allylic ester (scheme 1). sosnovsky reaction, but the starting material is an alkane, rather than an alkene. the reactions occur by oxidative dehydrogenation of an alkane and oxidation of the resulting allylic c h bond. detailed mechanistic studies show that the tert - butoxy radical abstracts a c h bond of cyclohexane to generate a transient cyclohexyl radical, and this radical is converted to cyclohexene by a copper benzoate complex. the relative rates for trapping of the radical by the ligand on copper versus conversion of the radical to an alkene control the selectivity for the formation of allylic vs alkyl ester products. to extend our recently published copper - catalyzed amidation of cyclohexane to the acetoxylation or benzyloxylation of cycloalkanes, we conducted the reaction of cyclohexane with benzoic acid and tbuootbu in the presence of [(phen)cu](2-i)2 (phen = 1,10-phenanthroline). we envisioned that the combination of the cu(i) and tbuootbu should generate tbuo, which could generate cyclohexyl radical, and this radical could combine with [(phen)cu(o2cph)2 ] to form cyclohexyl benzoate. although benzoic acid did react with cyclohexane and tbuootbu in the presence of 2.5 mol% of [(phen)cu](2-i)2, this combination of materials yielded the allylic ester cyclohex-2-en-1-yl benzoate (21%) and methyl benzoate (16%), not the alkyl benzoate (scheme 2). thus, the reaction of cyclohexane with benzoic acid and tbuootbu in the presence of copper occurs by a combination of dehydrogenation and c h bond carboxylation.scheme 2initial studies of odc of cyclohexane to increase the yield of the allylic ester from this reaction, we evaluated the reactivity of benzoic acid (0.5 mmol) and cyclohexane (10 equiv) with a series of copper salts and discrete copper complexes. simple cu(i) and cu(ii) halides catalyzed the coupling of benzoic acid and cyclohexane to produce cyclohex-2-en-1-yl benzoate in moderate to good yields (entries 17). for example, the combination of cucl (5 mol%) and tbuootbu (3 equiv relative to benzoic acid) gave a high yield of product (76%). reactions conducted with a higher 10 mol% loading of cu occurred in a lower yield (54%) than did the reaction with 5 mol% copper (entry 4). a similar trend of lower yield with higher loadings of catalyst was observed for the copper - catalyzed amidation of cyclohexane. the lower yield of product from reactions containing higher concentrations of copper presumably results from quenching of the transient tert - butoxy radical by cu(i) to form cu(ii)-otbu species. conditions : 0.5 mmol of acid, 5.0 mmol of cyclohexane, 0.025 mmol of catalyst, 1.0 mmol of oxidant, 1 ml of c6h6 at 100 c for 24 h. h nmr yield with meno2 as the internal standard added after reaction. l1, me2nch2ch2n = ch (2-oh - c6h4) ; bpi, bis(2-pyridylimino)isoindole ; phth, phthalimide. the reaction also occurred when catalyzed by cu(i) complexes containing neutral bidentate nitrogen ligands, such as 4,7-dichloro-1,10-phenanthroline, 3,4,7,8-tetramethyl-1,10-phenanthroline, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, 4,5-diazafluoren-9-one, and 1,10-phenanthroline-5,6-dione. the reactions catalyzed by these cu(i) complexes ligated by dative nitrogen ligands formed cyclohex-2-en-1-yl benzoate (2364%) in modest yields. reactions catalyzed by the well - defined [(l1)cucl ] (l1 = me2nch2ch2n = ch(2-o - c6h4), [(bpi)cu(pph3)2 ], and [(bpi)cucl ] (bpi = bis(2-pyridylimino)isoindole) reproducibly produced the product in 4050% yields (entries 913). the reactions catalyzed by ligated copper complexes gave larger amounts of methyl benzoate than did reactions with unligated copper. consistent with this observation, kochi reported that nitrogen - ligated cu(ii) complexes oxidize alkyl radicals to alkenes more slowly than do simple cu(ii) salts. although these reactions with ligated copper occurred in lower yield than those with simple copper halides, they did give substantial amounts of product and were valuable for studying the mechanism of this reaction (vide infra). the scope of the odc of cyclohexane with carboxylic acids to form allylic esters is presented in table 2. the yields of these reactions are based on carboxylic acid. the mass balance consisted of unreacted carboxylic acid and methyl benzoate, the origin of which the reaction is tolerant of halogens on the benzoic acid 4-x - c6h4-co2h (x = f (1a), cl (2a), br (3a)), forming the corresponding allylic esters in 5779% yields in these cases. the reaction is also tolerant of a halide (1c, 2c), methoxy (4c), and acetyl group (5c) in the ortho position. carboxylic acids containing electron - donating substituents on the aromatic system, such as methyl (1b, d, e), tert - butyl (2b), methoxy (1f), 4-phenoxy (2f), and phthalimido (l) groups, generated the corresponding allylic ester products in 5671% yields. substrates containing electron - withdrawing substituents, such as acetyl (m), trifluoromethyl (j), cyano (p), and carboalkoxy (n) groups, also gave the corresponding products in moderate to good yields (5276%). even a thioether (h) is tolerated, despite the oxidizing conditions of the catalytic reaction ; cyclohex-2-en-1-yl 4-(methylthio)benzoate was produced in 69% yield. yields were determined by h nmr spectroscopy with meno2 as internal standard, added after the reaction, and reported as an average of two reactions. the reactions with heteroaryl carboxylic acids, such as furan (q) and thiophene (r), also gave substantial yields of allylic esters ; however, pyridine carboxylic acids did not yield allylic oxidation products. finally, vinyl and aliphatic carboxylic acids reacted to form allylic esters. specifically, the odc of cyclohexane with cyclohexanecarboxylic acid (s), (e)-2-methyl-3-phenylacrylic acid (t), octanoic acid (1u), and phenylacetic acid (2u) gave cyclohex-2-en-1-yl cyclohexanecarboxylate (62%), cyclohex-2-en-1-yl (e)-2-methyl-3-phenylacrylate (57%), cyclohex-2-en-1-yl octanoate (69%), and cyclohex-2-en-1-yl 2-phenylacetate (69%), respectively. the reaction also occurred with smaller or larger cycloalkanes and, to an extent, with acyclic alkanes. reactions of benzoic acids with cyclopentane and cycloheptane in the presence of 5 mol% of cucl yielded the corresponding products in good yield (cyclopent-2-en - yl benzoate (1v, 75%), cyclohept-2-en - yl benzoate (2v 75%)), but the reaction with cyclooctane formed cyclooct-2-en - yl benzoate (3v, 12%) in modest yield. in addition to reactions of cyclic alkanes, reactions of linear alkanes (i.e., pentane) containing multiple c h bonds were performed to assess the selectivity of the catalytic odc. the reaction of pentane and benzoic acid in the presence of 2.5 mol% of 1-pph3 and tbuootbu produced two products : pent - en-2-yl benzoate (1w, 26%) and pent-1-en-3-yl benzoate (2w, 10%). the potential product of pen-2-en-1-yl benzoate, which would be obtained from the oxidation of the pent-2-ene intermediate at the primary c h bond, was not observed. h bond is favored over oxidation of a primary allylic c h bond. the reaction of 2,2-dimethylpentane and benzoic acid produced 4,4-dimethylpent-1-en-3-yl benzoate (y, 16%) and methyl benzoate (80%) as the major byproduct. although most of the catalytic reactions were performed with cucl as catalyst, copper complexes ligated by the imidobipyridine ligand bpi did catalyze the reaction, and the molecular complex [(bpi)cu(o2cph) ] (1-o2cph) was amenable to isolation. the soluble, single - component cu(ii) species 1-o2cph was prepared in 80% yield as a green solid by salt metathesis between [(bpi)cucl ] (1-cl) and nao2cph in meoh at room temperature for 3 h (scheme 3). elemental analysis of the product was consistent with the proposed atomic composition for 1-o2cph. we suspect that the molecular structure of 1-o2cph is similar to that of the derivatives of [(bpi)cux ] (x = 2,6-dimethoxybenzoate and 3,4-dimethoxybenzoate) (vide infra). to assign the oxidation state of the copper center, we performed x - band epr measurement on 1-o2cph in toluene at 25 k. the x - band epr spectrum of 1-o2cph revealed an axial pattern, consistent with a cu(ii) (s = 1/2) center. to isolate a discrete cu(i) complex, [(pph3)2cu(oac) ] the reaction in toluene at room temperature formed [(bpi)cu(pph3)2 ] (1-pph3) in 58% yield as an orange crystalline solid (scheme 3). compound 1-pph3 was characterized by multinuclear (h, c, p) nmr spectroscopy, ft - ir spectroscopy, and elemental analysis. with discrete cu(i) and cu(ii) complexes in hand, we investigated the resting state of the catalyst. the resting state of the copper species in the reaction between cyclohexane and benzoic acid catalyzed by 1-pph3 with tbuootbu as oxidant was determined by uv vis spectroscopy, x - band epr spectroscopy, and independent synthesis of copper complexes. a mixture of benzoic acid, cyclohexane, and tbuootbu with 5 mol% 1-pph3 in benzene was allowed to react for 2 h at 100 c. the uv vis spectrum of this reaction mixture was identical to that of independently synthesized 1-o2cph recorded in benzene. likewise, the x - band epr spectrum of the reaction mixture collected at 25 k was identical to that of an authentic sample of 1-o2cph. to assess the identity of this complex further, a stoichiometric reaction of 1-pph3 with benzoic acid (1.5 equiv) and tbuootbu in the absence of cyclohexane was conducted at 100 c for 0.5 h in benzene. this reaction afforded 1-o2cph in 64% isolated yield (scheme 4), as determined by ft - ir, x - band epr spectroscopy, elemental analysis, and comparison of the material to 1-o2cph synthesized independently from the salt metathesis reaction of 1-cl with nao2cph. these data from the spectroscopic measurement of the copper species in the catalytic reactions and of the species formed independently from stoichiometric reactions strongly indicate that a copper(ii)benzoate complex is the resting state of the catalyst. to assess the role of 1-o2cph in the catalytic reaction the reaction of 1-o2cph with cyclohexane was conducted in the presence of tbuootbu at 100 c for 21 h in acetonitrile (scheme 5). the products consisted of cyclohex-2-en-1-yl benzoate (44%) and methyl benzoate (49%). these results are consistent with competitive reactions of a cyclohexenyl radical and a methyl radical with 1-o2cph to produce cyclohex-2-en-1-yl benzoate and methyl benzoate, respectively. the analogous reaction performed in the absence of tbuootbu gave no product from reaction of the cyclohexane. these results show that the copper benzoate does not react directly with the alkane. instead, a species generated from copper and tbuootbu reacts with the alkane. to assess the sequence of bond - forming events in the catalytic odc of cyclohexane, we conducted the reaction of cyclohexyl benzoate and cyclohexene (separately) (scheme 6,a) with tbuootbu and the copper catalyst. these two reactions reveal whether formation of the alkene occurs before or after formation of the c the reaction of cyclohexyl benzoate, benzoic acid, and tbuootbu with 2.5 mol% of 1-o2cph at 100 c for 24 h did not form cyclohex-2-en-1-yl benzoate. the detection of methyl benzoate indicates that tert - butoxy radical was generated from the reaction of tbuootbu with copper, but that this radical reacts more slowly with the cyclohexyl benzoate than it undergoes -methyl scission to generate the methyl radical (which reacts with the copper benzoate complex to form methyl benzoate). in contrast to the reaction of cyclohexyl benzoate, the reaction of cyclohexene with benzoic acid and tbuootbu in the presence of 2.5 mol% 1-o2cph generated the allylic ester. this reaction formed cyclohex-2-en-1-yl benzoate in 58% yield and methyl benzoate in 27% yield after 24 h at 100 c (scheme 6b). moreover, the reaction of cyclohexane with tbuootbu in the presence of 1 mol% of 1-o2cph (based on tbuootbu) in benzene - d6 at 100 c for 20 h (scheme 6c) formed cyclohexene in 12% yield, with respect to cyclohexane, as determined by h nmr spectroscopy. these results clearly indicate that odc of cyclohexane to cyclohex-2-en-1-yl benzoate proceeds by initial conversion of the cycloalkane to the cycloalkene, followed by oxidation of the cycloalkene to the final allylic ester product. the mechanism for the initial conversion of cyclohexane to cyclohexene likely proceeds by abstraction of a hydrogen atom from cyclohexane by a tert - butoxy radical to generate a cyclohexyl radical, which undergoes oxidation to the alkene. the oxidation of alkyl radicals to olefins by copper peroxide systems has been studied by kochi and walling. their studies imply that oxidation of the cyclohexyl radical formed in the current system likely generates cyclohexyl cation, which undergoes deprotonation to form the alkene. the deprotonation could occur by the anionic cu(i) complex [(bpi)cu(o2cph) ] (1-o2cph) (scheme 7). the yield of allylic ester would then be a function of the relative rate of oxidation of the alkyl radical versus reaction of the alkyl radical with the copper carboxylate. after formation of cyclohexene, oxidation at the allylic position to form cyclohex-2-en-1-yl benzoate would occur through the mechanism of the kharasch sosnovsky reaction. in this pathway, the allylic hydrogen is abstracted by a tert - butoxy radical, and the resulting allylic radical reacts with the copper carboxylate to form the allylic ester. to detect for the possible formation of 1,3-cyclohexadiene or benzene from cyclohexene through a series of steps involving abstraction of the allylic hydrogen, oxidation of the allyl radical, and deprotonation of the allyl cation, the catalytic reaction of benzoic acid, cyclohexane, and tbuootbu in the presence of 1-pph3 (2.5 mol%) in d6-benzene (or d3-mecn) at 100 c for 16 h was monitored by h nmr spectroscopy. the result of the reaction revealed only cyclohex-2-en-1-yl benzoate and methyl benzoate as products in 40% and 14% yields, with respect to benzoic acid. the stoichiometric and catalytic odc of benzoic acid with cyclohexane forms methyl benzoate as the major side product. the observation of this product is consistent with the intermediacy of tert - butoxy radical. -methyl scission of a tert - butoxy radical is known to produce a methyl radical, and this radical would react with the resting - state 1-o2cph to give methyl benzoate. to evaluate the potential generation of tert - butoxy radical in the system, the standard catalytic reaction of benzoic acid in c6d6 was performed in the absence of cyclohexane (scheme 8a). without a source of an alkyl radical besides the one formed by -methyl scission of otbu, the reaction produced a quantitative yield of methyl benzoate and acetone (based on benzoic acid as limiting reagent). this high - yield formation of methyl benzoate from benzoic acid and tbuootbu in the presence of 1-pph3 further supports the intermediacy of a transient tert - butoxy radical in the catalytic reaction. as a final test of the potential intermediacy of tert - butoxy radical in the catalytic process, we conducted reactions in the presence of diphenylmethanol, a known trap for tert - butoxy radical, and in the presence of 9,10-dihydroanthracene, which forms anthracene via hydrogen atom abstraction by alkoxy radicals. the catalytic reaction of cyclohexane, benzoic acid, and diphenylmethanol in the presence of 1-o2cph produced methyl benzoate (18%), benzophenone, and diphenylmethanol in a ratio of 1:10:7.3 (scheme 8b). the same reaction between cyclohexane and benzoic acid at 100 c for 24 h in the presence of 9,10-dihydroanthracene produced anthracene as the exclusive product from the hydrocarbon reactants (scheme 8c). the formation of benzophenone and anthracene is consistent with h - atom abstraction of the methine c h bond of diphenylmethanol and a methylene c h bond of dihydroanthracene by tert - butoxy radical to produce the organic products. the detection of methyl benzoate as an additional product, again, is consistent with -methyl scission of a tert - butoxy radical under the catalytic conditions. to assess the potential intermediacy of a cyclohexyl radical, the catalytic reaction of cyclohexane with benzoic acid was performed in the presence of cbr4. the observation of bromocyclohexane is further consistent with the formation of cyclohexyl radical by abstraction of a hydrogen atom from cyclohexane by a tert - butoxy radical in the catalytic reaction. in this case parallel reactions were performed with cyclohexane and cyclohexane - d12 to determine if cleavage of the c h bond of the alkane is the turnover - limiting step of the copper - catalyzed odc of cyclohexane. a comparison of the initial rates for catalytic odc of octanoic acid with cyclohexane and cyclohexane - d12 in separate vessels revealed a kie value of 2.8 0.2 (scheme 10). in addition, a comparison of the rates of the catalytic reaction of octanoic acid with cyclohexane and cyclohexene revealed that the conversion of cyclohexene to cyclohex-2-en-1-yl octanoate is faster than the conversion of cyclohexane. after 1 h, the reaction of octanoic acid and cyclohexene cleanly produced 40% of cyclohex-2-en-1-yl octanoate, whereas the reaction of octanoic acid with cyclohexane produced only 4% of cyclohex-2-en-1-yl octanoate and 2% of methyl octanoate. h bond from cyclohexane, not from cyclohexene, is the turnover - limiting step in the catalytic odc. the roles of copper in the catalytic odc of cyclohexane are closely related to those of copper in the catalytic amidation of cyclohexane we reported recently. however, the two reactions form products containing different hydrocarbyl groups (alkyl vs allylic), and the difference between these groups likely stems from a difference in relative rates for reaction of the alkyl radical with the copper benzoate and copper the alkyl radical can undergo electron transfer, or it can combine with a ligand at copper to form a product containing a new carbon heteroatom bond (scheme 11). apparently, oxidation of the alkyl radical by the copper benzoate is faster than reaction of the alkyl radical with the benzoate ligand, whereas oxidation of the alkyl radical by the copper amidate is slower than reaction of the alkyl radical with the amidate ligand (scheme 11). this difference in relative rates could arise from a difference in redox potential of the benzoate and amidate complexes. a cu(ii)benzoate complex, presumably, is less electron - rich than a cu(ii)amidate complex. therefore, the former complex could oxidize the alkyl radical to an alkyl cation faster than the latter complex. alternatively, the difference in relative rates could arise from differences in the rates of reaction of alkyl radicals with the cu(ii)carboxylate and cu(ii)amidate complexes. to reveal the origin of the difference in formation of alkyl and allyl products with amide and carboxylic acid reagents we conducted a series of reactions in which a methyl radical is generated in the presence of a copper carboxylate, amidate, or imidate complex. first, the reaction of a source of methyl radical (tbuootbu) with a combination of [(bpi)cu(nhc(o)[heptyl ]) (1-nhc(o)hept) and [(bpi)cu(o2c[heptyl ]) ] (1-o2chept) at 100 c (scheme 12a) was performed to assess the ratio of products resulting from reactions of alkyl radicals with cu(ii)amidate and cu(ii)carboxylate complexes together. this reaction produced only menhc(o)[heptyl ] (52% at 24 h) ; meo2c[heptyl ] was not observed by gc. thus, the rate of reaction of methyl radical with 1-nhc(o)hept is faster than that with 1-o2chept. analogous reactions of methyl radical with a combination of 1-o2cph and either [(bpi)cu(nhc(o)ph) ] (1-nhc(o)ph) or [(bpi)cu(phth) ] (1-phth) in the presence of tbuootbu (20 equiv) in benzene at 100 c also showed that the reaction of methyl radical with 1-nhc(o)ph and 1-phth is faster than that with 1-o2cph. second, a competition reaction was performed between unligated [cu(nhc(o)[heptyl ]) ] and [cu(o2c[heptyl ]) ] in the presence of tbuootbu at 100 c in benzene (scheme 12b) to probe the effect of the bpi ancillary ligand on the rate of reaction of the methyl radical with the cu(ii)amidate and cu(ii)carboxylate complexes. like the reactions with the ligated copper complexes, the reaction of the unligated complexes with the source of me produced higher yields of menhc(o)[heptyl ] than of meo2c[heptyl ] throughout the reaction. the result of this experiment indicates that the rate of reaction of a methyl radical with a ligandless copper amidate is faster than that with a ligandless copper carboxylate. third, a competition reaction was performed with 1-o2chept and 1-nhc(o)hept in the presence of cyclohexane and tbuootbu at 100 c in benzene (scheme 13). unlike a methyl radical heteroatom bond or convert to cyclohexene ; carboxylation of the resulting alkene then forms an allylic ester. the reaction of the two copper complexes formed n - cyclohexyloctanamide in 92% yield and the allylic ester product cyclohex-2-en-1-yl benzoate in 30% yield. this result is consistent with faster reaction of an alkyl radical with a copper amidate than with a copper benzoate, but the origin of the absence of product from reaction of the allylic radical with the copper amidate is unclear. we also observed the odc of cyclohexane to cyclohex-2-en-1-yl benzoate only after an appreciable amount of 1-nhc(o)hept is consumed because 1-nhc(o)hept would react rapidly with the transient alkyl radical. to investigate the effects of the nitrogen substituents on the reactions with alkyl radicals, we conducted the reaction of 1-nhc(o)ph and 1-phth with tbuootbu (20 equiv) in benzene at 100 c for 24 h. this reaction formed higher yields of the menhc(o)ph (70%) than of me - phth (53%) (scheme 14). the result of this competition experiment indicates that the reaction of the alkyl radical with 1-nhc(o)ph is faster than that with 1-phth. this trend is consistent with faster reaction of an alkyl radical with the more electron - rich anionic ligand on copper. to gain more systematic data conerning the electronic effects on the rates of reactions of alkyl radicals with the copper complexes, we studied reactions with a series of substituted benzoate complexes. reactions of a methyl radical generated from tbuootbu with a mixture of [(bpi)cu(o2c[c6h4 - 4-ome ]) (1-ome) and [(bpi)cu(o2c[c6h4 - 4-cn ]) (1-cn) were conducted at 100 c in benzene (scheme 15a). the result showed that methyl radical reacted faster with the electron - rich 1-ome to produce the corresponding methyl 4-methoxybenzoate than with the more electron - poor 1-cn to produce methyl 4-cyanobenzoate. for example, at 24 h the reaction produced methyl 4-methoxybenzoate in 76% yield and methyl 4-cyanobenzoate in 10% yield. to gain analogous information on the reaction of an alkyl radical with copper amidates, the analogous experiment was conducted with [(bpi)cu(nhc(o)[c6h4 - 4-ome ]) (1-nhome) and [(bpi)cu(nhc(o)[c6h4 - 4-cf3 ]) (1-nhcf3). the reaction of tbuootbu with these complexes at 100 c in benzene (scheme 15b) showed that the methyl radical reacts faster with the more electron - rich 1-nhome than with the more electron - poor 1-nhcf3 to produce the corresponding product of n - methyl-4-methoxybenzamide (53%) at 24 h. the results of these competition reactions clearly demonstrate that alkyl radicals react faster with the more electron - rich copper benzoate and amidate complexes than with the more electron - deficient copper benzoate and amidate complexes to form the corresponding n - alkyl and o - alkyl products, respectively. to elucidate the steric effect of aromatic ring of the carboxylate ligand in copper benzoate complexes on the reactivity, we performed reactions of cyclohexane and tbuootbu in the presence of a series of copper benzoates containing methyl groups in the ortho, meta, and para positions : [(bpi)cux ], with x = 2,6-dimethylbenzoate (2,6-me2), 2,4-dimethylbenzoate (2,4-me2), and 3,4-dimethylbenzoate (3,4-me2)). the reaction with the combination of 2,4-me2 and 2,6-me2 formed a higher yield of cyclohex-2-en-1-yl 2,4-dimethylbenzoate than cyclohex-2-en-1-yl 2,6-dimethylbenzoate (scheme 16a). the reaction with a combination of 2,4-me2 and 3,4-me2 formed the corresponding cyclohex-2-en-1-yl 3,4-dimethylbenzoate in higher yields than it formed cyclohex-2-en-1-yl 2,4-dimethylbenzoate (scheme 16b). lastly, the reaction with the combination of 3,4-me2 and 2,6-me2 produced higher yield of cyclohex-2-en-1-yl 3,4-dimethylbenzoate (40%) than of cyclohex-2-en-1-yl 2,6-dimethylbenzoate (20%) at 24 h (scheme 16c). similar results were obtained from competition reactions between cyclohexane and tbuootbu with a series of dimethoxybenzoate the difference in rates of reaction of the carboxylate complexes as a function of the steric properties could result from the effect on the conformation of these copper benzoate complexes and overlap of the aryl system with the carbonyl group. the molecular structures of 2,6-ome2 and 3,4-ome2 were determined by x - ray diffraction (figure 1). the aryl ring of the 2,6-dimethoxybenzoate is nearly orthogonal to the carbonyl group with a torsion angle of 76, whereas the benzene ring of the 3,4-dimethoxybenzoate lies nearly in plane with the carbonyl group with a torsion angle of 163. thus, the aryl ring of 3,4-ome2 has more orbital overlap between the carbonyl and benzene -systems than does that of the 2,6-dimethoxylbenzoate. this distortion from planarity of the 2,6-disubstituted benzoate makes it less nucleophilic (scheme 17). this reduced nucleophilicity is then responsible for the difference in the reactivity of copper benzoate with the allylic radical to generate allylic benzoate products. molecular structures of [(bpi)cu(3,4-ome2-c6h4) ] (3,4-ome2) (top) and [(bpi)cu(2,6-ome2-c6h4) ] (2,6-ome2) (bottom) are shown with 50% thermal ellipsoid. selected bond lengths () and angles () for 3,4-ome2 : cu1n1 = 2.019(2) ; cu1n3 = 1.892(2) ; cu1o1 = 1.9443(18) ; n1cu1n3 = 90.55(9) ; n3cu1o1 = 168.23(9). selected bond lengths () and angles () for 2,6-ome2 : cu1n1 = 20074(17) ; cu1n3 = 1.9017(17) ; cu1o1 = 1.9721(14) ; n1cu1n3 = 90.94(7) ; n3cu1o1 = 167.63(7). the step that distinguishes the reactivity of copper - catalyzed odc of cyclohexane and copper - catalyzed amidation of cyclohexane is the reaction of the transient cyclohexyl radical with a copper benzoate versus a copper amidate or copper imidate intermediate. moreover, more electron - rich copper amidate and benzoate complexes react faster with alkyl radicals than more electron - deficient copper amidate and benzoate complexes. benzoate allows electron transfer to occur faster than ligand transfer, unless the alkyl radical is not able to form an alkene (i.e., a methyl radical). when the alkyl radical can not form an alkene, ligand transfer occurs to form an o - alkyl product. based on these hypotheses, we investigated copper - catalyzed oxidative dehydrogenative amination (oda) of cyclohexane with electron - deficient amides. more electron - deficient copper amidate and imidate complexes would undergo slower reactions with an alkyl radical and faster electron transfer. to this end, we conducted catalytic reactions of phthalimide and cyclohexane (10 equiv) in the presence of 2.5 mol% of [(phen)cu(phth) ], 1-pph3, and [cu(phth) ] (scheme 18). the reactions in the presence of 1-pph3 and [cu(phth) ] produced 1825% of cyclohex-2-en-1-yl pthalimidate and roughly 70% of n - cyclohexylphthalimide. the reactions conducted with 1-pph3 and [cu(phth) ] apparently occur by competitive ligand transfer and electron transfer to form a mixture of n - cyclohexylphthalimide and cyclohex-2-en-1-yl pthalimidate, respectively. conversely, the reaction catalyzed by [(phen)cu(phth) ] formed n - cyclohexylphthalimide (63%) and n - methylphthalimide (19%), and no cyclohex-2-en-1-yl pthalimidate, as determined by gas chromatography, mass spectrometry, and h nmr spectroscopy. the catalytic reaction in the presence of [(phen)cu(phth) ] exclusively produced n - cyclohexylphthalimide and n - methylphthalimide, presumably from reaction of the methyl and cyclohexyl radicals with the resting - state species [(phen)cu(phth)2 ]. the preference of [(phen)cu(phth)2 ] to react with cyclohexyl radical by ligand - transfer is presumably because [(phen)cu(phth)2 ] is more electron - rich than the complexes 1-phth and [cu(phth)2]n ; thus, reaction of the alkyl radical with [(phen)cu(phth)2 ] occurs faster than electron transfer. previously, we reported the copper - catalyzed oxidative coupling of p - toluenesulfonamide with cyclohexane to generate the corresponding n - cyclohexyl - p - toluenesulfonamide. to achieve oda of cyclohexane, we reasoned that replacing p - toluenesulfonamide with the more electron - deficient 4-cf3-benzenesulfonamide should decrease the rate of reaction of the alkyl radical with the ligand and increase the rate of oxidation of the alkyl radical. the reaction of 4-cf3-benzenesulfonamide with cyclohexane and tbuootbu in the presence of 2.5 mol% of 1-pph3 in acetonitrile produced n - cyclohexyl-4-cf3-benzenesulfonamide (50%) and n-(cyclohex-2-en-1-yl)-4-cf3-benzenesulfonamide (38%) (scheme 19). this result demonstrates that electron - deficient sulfonamides can form substantial amount of product from oda of cyclohexane by the electron - transfer pathway. the selectivity of the reaction of cyclohexane with cf3 - 4-benzenesulfonamide in the presence of 1-pph3 depended on solvent and supporting ligand (scheme 19). the reaction in acetonitrile formed the mixture of alkyl and allylic amides just described, but the reaction in benzene generated exclusively cyclohexyl-4-cf3-benzenesulfonamide (57%). the distribution of products from the reaction of cf3 - 4-benzenesulfonamide and cyclohexane in the presence of ligandless copper generated from [cu(mes) ] also depended on solvent, but with the opposite trend. the products of oda were observed in benzene, but not in acetonitrile (scheme 19). like the reactions of phthalimide, the reactions of cf3 - 4-benzenesulfonamide with cyclohexane in the presence of phen - ligated copper provided only the n - alkyl product (scheme 19). this result again highlights the effect of the electronic properties of the supporting ligand on the relative rates to form n - alkyl and n - allyl products. this effect of ligand (i.e., bpi vs phen) on this selectivity reflects an opportunity to design ligands that favor oda of unactivated alkanes. a proposed mechanism for the catalytic odc of cyclohexane to cyclohex-2-en-1-yl benzoate is presented in scheme 20. in this pathway, catalysis is initiated by the decomposition of tbuootbu by 1-pph3 to produce a tert - butoxy radical and [(bpi)cuotbu ], which rapidly reacts with benzoic acid to form 1-o2cph and tbuoh. the tert - butoxy radical can undergo reversible, secondary internal return to regenerate tbuootbu or abstract a hydrogen atom from cyclohexane to generate a cyclohexyl radical. to form the unsaturated product, 1-o2cph would oxidize the alkyl radical by one electron to form a carbocation, and the carbocation would undergo deprotonation by an anionic cu(i) species [(bpi)cu(o2cph) ] to give cyclohexene and benzoic acid. the resulting cyclohexene would then undergo a second c h abstraction by a tert - butoxy radical to give an allylic radical that reacts with 1-o2cph to release the allylic ester product and regenerate a (bpi)cu(i) species to complete the catalytic cycle. in a side reaction, the tert - butoxy radical would decompose to a methyl radical and acetone. the methyl radical would then combine with 1-o2cph to produce methyl benzoate and a (bpi)cu(i) species. the catalytic cycle for oda of cyclohexane to form n - allyl products is presumably analogous to that of the catalytic odc. this catalytic cycle would contain a copper amidate resting state, based on mechanistic investigations of a related copper - catalyzed amidation of unactivated alkanes. in summary, we have described a copper - catalyzed oxidative dehydrogenative carboxylation (odc) of unactivated alkanes with a variety of benzoic acids to produce the corresponding allylic ester products. a measurement of kinetic isotope effects showed that the turnover - limiting step is c h bond cleavage, and experiments to trap radical intermediates revealed that a transient tert - butoxy radical cleaves the c h bond of the alkane to generate an alkyl radical. reactions of alkyl radicals with a combination of cu(ii)amidates and cu(ii)benzoates revealed that the alkyl radical reacts faster with a cu(ii)amidate than with a cu(ii)benzoate to form n - alkyl products. additional mechanistic investigations indicated that the electronic properties of the cu(ii)x (x = amidate, benzoate) resting state contributes to the partitioning of the alkyl radical between ligand transfer to form the alkyl heteroatom bond and electron transfer to oxidize the alkyl radical to an olefin, followed by oxidative carboxylation to produce an allylic ester. the reaction of the alkyl radical with a cu(ii)amidate versus a cu(ii)benzoate is the step that distinguishes copper - catalyzed amidation and copper - catalyzed odc of the alkane. this insight into the mechanism of odc of cyclohexane led to preliminary observations of copper - catalyzed oxidative dehydrogenative amination of cyclohexane with electron - deficient nitrogen sources (i.e., phthalimide and an electron - deficient sulfonamide) to form n - allyl products. current efforts are underway to discover reaction conditions to suppress ligand transfer and favor electron transfer to achieve high selectivity for a copper - catalyzed oda of unactivated alkanes. | we report copper - catalyzed oxidative dehydrogenative carboxylation (odc) of unactivated alkanes with various substituted benzoic acids to produce the corresponding allylic esters. spectroscopic studies (epr, uv vis) revealed that the resting state of the catalyst is [(bpi)cu(o2cph) ] (1-o2cph), formed from [(bpi)cu(pph3)2 ], oxidant, and benzoic acid. catalytic and stoichiometric reactions of 1-o2cph with alkyl radicals and radical probes imply that c h bond cleavage occurs by a tert - butoxy radical. in addition, the deuterium kinetic isotope effect from reactions of cyclohexane and d12-cyclohexane in separate vessels showed that the turnover - limiting step for the odc of cyclohexane is c h bond cleavage. to understand the origin of the difference in products formed from copper - catalyzed amidation and copper - catalyzed odc, reactions of an alkyl radical with a series of copper carboxylate, copper amidate, and copper imidate complexes were performed. the results of competition experiments revealed that the relative rate of reaction of alkyl radicals with the copper complexes follows the trend cu(ii)amidate > cu(ii)imidate > cu(ii)benzoate. consistent with this trend, cu(ii)amidates and cu(ii)benzoates containing more electron - rich aryl groups on the benzamidate and benzoate react faster with the alkyl radical than do those with more electron - poor aryl groups on these ligands to produce the corresponding products. these data on the odc of cyclohexane led to preliminary investigation of copper - catalyzed oxidative dehydrogenative amination of cyclohexane to generate a mixture of n - alkyl and n - allylic products. |
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